Société Belge de Physiologie et de Pharmacologie: Belgische Genootschap Voor Fysiologie En...

Archives Internationales de Physiologie el de Biochimie, 1986, 94, P45-Pl2 P45

SOCIETE BELGE DE PHYSIOLOGIE ET DE PHARMACOLOGIE

BELGISCHE GENOOTSCHAP VOOR FYSIOLOGIE EN FARMAKOLOGIE

REUNION DE GAND, 19 AVRIL 1986")

COMMUNICATIONS

0. GURNE, C. HANET, B. HABIB, H. VAN MECHELEN, A. A. CHARLIER and H.

Haemodynamic effects of the benzimidazolinone derivative Men-935 in conscious dogs with chronic renal hypertension (1 figure).

MEN-935 (1- (3- ( (3- (l-naphtoxy)-2-hydroxypropyl)amino)-3,3-dimethylpropy~) -2-benzimidazolinone-hydrochloride monohydrate, adimolol) is a long acting, non cardioselective P-adrenergic blocking drug which also exhibits a weak cY-blocking activity in vitro. The aim of the study was to assess the effects of MEN-935 on the left ventricular (LV) function and regional blood flow distribution in dogs with chronic renal hypertension. Renal hypertension was produced by wrapping the left kidney with silk 2-4 months before study. This resulted in an average increase in mean arterial blood pressure of 38 mmHg (P<O.OOl ) .

Haemodynamic data were first followed by telemetry in 8 conscious, unrestrained hypertensive dogs, during 4 hours after oral administration of MEN-935 (2 mg/kg) or placebo. After the telemetry study, the animals were then sedated with morphine and the effects of MEN-935 on regional blood flow distribution were determined, using radiolabelled microspheres. Regional blood flow determinations were performed in basal conditions and immediately after the end of a phenylephrine infusion (0.3 mg in 2 min).

During telemetry, significant reductions in heart rate (102+- 17 SD after 2 h vs. 122+ 17 beats/min in control, P<O.Ol), in peak( +) dP/dt (3 438 +682 after 2 h vs. 4 076k868 mmHg/s in control, P<O.Ol), accompanied by a slowing of the rate of isovolumic pressure fall (32 k 6 vs. 27 k 3 ms, P< 0.05) were observed rapidly after oral administration of MEN-935. These changes remained significant after 24 h and were not seen after placebo. The LV systolic pressure was slightly reduced 4 h after drug intake but was more reduced after 24 h (154k 17 vs. 180+ 17 mmHg before drug, P< 0.01). The LV end-diastolic pressure remained unchanged.

POULEUR (Departments of Physiology and Cardiology, University of Louvain, Brussels, Belgium).

(I) Publication subsidiee par le MinistPre de I'Education Nationale et de la Culture. Publicatie gesubsidieerd door het Ministerie van Nationale Opvoeding en Culluur.

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P46 SOC. BELGE DE PHYSIOLOGIE ET DE PHARMACOLOGIE, GAND, AVRLL 1986

In morphine-sedated dogs, phenylephrine infusion resulted in significant increases in LV systolic, end-diastolic and mean arterial pressures, and tended to provoke a reflex bradycardia, not only in placebo-treated dogs, but also in dogs pretreated with MEN-935. However, the LV end-diastolic pressure, which was already slightly reduced in basal conditions after MEN-935 administration (17.4 f 14.8 vs. 21.9 f 10.4 mmHg, NS), remained significantly lower during phenylephrine infusion in dogs pretreated with MEN-935 (22.6k 14.8 vs. 32.1 f 13.5 mmHg, Pe0.05).

Before phenylephrine infusion, the average regional blood flows were essentially unaffected by MEN-935 administration. After phenylephrine infusion, in the absence of MEN-935 administration, splenic blood flow decreased by 36% (PeO.05). All other regional blood flows were unchanged or decreased moderately, except for the endocardia1 and epicardial flows which tended to increase (NS). In dogs pretreated with MEN-935, coronary blood flow increased ( P e 0.001) after phenylephrine infusion. Most others regional blood flows, such as splenic, cerebral or skeletal muscles flows were significantly improved when compared to the control phenylephrine infusion. The renal cortical blood flow also improved by 19% (Pe0.05) for the left kidney, and by 17% (P<O.l) for the right one. Since the changes in mean arterial pressure were comparable in the presence or in the absence of MEN-935, this im- provement in regional blood flows indicated that MEN-935 was able to significantly reduce the increase in regional resistances induced by phenylephrine (Fig. 1).

In conclusion, in dogs with chronic renal hypertension, a single oral administration of MEN-935 lowered heart rate, myocardial contractility and arterial pressure, without increasing LV end-diastolic pressure. MEN-935 was also able to significantly reduce the a- mediated vasoconstriction induced by phenylephrine, suggesting that the a-antagonist action of MEN-935 might be clinically relevant.

RK CORTEX LK CORTEX ENDO

5 0 111 50L 0

-15 & 0

-S - 50 L - 30 Resistances are expressed in dynes s cm-5 * P<0.05

** P<0.01

EPI

-15

- 30

t

SPLEEN r look 0

-100 II

FIG. 1 . Effect of MEN-935 on the changes in region6 tascular resistances duringphenylephrine infusion. Changes in resistances are expressed in dynes s cm-5. C : control conditions, M : pretreatment with MEN-935, RK : right kidney, LK : left kidney, ENDO : endocardium, EPI : epicardium.

Arch. int. Physiol, Biochim. 1986, 94 ( 5 )

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SOC. BELGE DE PHYSIOLOGIE ET DE PHARMACOLOGIE, GAND, AVRIL 1986 P47

P. CARMELIET and c. DENEF (Laboratory of Cell Pharmacology, School of Medicine, University of Leuven, Campus Gasthuisberg, 8-3000 Leuven)

The involvement of intercellular communication in the cholinergic stimulation and inhibition of GH release in vitro.

It is well known that acetylcholine can stimulate G H release and inhibit PRL release of rat and bovine anterior pituitary (AP) cells in vitro (MUKHERJEE et at., 1980; YOUNG et at., 1979). These muscarinic effects, however, have been mainly studied in cells cultured as monolayers in a serum-supplemented medium or on freshly dissected A P slices. In this study, AP cell aggregates prepared from immature female rats and cultured for 5 days in a chemical- ly defined medium without serum, were used (DENEF & ANDRIES, 1983). The secretory responses were studied in a superfusion system (DENEF & ANDRIES, 1983).

When the aggregates were cultured in a culture medium without the hormones T, and dexa- methasone (DEX), 10 p~ carbachol had almost no effect on G H release. On the other hand, a marked inhibition ( - 70%) followed by a rebound release of G H was seen when 80 nM DEX was added to the culture medium. In contrast, addition of 1 nM TI t o the culture medium resulted in a stimulation ( f 220%) of G H release while addition of both T, and DEX resulted in an inhibition ( - SOVo), again followed by a rebound.

In order to test the hypothesis that these reciprocal effects of carbachol were based on intercellular communication, aggregates which had been cultured for 5 days in a medium with T, or DEX were redispersed; these redispersed single cells were placed in the superfusion chamber together with biogel P,-beads so that the distance between the individual cells was large enough to eliminate close contacts and intercellular communication between them.

In individual cells, which had been cultured in a I nM T,-supplemented medium, 10 p~ carbachol evoked a stimulation of the G H release (+ 50%), but this stimulation was 3-4 x smaller than that seen in aggregates. The inhibition of G H release as seen in aggregates when they were cultured in a 80 nM DEX-supplemented medium, was completely lost in the redispersed individual cells. Instead, a small stimulation (+ 50%) was noticed.

These data suggest that the acetylcholine agonist carbachol has a direct stimulatory effect on the individual G H cell in both T3-or DEX-supplemented culture media. However, when the GH cell can make close contacts with other A P cells as is the case in the aggregates, a marked potentiation of this stirnulation is seen in the T,-supplemented medium, suggesting that an as yet undefined A P cell type facilitates the carbachol-induced stimulation of G H release and that this facilitation depends on the presence of T,.

In contrast when the GH cell interacts with other A P cells, which were cultured in a DEX- supplemented medium, no stimulation but a marked inhibition of the G H release occurs in response to carbachol, suggesting the existence of an inter-cell in close contact with the G H cell, which is mediating this inhibitory carbachol effect. The presence and/or the responsiveness or activity of this inter-cell seems to be dependent on a glucocorticoid (DEX).

The use of a system in vitro in which intercellular communication is optimalized (cell ag- gegates) (VANDERSCHUEREN ef at., 1982), in combination with the selective manipulation of the hormone condition of the culture medium (T,/DEX) may explain why both stirnulatory and inhibitory actions of the cholinergic agonist carbachol were observed in the present study, in contrast to previous studies, in which only a stimulation of G H release was seen.

References

DENEF, C. & ANDRIES, M . (1983) Endocrinology 112, 813-822. MUKHERJEE, A., SNYDER, 0. & MCCANN, S. M. (1980) Life Sci. 27, 475-482. VANDERSCHUEREN, B., DENEF, C. & CASSIMAN, J . 4 . (1982) Endocrinology 110, 513-523. YOUNG, P . , BICKNELL, R . J . & SCHOFIELD, D. (1979) J. Endocrinol. 80, 203-213.

Arch, int. Physiol. Biochim. 1986, 94 (5)

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P48 SOC. BELGE DE PHYSIOLOGIE ET DE PHARMACOLOGIE, GAND, AVRIL 1986

H. VANDERSTICHELE, W. EECHAUTE and E. LACROIX (Laboratorium voor Normale en Pathologische Fysiologie, Universiteil Gent, 8-9000 Gent).

The testicular metabolic activity of neonatal androgenized rats. The consequences of androgenization of male rats with a single dose of testosterone before the

fifth day of life are discrete. In adult, neonatally androgenized rats the testes, seminal vesicles and ventral prostate are smaller (SWANSON & VAN DER WERFF TEN BOSCH, 1963) but there are no histological alterations and fertility appears to be normal (JOHNSON & WITSCHI, 1963; KINCL ef al., 1965; BARANAO et al., 1981). The plasma levels of FSH and, to a less extent, of LH are increased while the plasma levels of testosterone are normal (VANDERSTICHELE et al., 1986).

In the present experiments, the capacity of the testes of adult neonatal androgenized rats to produce androgenic steroids was investigated.

Mefhods

Androgenization was accomplished by a subcutaneous injection of 1 mg testosterone propionate on the third postnatal day. The rats were sacrificed at 100 days of age. Production and metabolism experiments were performed as already described (LACROIX et al., 1984; EECHAUTE ef al., 1984).

In the production experiments, the decapsulated pairs of testes were incubated at 37°C for 2 h. The amount of testosterone, 7a-OH-testosterone and 5a-androstanediol was estimated in the in- cubates (testes + medium), after Sephadex and silicagel chromatography, respectively by RIA, fluorimetry and gas chromatography. The reactivity towards an acute hCG stimulus was tested by the i.p. injection of 10 IU hCG (PregnyP ) 2 h bebre sacrifice.

In the metabolism experiments, homogenates of pairs of testes were incubated for various times, depending on the substrate used, at 37°C in the presence of different [4-',C] labelled steroid precursors (testosterone, A,-androstenedione, progesterone, 17a-OH-progesterone and DHEA). The produced metabolites were separated by chromatography and measured by liquid scintillation counting. The yield of metabolites in function of the incubated precursor allows an estimation of the activity of the main enzymes involved in the synthesis of testosterone (30-steroid dehydrogenase-isomerase, 17a-hydroxylase and 17-20 desmolase), as well as in its further metabolism (7a-hydroxylase and 5a-reductase).

Results

The testes of the neonatally androgenized rats were some 25% smaller than the testes of the untreated controls; the plasma testosterone levels were not different.

The production experiments clearly show that the smaller testes of the androgenized rats produce, in basal conditions, the same amounts of testosterone, 7a-OH-testosterone and 5a- androstanediol as the controls. The injection of 10 IU hCG 2 h before sacrifice, which produces a maximum stimulation of the testes, increases very strongly the steroid production and the plasma levels of testosterone, both in the androgenized and the control rats. The increase of steroid production and of testosterone plasma level is, however, markedly less in the androgenized rats.

The metabolism studies show that in the testes of the neonatal androgenized rats, the activity of the enzymes involved in the testosterone synthesis (30-steroid dehydrogenase-isomerase, 1 7 ~ - hydroxylase and 170-steroid dehydrogenase) is significantly increased, without modification of the activity of the 5a-reductase and the 7a-hydroxylase.

The experiments indicate that the testes of the neonatal androgenized rats have a reduced maximum steroidogenic capacity, and that the normal plasma levels of testosterone observed in basal conditions are maintained by an increased testosterone production per unit of steroidogenic tissue in these testes.

References BARANAO, J. L. S., CHEMES, H. E., TESONE, M . , CHIAUZZI, V. A., SCACCHI, P., CALVO, J. C., FAIGON, M .

R . , MOGUILEVSKY, J . A., CHARREAU, E. H. & CALANDRA, R. S. (1981) Biol. Reprod. 25, 851-858. EECHAUTE, W. , LACROIX, E. & LEUSEN, I . (1984) J . Steroid Biochem. 20, 1201-1205. JOHNSON, D. C . & WITSCHI, E. (1963) Acta Endocrinol. (Copenhague) 44, 119-127. KINCL. F. A., FOLCHI PI, A., MAQUEO, M. , HERRERA LASSO, L., ORIOL, A. & DORFMAN, R. 1. (1965) Acta En-

LACROIX, E., EECHAUTE, W. & LEUSEN, I . (1984) J . Steroid Biochem. 20, 633-637. SWANSON, H. E. & VAN DER WERFF TEN BOSCH, J. J . (1963) J. Endocrinol. 26, 197-207. VANDERSTICHELE, H. , EECHAUTE, W., LACROIX, E . & LEUSEN, I . (1986) Arch. int. Physiol. Biochim. 94, P27.

docrinol. (Copenhague) 49, 193-206.

Arch. inl. Physiol. Biochim. 1986. 94 ( 5 )

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S O C . BELGE DE PHYSIOLOGIE ET DE PHARMACOLOGIE, GAND, AVRIL 1986 P49

H. HEIDBUCHEL, J. VEREECKE and E. CARMELIET (Laboratorium voor Fysiologie, KULeuven, Campus Gasthuisberg, 8-3000 Leuven).

Acetylcholine induces an inwardly rectifying K current in human atrial cells.

Single human atrial cells were isolated from pieces of atrial appendage tissue removed during surgical intervention. The isolation procedure was based on en- zymatical dissociation with collagenase and protease in Ca-free medium. The isolated cells were voltage clamped using the whole cell recording technique. The pipette solution concentrations were in mM : 140 KCl, 6 MgCl,, 5 Na2ATP, 10 Hepes, 5 EDTA, 0.154 CaCl, at pH 7.2. Experiments were performed at room temperature.

When the cells are superfused with normal Tyrode's solution containing 5.4 mM KCl, 1 s hyperpolarizing clamp steps from a holding potential at - 50 mV to potentials less negative than - 110 mV result in time-dependent inward current-shifts. For stronger hyperpolarizations current-shifts remain time-independent in a number of cells. In other cells however the inward current can either decrease or increase during the hyperpolarizing step. Sometimes a biphasic response can be observed : after an initial decay the inward current rises again, to reach a steady-state within 1 or 2 s. Experiments using Ba2+ and Cs' enable us to conclude that the decaying inward current component corresponds to the inward rectifying K+-current i,,, which largely determines the resting potential. The component which increases as a function of time has properties similar to the i, pacemaker current.

Addition of Ach (2 x 1 0 - 6 ~ ) shifts the steady-state current in the outward direction at potentials positive to the K equilibrium potential and in the inward direction at more negative potentials. The Ach-induced current therefore reverses at E,. It shows inward rectification and is blocked by millimolar concentrations of Ba2+. A number of properties however, dissociate the Ach-induced current from i,, : a) the Ach-induced current increases as a function of time during hyperpolarizing clamp steps, whereas i,, decreases under those conditions; b) the degree of inward rectification is less pronounced for the Ach-induced current than for i,,; c) i,, is more sensitive to BaZ+ than the Ach-induced current.

The effect of Ach on the K+ current reaches a maximum in about 30 s after application of the drug to the bathing solution, and shows desensitization, to reach a steady-state within 10 min. When Ach is washed out, complete reversibility is obtained. A second application of the drug however, results in a markedly reduced response.

In conclusion, our experiments indicate that in human atrial cells, Ach induces an inwardly rectifying K current by modifying the properties of channels which appear to be different from the i,, channels. This effect desensitizes and is less pronounced during a second application of the drug.

Arch. int. Physiol. Biochim. 1986, 94 ( 5 )

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G . A. ORBAN, B. GULYAS, W. SPILEERS and J. DUYSENS (Laboratorium voor Neuro- en Psychofysiologie, KULeuven, Campus Gasthuisberg, B-3000 Leuven).

Velocity tuning of cat cortical cells for light and dark bars compared.

In a previous study (DUYSENS et al., 1985), we showed that the velocity tuning of cortical cells depends on spatio-temporal interactions, since masking most of the receptive field (RF) except a 0.3" wide strip abolishes the velocity tuning. The same study also suggested a way of achieving these interactions since the spatial lag-velocity relationship of velocity tuned cells with separate ON and-OFF subregions consisted of two parts : at low velocity the response originated from the ON subregion with short latency and at faster speed responses arose from the OFF subregion with longer latency. This then suggests that one way of producing a velocity tuning is by mutual facilitation of a long latency excitation produced by leaving an OFF subregion and a short latency excitation produced by subsequently entering an ON subregion. Since by changing the polarity of the stimulus the latencies of responses from ON and OFF centre geniculate cells are inverted (ORBAN et al., 1985), one would expect cells in which velocity tuning is produced by such a mechanism, to change their velocity characteristics with the polarity of the stimulus. We therefore compared the velocity for light and dark bars and tested to what extent the dependence of velocity characteristics on stimulus polarity could be explained by the RF structure. Out of a larger sample of area 17 and 18 neurons recorded in anaesthetized and paralysed cats, 38 cells were available which were velocity tuned for light or dark bars or for both. Of these 38 cells, 7 cells differed in response strength for light and dark bars by at least a factor 3. Of the remaining 31 cells, 12 were either tuned for the light or the dark bar but not for both. In the group of 19 cells tuned for both light and dark bars, there was a strong correlation between the direction selectivity for light and dark bars ( e = 0.77, P<0.00015) and an even stronger correlation between the preferred speeds for both bars (e = 0.86, P<O.OOOOl). Only 3 of the 19 cells preferred opposite directions for light and dark bars. Hence 16 out of 38 cells were completely invariant in their velocity characteristics for changes in polarity of the moving bar.

Twenty of the 38 cells had a RF consisting of non-overlapping ON and OFF subregions (A and S families) and for these cells we could test whether the velocity characteristics depend on interactions between ON and OFF subregions. In two out of five cells differing in response strength, and in four out of five cells differing in degree of selectivity, the difference could be explained by an asymmetry in the RF structure. For two out of two cells the difference in preferred direction could be explained by the RF structure. Finally in 6 out of 8 cells the similarity in tuning for light and dark bars could be explained by the symmetry of the RF consisting of 3 subregions. Thus in total, in 14 out of 20 cells the velocity tuning characteristics for light and dark bars could be explained by the RF structure. These results show that the facilitatory interactions between ON and OFF subregions are an important, but not the only mechanism to produce velocity tuning in cortical cells.

References

DUYSENS, J., ORBAN, G . A. & CREMIEUX, J. (1985) J . Neurophysiol. 54, 1050-1067. ORBAN, G. A. , HOFFMANN, K.-P. & DUYSENS, J. (1985) J. Neurophysiol. 54, 1026-1049.


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SOC. BELGE DE PHYSIOLOGIE ET DE PHARMACOLOGIE, GAND, AVRIL 1986 P5 1

J. L. MATHIAS, L. PLAGHKI et G . M & C W (') (Service de Mddecine Physique et Rdodop- tation, UCL et ( I ) Service de Physiologie Gdndrole des Muscles, UCL).

Effets du travail ndgatif sur la force isomktnque de 1'E.D.L. de rat.

Le travail musculaire ntgatif (contractions excentriques), realist au cours d'exer- cices rtpttts, entraine des lesions musculaires (DAVIES & WHITE, 1981; ARMSTRONG et'ul., 1983; Mc CULLY & FAULKNER, 1985). Le but de notre travail est de vtrifier sur une preparation in vivo que le travail ntgatif perturbe les propriktes contractiles davantage que le travail positif ou les contractions isomttriques. Comme variable, nous avons choisi la force isomttrique tttanique.

Des rats Wistar mdles de deux mois sont anesthesits au Thalamonal (2 ml/kg en s.c.). Le muscle E.D.L. droit est dissequt en respectant son ptdicule vasculo- nerveux et son insertion tendineuse proximale. Le nerf sciatique droit est sectionnt B la racine de la cuisse. Le muscle est montt sur un ergometre tlectromagnttique (M&CHAL & PLAGHKI, 1979). Sa temptrature superficielle est maintenue 36°C par un goutte B goutte d'une solution de Krebs oxygtnte. Deux tlectrodes de stimulation en platine sont dispostes sur les extrtmitts charnues du muscle.

Chaque muscle recoit dix stimulations tttaniques supramaximales de 0.4 s B 250 Hz avec un repos de 2 min entre chaque contraction. Dix-huit rats sont repartis en trois groupes de six. Dans le premier groupe (ISO), la contraction est isometrique B la longueur Lo. Dans le deuxidme groupe (W +), la contraction est isomttrique durant 100 ms B la longueur Lo + 3.6% Lo, puis le muscle est raccburci de 7.2% Lo B une vitesse de 12 mm/s soit environ 40% Lo/s . Une minute plus tard, le muscle est passivement ramen6 B la longueur initiale. Dans le troisikme groupe (W-), la contraction est tgalement isomttrique durant 100 ms mais B une longueur Lo - 3.6% Lo. Aprb quoi le muscle est ttirt de la mCme amplitude et B la mCme vitesse que dans le groupe prtctdent.

Nous mesurons la force tttanique isomttrique F,,, 100 ms apres le dtbut de chaque stimulation. Dans les groupes IS0 et w + , F, diminue respectivement de 5.2% f 2.2 et de 2.4% f 5.5 entre le premier et le 10eme tttanos. Cette difftrence n'est pas statistiquement significative. Par contre, dans le groupe W-, F, diminue de maniere trks significative aprts chaque contraction. A la l*me contraction, le muscle a perdu en moyenne 35% + 6 de sa force isomttrique F,.

Nous pouvons conclure que dans les limites du protocole exptrimental, seules les contractions avec travail ntgatif ont pour constquence une diminution impor- tante de la force maximale isomttrique des tttanos ulttrieurs.

References ARMSTRONG, R. B. , Oonvm, R . W. & SCHWANE, J . A. (1983) J. Appl. Physiol. Respirot. Environ. Exer-

DAVLES, C . T. M . &WHITE, M. J . (1981) Pfliigers Arch. Eur. J . Physiol. 392, 168-171. Mc CULLY, K . K . & FAULKNER, J. A. (1985) J. Appl. Physiol. Respirat. Environ. Exercise Physiol. 59,

MAR~CHAL, G . & PLAGHKI, L. (1979) J. Gen. Physiol73, 453-467.

cise Physiol. 54, 80-93.

1 19- 126.


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G . DE LEY and I. LEUSEN (Laboratorium voor Normale en Pathologische Fysiologie, Univer- siteit Genf, 8-9000 Gent).

Cerebrovascular reserve in the cat after bilateral common carotid occlusion.

The effect of acute occlusion of a major blood supply to the brain on cerebral function and metabolism largely depends upon the existence of functional collateral vessels able to keep nutritional flow to the brain tissue above the critical level for functional and morphological integrity (HEISS, 1983).

Acute bilateral carotid occlusion leads to a high mortality rate in some animal species (up to 80% in rats) (DE LEY et af., 1985), whereas it is well supported in dogs, due to the existence of a very extensive collateral vascular network.

The present study investigates the locoregional haemodynamic adaptations in the cat brain in the acute phase after bilateral common carotid occlusion.

Methods

Two types of experiments were carried out on cats weighing approximately 3 kg. In a first type (microsphere study) the effect of two Pa,,, levels on cerebral haemodynamics was studied shortly after occlusion of both common carotid arteries. In a second type of experiments (H, polarography) the response of cerebral blood flow (CBF) to hypercapnia in both caudate nuclei was examined before and after consecutive ligation of both carotid arteries.

Results The results of the first series of experiments are summarized in Table I.

TABLE 1. Microsphere study of haemodynamic parameters in the cat under normo- and hypercapnic con- dif ions.

I 1 I

n Blood flow ml 100 g-1 min-1 Pacoz 1 mm H~ ~ Cerebrum

C : control animals with both carotid arteries intact. E : acute bilateral carotid ligation. Observations of right and left cerebrum (herni) were pooled.

Med. Obl.

51.4 k 4.96

216.8 f 18.49

43.8 * 7.44

191.6 f 5.56

Cerebell.

71.4 f 7.20

219.9 f 30.51

70.9 f 10.89

181.4 f 29.86

CBF in the left and the right side of the cerebrum in the control group showed l a nearly 3.5 fold increase under hypercapnic conditions (12 observations in 6 cats).

Arch. int. Physiol. Biochim. 1986. 94 ( 5 )

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Posterior brain regions (medulla oblongata and cerebellum) had a 4 and 3 fold increase of CBF in hypercapnia.

Acute occlusion of both carotids in normocapnia significantly lowered blood flow with 30% (PcO.05, n = 12/6) in the cerebrum, but the flow in the posterior brain regions was virtually unchanged. Under hypercapnia blood flow in the cerebrum hard- ly increased. The posterior brain regions in the occluded animals had hypercapnic flow values as high as in the control group.

The results of the H,-polarography are represented in Table 11. During the control phase (both carotid arteries intact) blood flow in the caudate nuclei behaved qualitatively in a very similar way as observed in the microsphere study. After bilateral common carotid occlusion blood flow in normocapnia was significantly reduced with 30% (PcO.02, n = 1015). Hypercapnia further reduced this local flow to less than half its normocapnic level (P<O.OOl, n = 10/5).

From these experiments it may be concluded that collateral vessels between the vertebrobasilar and carotid system (posterior communicating arteries) in the cat brain may acutely protect anterior brain regions against ischaemic damage under resting conditions after bilateral common carotid occlusion. Under conditions of additional cerebrovascular stress, however, posterior brain regions seem to be preferentially pro- tected.

TABLE 11. H2-polarographic study of local blood f low in the caudate nucleus of the cat before ( C ) and after bilateral common carotid ligation (E).

n = 10/5"

C

E

Pa,,, mm Hg

31.7 f 1.9 28.9 f 0.6

f 1.6 k3.1

k 10.6 k 0.5

Blood flow

ml 100 g-1 g min-l

184 f 10.5

35 k 9.2

"Observations of right and left side were pooled.

References

DE LEY, G., NSHIMYUMUREMYI, J. B. & LEUSEN, I . (1985) Arch. int. Physiol. Biochim. 93, P22-P23. HEISS, W. D. (1983) Stroke 14, 329-331.


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c. DE MEY, D. ENTERLING and H. WESCHE (SK & F-Institute for Applied Clinical Pharmacology, Hildebrandstr. 10, 0-3400 Gottingen, FRG).

Methodological considerations in prolonged cardiovascular monitoring : Postprandial changes in systemic blood pressure and heart rate in normal subjects.

Fluctuations in blood pressure, heart rate and cardiac pump function during cardiovascular monitoring in subjects not maintained in a fasting condition have been suspected to be related to food intake (PACKER et al., 1985).

The present study was designed to assess whether, and to what extent, the systemic blood pressure and heart rate in normotensive subjects are affected by a standard lunch.

Materials and Methods

Eight male normal subjects (age : 25 years, SD : 3 . ; body weight : 78 kg, SD : 8) participated in this study. Each subject was studied on two occasions, one week apart. Subjects has been fasting on each study day since 10 pm of the preceding even- ing. On each study day a placebo was administered after 1 h of base-line measurements. The subjects were then monitored for 8 h after dosing. On one of the two study days a standard 740 Kcal meal was administered 4 h after dosing. The meal was ingested in sitting position in 15 min. The subjects not receiving a meal assumed a sitting position of the same duration at the same time. The lunch/no lunch allocation was randomized in balanced cross-over design.

The monitoring consisted of a base-line period of 60 min and a further 8 h after placebo dosing, in predefined cycles of 1 h duration, with measurements every 10 min in supine position during 50 min, a further measurement in supine position 5 min later, and measurements every minute during 5 min of erect immobile standing. Heart rate was calculated from the ECG-RR intervals of 12 consecutive cycles. Blood pressure was measured non-invasively. On both study days the same equipment was used for each subject. On each of the 101 time points when blood pressure was measured following parameters were available : heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure at sound disappearance (DBP, Korotkoff V), mean blood pressure (MBP) and pulse pressure (PP). For each 60-min cycle, a 55-min supine time zone and a 5-min erect time zone were considered. The parameters in each time zone were summarized by the area under the parameter vs. time curve divided by the duration of the time zone.

The summary values of heart rate and blood pressure for each time zone separately were assessed for subject, period and treatment ( lunchho lunch) related variance and for treatment x period interaction by 3-way ANOVA. The population means were contrasted for treatment by the 95% confidence interval around the difference of the means for fed-fasting.

Results

One subject felt faint on standing at all four occasions after ingestion of the meal (on his first study day). For data analysis only those measurements were taken into account that were registered when the subject was actually standing and symptom-free.

The mean supine and erect HR in base-line prior to placebo dosing were similar for both treatments (fasting vs. fed). A period effect was identified for supine heart rate, the mean on the first study day being statistically significantly larger than the mean for the second study day (difference of the means : - 8.7 bpm, 95% CI : - 14.9

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to - 2.6). No statistically significant difference was identified between the treatments, for erect HR at any of the zones considered. The mean supine HR after lunch was statistically significantly larger than the mean supine HR at the corresponding time zones on the fasting study day. The magnitude of these differences however was small (difference of the means for the total post-prandial phase, fed-fasting : 4.0 bpm, 95% CI : 1.3 to 6.7) .

The mean supine and erect SBP in base-line prior to placebo dosing were similar for both treatments (fasting vs. fed). Only a very small but statistically significant difference was identified for supine SBP during the 2nd h after lunch (difference of the means of fed-fasting : 2.0 mmHg, 95% CI : 0.1 to 4.0).

The mean supine and erect DBP in base-line prior to placebo dosing were similar for both treatments (fasting vs. fed). A period effect was identified for supine DBP, the mean on the first study day being statistically significantly larger on the first study day (difference of the means : - 1.5 mmHg, 95% CI : -2.9 to -0.1). The mean supine DBP was statistically significantly smaller after lunch than the mean supine DBP at the corresponding time zones on the fasting study day (difference of means for post-prandial phase : -9.6 mmHg, 95% CI : - 13.5 to -6.9). This decrease was especially pronounced during the 2nd and 3rd h after lunch. The mean erect DBP was statistically significantly smaller after lunch than the mean erect DBP at the corresponding time zones on the fasting study day, up to 3 h after lunch (difference of means for total post-prandial phase : -9.8 mmHg, 95% CI : - 15.9 to - 3.7).

An increase in mean supine and erect pulse pressure and a decrease in mean supine and erect MBP were found to occur in parallel to the changes in mean DBP after lunch. The maximum shift again was located at 2 and 3 h after lunch.

Discussion and Conclusion

It is a standard method in cardiovascular research to monitor blood pressure and heart rate over several hours. It often is considered difficult to maintain the subjects fasting throughout. This inconvenience is to balanced with the eventual changes induced by eating. The present study demonstrated a moderately large meal to decrease supine and erect mean diastolic blood pressure to a large extent and over a long period of time in normotensive subjects. Mean systolic blood pressure was not changed, and mean heart rate was only slightly increased in supine position but not on immobile erect standing. The average mean blood pressure was decreased and the pulse pressure was increased accordingly. These changes unless accounted for by an ap- propriately controlled study-design might bias the data interpretation on prolonged cardiovascular measurements (REN et al., 1983; PACKER el at., 1985).

References

PACKER, M . , MEDINA, N . & YUSHAK, M . (1985). Circulation 71, 761-166. REN, J . , LEITHE, M . , Huss, P . , UNVERFERTH, D . & LEER, C . (1983) Curr. Ther. Res. 34, 667-675.


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J-L. THONNARD, P. WILLEMS and L. PLAGHKI (Unite de Readaptation, Universire Catholique de Louvain, 5375, Av . Mounier 53, 8-1200 Bruxelles).

Latency responses of the peroneus brevis and tibialis posterior muscles after a sudden inversion of the foot in man (1 figure).

Postural adjustments elicited in the leg muscles of subjects standing upon a movable platform have only been studied during perturbations in the sagittal plane (flexion and extension of the foot) by recording the EMG of the tibialis anterior and the triceps surae muscles (ALLUM & BUDINGEN, 1979; DIENER et al., 1984). Since the reflex latencies of the peroneus brevis muscle (PB) and the tibialis posterior muscle (TP) during inversion of the foot are unknown and since these informations appear essential for our understan- ding of the ankle stability, the experiments described here were performed.

Subjects for this study are 7 volunteers (age 23 to 32 years) without any history of ankle injury. They are instructed to maintain a comfortable upright posture on a platform with two individual footplates. The tilt of one footplate imposes the inversion movement to the corresponding ankle. The mean rate of motion of the footplate is around 250°/s according to the trials. The EMG of the PB and the T P muscles are recorded by indwelling electrodes.

Figure 1 presents the relative frequency (To) of occurrence of EMG activity for the 100 platform rotations performed with the 7 subjects. According to the classification pro- posed by ALLUM & BUDINGEN (1979) and DIENER et al. (1984), the first burst of activity in the stretched PB muscle corresponds to a medium-latedcy response or postmyotatic reaction. This response occurs with the highest frequency (66%) between 70 and 100 ms. I t is thus interesting to note that a sudden inversion of the foot is unable to evoke a myotatic stretch reflex or short-latency response in the PB muscle. Then we observe a long-latency response with a slightly lower occurrence frequency (45%) at latencies of 140 to 160 ms and a long lasting voluntary reaction starting from about 200 ms. In the antagonistic tibialis posterior muscle, we observe only a long-latency response with the highest probability of occurrence (86%) between 120 and 160 ms.

PERONEUS BREVIS TlBlALlS POSTERIOR

LL vc 90 ML LL f r 7 0

80 70

60

60 50 e . /

q ' u 40 50

30 40 30 20

0

" 20 v ' 0 10

100 200 100 200 Time (rnsl 0

I. I(; I Krln,ii,(,,/rc.qicc,iic:i, o c ' l ~ i i r i w w / ) lo / o J'E,tfC; i ic , / i i , i / ! , iii i lw p c v w i c w . \ hrc~i~is t i t i d /lie /il)iuli.$ p o ~ i l ~ r u ~ i iriuscles uJier u sudden inversion oJ/heJoo/. Data are collected from 7 subjects. (ML = medium-latency; LL = long-latency; VC = voluntary contraction).

References

ALLUM, J. H . J. & WDINCEN, H . J. (1979) Progr. Brain Res. 50, 185-195. DIENER, H . C. , DICHGANS, J . , BOOTZ, F. & BACHER, M. (1984) Exp. Brain Res. 56, 126-134.


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J . V A N DE VOORDE and I. LEUSEN (Laboratorium voor Normale en Pathologische Fysiologie, Universiteit Gent, 8-9000 Gent).

Endothelium-dependent relaxation on aorta of nitroglycerin-tolerant rats.

It is known that many physiological important substances elicit a relaxing effect on blood vessels which is mediated by (a) substance(s) released from the endothelial cells (for review : FURCHGOTT, 1983). This mediator is called endothelium-derived relaxing factor (EDRF). In a previous study we demonstrated that the relaxing effects of acetylcholine and histamine on pre-contracted aorta preparations of the rat are mediated by this EDRF (VAN DE VOORDE & LEUSEN, 1983).

Recently many studies have indicated that the EDRF-induced relaxation effect is the consequence of guanylate cyclase-stimulation, resulting in increased cGMP and relaxation at the smooth muscle level. This mechanism of action of EDRF shows many similarities with that of nitrovasodilator substances such as nitroglycerin, nitroprusside, isosorbide dinitrate (RAPOPORT & MURAD, 1983; MARTIN et al., 1985). In this respect, EDRF could be considered as an endogenous nitrate-like substance. This study was designed to investigate if EDRF would act on the organic nitrate receptor, which is thought to be the common site of action of organic nitrates (NEEDLEMAN & JOHNSON, 1973).

To approach this problem, the affinity of the organic nitrate receptor was lowered by administration of high doses of nitroglycerin. If EDRF acts on this receptor, an impaired relaxation effect of EDRF-releasing agents can be expected on aorta preparations of nitroglycerin-treated rats.

Tolerance was induced in male rats (300 g) by subcutaneous administration of 100 mg/kg of nitroglycerin 3 times a day for 2 days. Control rats were injected with the solvent (ethanol 0.2 ml). Thoracic aortic rings of control and pre-treated rats were mounted in parallel for isometric tension recording in Krebs-Ringer-bicarbonate solution, gassed with 95% O2 and 5 % C02 , at 37°C. After precontraction with norepinephrine ( ~O-’M), dose-response curves for acetylcholine, histamine and nitroglycerin were established on each preparation.

The dose-relaxation curve of nitroglycerin on aorta preparations of pre-treated animals showed a significant shift to the right compared to preparations of control rats, proving the efficient induction of nitrate-receptor tolerance. However, when the same preparations were tested for their reactivity to acetylcholine and histamine, substances whose effects are mediated by EDRF, no differences were obtained between the dose-relaxation curves of control and pre-treated animals.

These results indicate that although organic nitrates and EDRF relax vascular smooth muscle cells by stimulating guanylate cyclase, this stimulation is mediated by a different mechanism.

References

~ U R C H G O T T , R. F. (1983) Circ. Res. 53, 557-573. MARTIN. W . . VILLANI. G . M.. JOTHIANANDAN. D. & FURCHGOTT. R. F. (1985) J. Pharniacol. Exu. Ther.

232, 708-716. NEEDLEMAN. P. & JOHNSON. E. M. J r (1973) J . Phurrnucol. Exu. Ther. 184. 709-715. RAPOPORT, k. M. & MURAD, F. (1983jCirc: Res. 52, 352-357.’ VAN DE VOORDE, J . & LEUSEN, I . (1983) Eur. J . Pharmacol. 87, 113-120.

Arch. inr. Physiol. Biochim. 1986, 94 ( 5 )

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P58 SOC. BELGE DE PHYSIOLOGJE ET DE PHARMACOLOGIE, GAND, AVRIL 1986

P. P. VAN BOGAERT, G . A. MATRICALI & Y. JACQUEMYN [Laboratorium voor Fysiologie, Universiteit Antwerpen, (RUCA), Antwerpen].

Chloride-free solutions induce an increase in steady-state sodium current in sheep cardiac Purkyni! fibres.

In zero C1- -Tyrode where impermeant anions replace C1-, the action potential duration frequently increases and a number of preparations depolarize to the level of the plateau potential (CARMELIET, 1961; KENYON & GIBBONS, 1977). Short ( - 1 mm) PurkynE fibres were voltage clamped with a conventional two microelectrodes technique. The membrane currents, measured in normal Tyrode solution buffered at pH 7.4 with Hepes, were compared with the currents obtained after 30-min perfusion with C1- -free Tyrode. This solution contained Ca-gluconate, MgSO., and the Na’ and K’ salts of acetylglycinate, glucuronate or methanesulphonate.

Irrespective of the major substituting anions, similar current changes were observed in C1- -free media : between - 70 and - 30 mV the steady-state membrane current shifted in inward direction, becoming sometimes net inward and inducing the appearance of a second stable resting potential at plateau level. In a number of fibres this was accompanied by oscillatory transient inward currents (LEDERER & TSJEN, 1976). TTX (15 x 10-6~) reversibly suppressed these effects. By comparing the TTX- sensitive currents, calculated by subtraction of membrane currents from currents measured in the presence of TTX in normal and C1-free solutions, it can be concluded that in C1- -free solutions the TTX-sensitive, steady state Na’ “window” current is increased (ATTWELL et al., 1979). This causes the inward shift of the membrane currents in the plateau voltage range and explains the increase in action-potential duration as well as the tendency of PurkynE fibres to depolarize in Cl--free solutions (VAUGHAN-JONES, 1979).

References

ATTWELL, D., COHEN, I . , EISNER, D., OHBA, M. & OJEDA, C. (1979) Pfliigers Arch. Eur. J . Physiol. 319, 137-142.

CARMELIET, E. (1961) J. Physiol. (London) 156, 375-388. KENYON, J. L. & GIBBONS, W. R. (1977) J. Gen. Physiol. 70, 635-660. LEDERER, W. J. & TSIEN, R. W. (1976) J. Physiol. (London) 263, 73-100. VAUOHAN-JONES, R. D. (1979) J. Physiol. (London) 295. 111-137.

Arch. int. Physiol. Biochim. 1986, 94 (5)

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G. DE LEY and I. LEUSEN (Laboratorium voor Normale en Pathologische Fysiologie, Universiteit Gent, B-9000 Gent).

Cerebrovascular reserve in normotensive and spontaneously hypertensive rats (SHR) after unilateral common carotid occlusion : practical implications.

Acute unilateral common carotid artery (CCA) occlusion in (normotensive) Wistar rats barely induces changes in hemispheric blood flow (HBF) under resting conditions; cerebrovascular reserve (measured as HBF response to hypercapnia) at the occluded side, however, is always strongly decreased. During the days following unilateral CCA occlusion, cerebrovascular reserve is gradually re-established (DE LEY et al., 1985a).

As a consequence, mortality rate after consecutive bilateral CCA occlusion decreases with increasing time interval between both ligations (DE LEY et al., 198%). In spontaneously hypertensive rats (SHR) structural alterations of the brain vessels develop during the establishment of hypertension (reduced lumen and increased media to diameter ratio). These morphological changes are at the basis of reduced cerebral perfusion pressure (due to increased vascular resistance) after bilateral CCA occlusion compared to normotensive animals (FREDRIKSSON et al., 1984). The present study investigates HBF and cerebrovascular reserve in SHR rats five days after unilateral CCA occlusion and its consequences on the survival rate after consecutive bilateral CCA occlusion.

Methods

Two series of experiments were undertaken on male, 6-month-old SHR rats weighing approximately 300 g.

Normocapnic and hypercapnic HBF were measured with radiolabelled microspheres under nitrous oxide anaesthesia in intact SHR animals and five days after unilateral (right) CCA occlusion.

SHR rats underwent consecutive bilateral CCA occlusion under halothane ( 1 070

in N,O/O, mixture) anaesthesia with a time interval of 1 , 5 , 15 and 30 days; survival rate was estimated.

The results of both series of experiments are compared to the results obtained in normotensive rats that underwent the same procedure.

Results and Discussion

The results of this study are represented in Table I. Five days after right CCA occlusion in SHR rats, right normocapnic HBF is still

47% lower than in the control group (P<O.Ol) resulting in a significantly (P<0.05) decreased right to left HBF ratio (R/L). In hypercapnia HBF at the ligated side reaches only 51% of HBF at the non-occluded side (P<O.O5) resulting in a strongly asym- metric HBF ratio (R/L) compared to the control group (P<O.Ol).

The cerebral haemodynamic situation of SHR rats, 5 days after right CCA occlusion is very similar to the 24-h situation in normotensive rats with the same kind of occlusion. Consecutive bilateral CCA occlusion in SHR rats resulted in 100% mortality up to 24 h of interocclusion interval. Survival rate increased to 30,90 and 100% with interocclusion intervals of 5 , 15 and 30 days respectively. In normotensive animals survival rate was 20% with acute bilateral ligation and increased to 30, 60, 90 and 100% with interocclusion intervals of 24 h, 5 , 15 and 30 days respectively.

Arch. inf. Physiol. Biochim. 1986. 94 ( 5 )

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PaCOZ H B F R H B F L R/L mm Hg ml 100 g-Imin- '

33.4 194 186 1.10 f.2.1 +21 f 2 7 i O . 0 9 28.2 102 134 0.79 f 1.5 i14 f 19 f0.08

29.9 141 146 0.96 f 1 . 3 + I7 f 1 4 lt0.02

29.5 I15 134 0.86 *0.8 i 8 f 9 f0.04

31.1 142 146 1.01 i 1.6 f 12 + I 8 i0.07

TABLE I . Microsphere study of right (HBF R) and left (HBF L) hemispheric bloodflow in SHR (upper part) and normotensive (lower part) rats in normocapnia (lefl panel) and hypercapnia (right panel). Values are mean f SEM.

n PaCO2 H B F R H B F L R/L mm Hg ml 100 g - l m i n - l

6 70.6 575 619 0.94 i 3 . 6 i l l 0 f l l O f0.07

4 71.1 248 485 0.44 f 4 . 9 +I05 i131 i O . 0 9

7 66.5 545 547 1.00 f 2 . 2 f 58 f 58 k0.02

7 65.1 188 4.08 0.46 f 3 . 4 i 26 i 3 2 f0.05

7 70.7 343 539 0.63 i l . 2 i 45 f 39 f0.07

S H R 1

5 days

The results of the present study suggest that normalization of resting hemispheric blood flow and of cerebrovascular reserve after unilateral CCA occlusion in SHR rats is delayed compared to normotensive animals. The retardation of cerebrovascular adaptation results in a higher mortality rate after consecutive bilateral CCA occlusion in SHR rats.

References

DE LEY, G., NSHIMYUMUREMYI, J. B. & LEUSEN, I. (1985,~) Stroke 16, 69-73. DE LEY, G., NSHIMYUMUREMYI, J. B. & LEUSEN, I. (1985,b) Arch. int. Physiol. Biochim. 93, P22-P23. FREDRIXSSON, K., NORDBORG, C. & JOHANSSON, B. B. (1984) Actu Physiol. Scund. 121, 241-247.


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G. VANDEPUTTE-VAN MESSOM, E. R. KUHN(I) and C. BURVENICH (Laboratory of Veterinary Physiology, RUG, Casinoplein 24, 8-9000 Gent and ( I ) Laboratory of Comparative En- docrinology, KUL, 8-3000 Leuven).

Differences between intracerebroventricular and intravenous administration of endotoxins on plasma cortisol and thyroid hormones in lactating goats.

Intravenous E. coli endotoxin administration produced an increase in glucocorticoid secretion in cows (LUMSDEN et al., 1974) and goats (KUHN & BURVENICH, 1986). Glucocorticoids are reported to suppress peripheral thyroid hormone concentrations in several species including cows (JOHKE & HODATE, 1979).

Methods Two series of experiments were carried out on 8 conscious, cross-bred goats

(average body weight 40 kg). In the first series, injections were made intracerebroven- triculary (i.c.v.) through a stainless steel cannula stereotaxically implanted into the rostral part of the 3rd cerebral ventricle (VANDEPUTTE-VAN MESSOM & PEETERS, 1974). In the 2nd series of experiments injections were given into the external jugular vein (i.v.) through a permanent catheter. In each series goats were divided ad random into two groups. One group was injected with endotoxins (lipopolysaccharides, LPS) from E. coli (026 : B6; Difco Laboratories, Detroit, USA) either i.c.v. (0.4 ng/kg b.wt) in a volume of 0.1 ml of pyrogen-free water (4 goats, 12 expts) or i.v. (200 ng/kg b.wt) in 5 ml of pyrogen-free water (4 goats, 12 expts). In the other group, injections with pyrogen-free water were made i.c.v. (4 goats, 8 expts) or i.v. (8 goats, 12 expts).

From each goat blood samples were taken hourly until the 10th h after injection for RIA determination of cortisol, 3,3',5-triiodothyronine (T,), thyroxine (T,) and 3,3',5'-triiodothyronine (rT,, reverse T,) (KUHN el al., 1983). Simultaneously rectal temperature (RT) was measured. Three control samples and measurements were made : samples 1 and 2 on the day before experimentation and sample 3 just before the injection.

Results and Discussion Preinjection control levels of cortisol, T,, T, and rT, in the 8 goats used in this

study amounted to 10.5, 68.0 and 1.81 ng/ml and to 306.5 pg/ml respectively. RT averaged 38.8 "C.

Experiments with i.c.v. injection RT was elevated after i.c.v. injection of LPS in lactating goats. The onset of fever

was delayed, peak values of 40.1 "C being observed only after 9 h. Immediately after injection cortisol increased significantly. Maximal values amounted to 57 ng/ml. On average thyroid hormones moderately declined, the decrease in T, being more pronounced and faster than that in T, or rT,. Only slight changes in the circulating concentrations of T, were observed. Maximum decreases in T, and T, amounted to 23% and 10% respectively. After a small fall (10%) rT, slowly rose (by 14%) above preinjection levels towards the 10th h. However, this increase was not significant.

Experiments with i.v. injection

After i.v. injection of LPS an increase in RT and cortisol occurred, whereas thyroid hormone levels were suppressed. The effects were more pronounced than those observed after i.c.v. LPS. Peak values of R T (40.2 "C) and cortisol (94.1 ng/ml) were obtained within 4 and 3 h after injection. T, and T, levels were gradually depressed by


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54% and 21 Yo respectively towards the 10th h. After an early fall (by 29%) rT, levels sharply rose from the 8th h after injection.

The time course of the effects observed suggests that after i.v. as well as i.c.v. administration of endotoxins into lactating goats thyroid gland is suppressed rather soon after the injection. The quick increase in cortisol probably contributes to that inhibition, since glucocorticoids are known to affect circulating concentrations of T, and T,. Conversion of T, to T, may be impaired while that to rT, may be enhanced. Those changes in T, metabolism could be reflected by the early decrease in T, and rT,, and by the late rise in rT, respectively.

The effects of endotoxin on cortisol and thyroid hormones are more pronounced after i.v. than after i.c.v. injection. The time lag between i.c.v. injection and the appearance of clinical symptoms (e.g. fever) suggests that endotoxin action within the central nervous system might be an indirect one. The involvement of endogenous mediators should be considered. Moreover, it seems that fever per se is not the trig- ger for the release of cortisol.

The delayed onset of fever and the time course in the thyroid hormone variations are consistent with the effects observed after i.mam. LPS ( K W & BURVENICH, 1986), however the effects after i.c.v. LPS being more pronounced.

Since hormone levels were not returned to preinjection level at the end of the present experiments, further studies were planned to investigate the course of variations in thyroid hormone levels in experiments with longer time schedules.

Acknowledgments. - This study was supported by a grant from the Belgian NFWO Foundation (grant number : 2.0031.85).

References

JOHKE, T. & HODATE, K. (1979) Endocrinol. Japon. 26, 533-539. KUHN, E. & BURVENICH, C. (1986) Arch. int. Physiol. Biochim. 94, P37-P38. KUHN, E., DECWPERE, E., HEMSCHOOTE, K., BERGHMAN, L. & PAULUSSEN, J . (1983) J . Endocrinol. 99,

LUMSDEN, J. H. , VALLI, V. E. O . , Mc SHERRY, B. J . & WILLOUGHBY, R . A. (1974) Can. J. Comp. Med.

VANDEPUTTE-VAN MESSOM, G . & PEETERS, G. (1974) Arch. int. Pharmacodyn. 211,80-98.

401 -407.

38, 56-64.


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SOC. BELGE DE PHYSIOLOGIE ET DE PHARMACOLOGJE, GAND, AVRIL 1986 P63

G. VANDEPUTTE-VAN MESSOM, E. ROETS and C. BURVENICH (Laboratory of Veterinary Physiology, RUG, Casinoplein 24, B-9OOO Ghenf).

Influence of the administration way of endotoxins on plasma tyrosine in lactating goats.

Inhibition of rumen motility has been observed after administration of endotoxins in small ruminants (VAN MIERT el al., 1978). Atropine injected subcutaneously, induces a decrease in free amino acid (AA) levels in arterial plasma of lactating cows (ROETS & PEETERS, 1981). It is known that atropine reduces rumen motility and sup- presses secretory processes of the digestive tract.

Methods The effects of E. coli endotoxin or lipopolysaccharides (LPS, 026 : B6, Difco

Laboratories, Detroit, USA) on plasma tyrosine levels were studied in conscious lactating goats after administration either by intravenous (i.v.), intracerebroventricular (i.c.v.) or intramammary (i.mam.) route. Sixty-eight experiments were carried out on 8 cross-bred goats. Three series of experiments were performed. In each series two groups of goats were selected ad random.

Goats of groups land IIwere slowly injected with LPS (0.4 ng/kg b.wt, 12 expts) or with pyrogen-free water (0.1 ml, 8 expts) respectively into the 3rd ventricle as described by VANDEPUTTE-VAN MESSOM & PEETERS (1974). In groups III and IVgoats were injected with endotoxin (200 ng/kg b.wt, 12 expts) or with pyrogen-free water (5 ml, 12 expts) into a permanently catheterized external jugular vein. In groups V and VI, injections with endotoxin (4 OOO ng/kg b.wt, 12 expts) or with pyrogen-free water (7 ml, 12 expts) were made into one mammary gland. From each goat venous blood samples up to 10 h after injection were taken hourly and rectal temperature (RT) was measured. Tyrosine in blood plasma was estimated by the fluorometric method of AMBROSE (1974). Three control samples and measurements were taken, two the day before experimentation and 1 just before endotoxin administration. Plasma tyrosine levels were expressed in percent with respect to mean control values.

Results and Discussion Injection of endotoxin induced fever in lactating goats. Although the magnitude

of the fever response (40 "C) was about the same in the three series of experiments, initiation of fever was delayed after i.c.v. injection with respect to that after i.mam. and i.v. LPS administration, peak RT being observed after 9,6 and 4 h respectively.

Experiments with i.v. injection

After i.v. injection of LPS, free tyrosine levels in plasma quickly decreased reaching minimum levels of 33% below control values around the 2nd h after injection. Then, levels remained low for about 5 h. From the 7th h on, plasma tyrosine increased. At the end of the experiment, the tyrosine level was about 40% higher than the control values. In the control experiments, tyrosine remained rather stable throughout the entire experiment and averaged 1.58 mg/100 ml.

Experiments with i.c.v. injection

When LPS was injected i.c.v., changes in plasma tyrosine concentration were of about the same magnitude as in the i.v. LPS experiments. The decline in free tyrosine

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levels started immediately after injection, but was much slower. Minimum levels (30% below control values) were observed from the 5th to the 7th h. Low tyrosine levels were maintained until the 10th h.

Experiments with i.mam. injection

The fall in plasma tyrosine observed after administration of LPS into one mammary gland was not so pronounced as after i.v. or i.c.v. injection. Maximal inhibition occurred at the 6th to 7th h after injection. Tyrosine decrease was followed by a rather quick recovery attaining a level being 14% above control values at the 10th h.

After i.v. LPS administration to ruminants, a striking inhibition of rumen motility (VAN MJERT et a!., 1978) and of abomasal emptying (VLAMINCK et al., 1985) is observed. Inhibition of rumen motility is also seen after i.c.v. LPS injection in goats (VAN MIERT et al., 1978). In contrast, i.mam. LPS administration has practically no effect on rumen motility (VERHEIJDEN, 1979).

After i.v., i.c.v. and imam. LPS administration, plasma tyrosine decreases. Time of latency, magnitude and duration of the effects seem to coincide with the data of gastro-intestinal inhibition under the same conditions. Some resemblance is observed between these phenomena and atropine treatment. Depending upon the dose, atropine is known to depress both gastro-intestinal motility and secretion, leading to an impairment of protein digestion and AA absorption. It therefore seems that in the present study the decrease in gastro-intestinal activity might be one of the mechanisms underlying the decrease in plasma tyrosine after LPS fever. However, it is not im- possible that other mechanisms, such as hormonal impairment, also might contribute to this decrease. The delayed rise in AA after i.v. LPS is probably not only the result of restoration of gastro-intestinal motility, since tyrosine increases above control level. At that moment, metabolic factors (such as acute phase reductants inducers) probably are involved as well.

Acknowledgments - This study was granted by the Belgian IRSIA and NFWO (grant number : 2.0031.85) Foundations.

References

AMBROSE, J . A. (1974) Clin. Chem. 20, 505-510. ROETS, E. & PEETERS, G . (1981) J. Dairy Res. 48, 23-34. VANDEPUTTE-VAN MESSOM, G . & PEETERS, 0. (1974) Arch. int. fharmacodyn. 211, 80-98. VAN MIERT, A.S.J.P.A.M., VAN DUIN, C. TH. M. & LEEK, B. F. (1978) Zbl. Vet. Med. A 25, 718-726. VERHEUDEN, J . H . M. (1979) Thesis, Utrecht. VLAMMCK, K . , VAN MEIRHAEGHE, H. , VAN DEN HENDE, C., OYAERT, W. & MUYLLE, E. (1985) Drsch.

Tierartzl. Wschr. 92, 392-393.


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E. ROETS, C. BURVENICH and G . PEETERS (Laboratory of Veterinary Physiology, RUG, Casinoplein 24, B-9wO Gent).

[3H] Rauwolscine binding to cow platelet membranes.

Longitudinal smooth muscles in the wall of bovine teats of lactating cows con- tain a,- and P,-adrenoceptors (ROETS et al., 1984; ROETS & PEETERS, 1986). It was recently found that milking time was positively correlated to the P,/a,-adrenoceptor density ratios in teat tissue (ROETS et al., 1986). In human blood, az-adrenoceptors (MOTULSKY et al., 1980) were found on platelets, whereas P,-adrenoceptors (WILLIAMS et al., 1976) were present on circulating lymphocytes. Quantitative investigations of these receptors might be used to predict milkability of dairy cows.

This paper reports the presence of saturable and specific binding sites for the a,-specific radioligand [3H] rauwolscine on bovine platelet membranes.

Blood was collected from the external jugular vein with 20% tri-sodium citrate 85 mM, citric acid 65 mM, 2% glucose, pH 5 as anticoagulant; platelet lysates were prepared as follows : platelet rich plasma (PRP) was obtained by centrifuging the blood at 3 800 x g for 20 min. To collect platelets, PRP was then centrifuged at 1 500 x g for 10 min. The platelet pellets were resuspended in isotonic buffer (Tris- HCI 50 mM, NaCl 150 mM, EDTA 20 mM, pH 7.35) and the number of platelets were counted with a Coulter counter. After centrifugation at 16 OOO x g for 10 rnin platelets were collected. The resulting pellet was washed once with isotonic buffer and platelets were obtained by centrifugation (16 OOOxg for 10 rnin). All the above steps were carried out at room temperature. Platelets were lysed by exposure in ice-cold hypotonic buffer (Tris-Na,EDTA 5 m ~ , pH 7.5) and homogenized using a teflon-glass homogenizer. The membranes were then precipitated by centrifugation at 39 O00 x g for 10 rnin at 4°C. Platelet membranes were collected and washed twice in Tris-EDTA 5 mM, pH 7.5 buffer and twice in incubation buffer (Tris-HC1 50 mM, EDTA 0.5 mM, pH 7.0) by successive centrifugations at 39 000xg for 10 rnin at 4°C. The final washed platelet membranes were suspended in incubation buffer in a concentration corresponding to f lo9 platelets/ml. Membrane proteins were incubated for 20 rnin at 25°C in Tris-HC1 50 mM, EDTA 0.5 rnM, pH 7.0 buffer with 0.25 - 7 nM [3H] rauwolscine for saturation binding experiments or with f 1 n~ [3H] rauwolscine and increasing concentrations of various drugs for competition binding experiments. Non- specific binding (NSB) was determined by incubation of membranes and [3H] rauwolscine in the presence of 1 0 - 5 ~ phentolamine. In order to obtain maximum specific binding (SB) and to minimize NSB, the pH of the incubation medium was investigated. Incubations were routinely performed at pH 7.0 : SB at this pH amounted to about 60% of total binding.

The association of [3H] rauwolscine to cow platelet membranes was rapid, equilibrium being reached within 15 min. Dissociation was complete within 20 rnin after the addition of phentolamine. Saturation experiments performed on platelets from one cow showed an equilibrium K,, value of 6.9 n~ and a maximum number of sites of 171 fmol/mg membrane protein corresponding to 23 sitedplatelet. N o cooperative interactions among the binding sites were observed.

Catecholamines competed for the binding sites with the generally accepted rank order of potency expected for a-adrenoceptors :(-)-epinephrine = (-)-norepinephrine > > (-) - isoproterenol. The a,-selective antagonist prazosin was about 20 times less potent than the specific a,-antagonists rauwolscine and yohimbine in inhibiting for [3H] rauwolscine binding, indicating that [3H] rauwolscine labelled the az-receptor. An interesting finding was the significant correlations found between K,-values


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of drugs, inhibiting 3H-ligands in teat and platelet membranes, indicating that similar a2-binding sites were detected in both tissues (Table I).

TABLE I. Ki-values (nM) of compounds for a,-binding sites in bovine teats and platelets.

Drug

Antagonists

Rauwolscine Yohimbine Phentolamine Prazosin

Agonists

Clonidine (-) - Epinephrine (-) - Norepinephrine (-) - lsoprenaline

['HI Rauwolscine ['HI Yohimbine Teat (1)

. . Platelet

25.2 39.5 31.1

732.0

193 2238 5105

22 1 423

6.8 6.0

23.9 121.0

97 2716 2570

174270

Teat

7.3 8.1

45.4 191 .O

328 3212 2382

137952

Ki-values were determined from the CHENO & PRUSOFF (1973) equation : Ki = ICs0/(1 + C/K,).C is the concentration of [3H] rauwolscine used in the assay, K , is the dissociation constant of ['HI rauwolscine derived from saturation experiments and IC,, is the molar concentration of drug causing 50% inhibition of specific binding. Spearman rank correlations between Ki-values : platelet vs. teat ([3H] rauwolscine) rs = 0.91, P<O.Ol; platelet vs. teat ([W] yohimbine) rs = 0.95, P<O.OOI.

From the present findings, it is concluded that [3H] rauwolscine is a suitable ligand to study orz-adrenoreceptors in bovine platelets. These easily accessible blood cells may represent a promising tool to investigate the receptor status in teat tissue in the future.

Acknowledgments. - This study was supported by a grant from the Belgian IRSIA Foundation.

References

CHENO, Y.-C. & PRUSOFF, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108. MOTULSKY, H. J., Swrrn, S. J . & INSEL, P. A. (1980) Biochem. Biophys. Res. Commun. 97, 1562- 1570. ROETS, E. & PEETERS, G. (1986) Arch. int. Pharmacodyn. 279, 212-221. ROETS, E., PEETERS, G. & LEYSEN, J . E. (1984) Arch. int. Pharmocodyn. 270, 203-214. ROETS, E., VANDEPUTTE-VAN MESSOM, G. & PEETERS, G. (1986) J. Dairy Sci., in press. WILLIAMS, L. T., SNYDERMAN, R. & LEFKOWITZ, R. J . (1976) J. Clin. Invest. 57, 149-155.

( I ) Data from ROETS & PEETERS (1986).


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J. BERNABE, G . VANDEPUTTE-VAN MESSOM and c. BURVENICH (INRA, 65, rue de Saint Brieuc, F-35042 Rennes, and Faculty of Veterinary Medicine, Physiological Department, Universi- ty of Ghent, Casinoplein 24, 8-9000 Gent).

Experimentally induced fever and plasma catecholamines in lactating goats : preliminary report.

It is generally accepted that the sympathetico-adrenomedullary system is involved in fever development. Thus fever is associated with vasoconstriction, increased muscular tone, inhibition of gastro-intestinal motility and secretion, and hyperglycaemia (VEFU-IEJJDEN, 1979). An a-lytic or antipyretic pretreatment inhibiting some of these effects may be an indirect demonstration of sympathetic activation. The interrelations in the secretion of sympathetic mediators, noradrenaline (NA), adrenaline (A) and dopamine (DA) during fever have so far been little studied.

The aim of the present preliminary report was to obtain more information about these interrelations by studying the plasma catecholamines during endotoxin-induced fever in the goat which often has been used as a model for studying ruminant physiopathology.

Methods

In an experimental herd, eleven cross bred goats (2 to 4 years old) in late lacta- tion were used for this study. Body weights ranged from 37 to 62 kg. Eleven trials were performed over 2 days.

Two groups of animals were selected ad random. The first group ( 5 goats - 5 trials) received 5 ml of 0.9% NaCl i.v.. The second group (6 goats - 6 trials) was injected i.v. with E. coli endotoxin (LPS; 026 : B6; 100 ng/kg) in 5 ml of 0.9% NaCI.

Injections were made into the external jugular vein through an indwelling catheter implanted 2 days before experiments.

Six control blood samples were collected : 2 on the day before (day -1) and 4 on the day of LPS-administration (day 0). Injections were performed at 9 h 30 (day 0). Samples were collected every 10 min between 10 h 00 and 10 h 30, every 30 min until 12 h 00 and every hour until 19 h 00. From each goat twenty blood samples (4 ml) were collected, which were immediately cooled and centrifuged. Plasma was frozen within 30 min after collection of blood samples. Rectal temperature (RT) was measured with a thermistor probe after each blood sampling.

All interventions were made in a minimally disturbell environment in order to reduce emotional stress. Room temperature varied between 18°C to 20°C.

Catecholamine (CA) levels were determined within 4 weeks. NA, A, DA were assayed in duplicate by the radio-enzymatic method (adapted from DA PRADA & Zm- CHER, 1976).

Results

1. Validation of the method

Modifications of the catechol-0-methyltransferase (COMT) assay allowed both high sensitivity and good precision. For NA, A and DA, the sensitivity was respectively 2.93, 1.93 and 2.75 pg/ml. The intra (and inter) assay coefficients of variation were 6.34% (12.87%) for NA, 6.97% (13.87%) for A and 8.6% (18.08%) for DA.

In group 1, plasma levels before injection reached 455 pg/ml for NA, 121 pg/ml for A and 72 pg/ml for DA. In group 2, the control plasma levels were slightly lower.


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After i.v. NaCI, plasma levels amounted to 372 pg/ml, 1 1 1 pg/ml and 105 pg/ml respectively.

Control levels in each goat showed a large variability. For example, 3 animals (goats 103, 104, 105) presented a higher plasma A-concentration (192, 219 and 452 pg/ml) than in the remaining 8 goats (less than 100 pg/ml). A very large variability was observed for DA.

In group 1 , there was a continuous decrease of NA and A levels during the day with a slight increase around 16 h 00, when the animals were housed.

2. Endotoxin fever

After LPS injection (9 h 30), fever developed over 7 h. Maximum pyrexia appeared at 13 h 00. Because of the animal variability of each amine, results are described individually.

Noradrenaline levels

In 5 goats, i.v. LPS injection elicited a clear and significant increase in plasma NA. This increase started 30 min after injection. In 4 goats, at the moment of maximum pyrexia there was a transitory drop in NA to basal values. In one other animal, the phenomenon was less important. In the 6th animal (goat 105), no significant alterations could be observed in plasma NA.

During fever increment, mean plasma NA was 34% higher (P<O.OOl) than after 0.9% NaCl injection. A similar NA pattern was observed during the fever decrement.

Adrenaline levels

In goats 103 and 105, judged as being very nervous, the A plasma level fluctuated largely. Consequently in these animals, no clear alterations could be detected. Two other goats showed a clear A increase with a maximum during fever increment (75% above control). As for NA levels, a drop in A release was observed during the fever peak in these animals.

In the other goats, the increase of A was less pronounced (17% above control). During the fever decrement, a delayed slight increase in A was observed in all goats (except 103 and 105), amounting to 12%.

Dopamine levels

No significant alterations of DA could be observed during the entire course of the experiment. After LPS injection, variations in DA levels were observed, which were comparable to those of the control group (93.77 + 10.32 and 105.08 +. 7.26 pg/ml respectively).

Acknowledgrnenfs. - This study is supported by grants of the French INRA and the Belgian NFWO Foundations.

References

DA PRADA, M . & ZURCHER, G . (1976) LifeSci 19, 1161-1174. VERHEUDEN, J. H. M . (1969) Thesis, Utrecht.

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E. JACQUES, J.-L. BISTER and R. PAQUAY (Laboratoire de Physiologie Animale, FacultPs Univer- sitaires de Namur, rue de Bruxelles 61, B-5000 Namur).

Follicular events during the cycle in ewes with low ovulation rate.

In order to investigate the mechanisms underlying the ovulation rate, we compare the follicular recruitment, growth and selection during luteal and follicular phases in ewes with low ovulation rate.

Animals and experimental design

Twelve Ile-de-France ewes (mean ovulation rate : 1.12 + 0.35) were allocated in two groups : 6 in group I for the follicular phase and 6 in group I1 for the luteal phase after hormonal synchronization of cycles (FGA impregnated sponges).

The group 1’s ewes underwent repeated laparotomies at day 13, 15 and 17. Ovarian follicles were measured on each ovary and the three largest’ ones of each ovary were ink-labelled by dots into the stroma surrounding the follicle (DUFOUR et al., 1972; DRIANCOURT & CAHILL, 1984).

Group 11’s ovaries were observed in the same way at day 3, 5, 7 of the cycle and at day 9 after slaughtering.

Means were compared by Mann and Whitney test (non parametric test).

Results

- The repeated laparotomies allowed us to observe the origin of the follicles reaching the preovulatory size in the two groups. In the case of Ile-de-France ewes, all the putative preovulatory follicules were actually labelled at day 13 or 3. - In Ile-de-France ewes, despite the greater number of recruited follicles in follicular phase,

the percentage of those reaching the preovulatory size was significantly lower in follicular than luteal phase linked to the higher selection intensity. But no significant difference was observed in the number of follicles reaching the preovulatory size a t day 7 or 17. - There was no significant difference between the size of the largest follicles measured

at day 7 or 17 in the two groups (just before regression). - The growth rate of the follicular populations did not differ as well for selected as non

selected ones between follicular and luteal phase. - Moreover, the growth rate in both early follicular and luteal phases appeared to be a

good criterion allowing us to discriminate between selected and non-selected follicles populations.

Conclusion

In summary, it appeared that the main features of follicular growth (growth rate of the two follicular populations, number of follicles reaching the preovulatory size, maximal size before regression) were similar in luteal and follicular phases.

These data were in agreements with previous results supporting the intra-ovarian control of the follicular selection hypothesis. Indeed, it has been already demonstrated that the hCG induced ovulation rate in Ile-de-France ewes was similar at day 7 of the cycle (mid luteal phase) and at day 16 of the cycle (DRIANCOURT, PHILIPPON & WEBB, unpublished data).

Acknowledgments. -This work was supported by a grant from the Fonds Nationalde la Recherche Scientifque. I t was realized at the Station de Physiologie de la Reproduction of the Institut National de la Recherche Agronomique, F-37380 Nouzilly.

References

DUFOUR, J . J . , WHITMORE, H . L., GINTHER, 0. J . & CASIDA, L. E. (1972) J. Anim. Sci. 34, 85-87. DRIANCOURT, M . A. & CAHILL, L. P. (1984) J. Reprod. Fert. 71, 205-21 I .


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TA

BL

E I. M

ain

feat

ures

(m

ean f SD

) of

the

folli

cula

r gr

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the

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the

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ewes

.

No

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(mm pe

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Folli

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r ph

ase

(gro

up 1

)

Lut

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e (g

roup

11)

betw

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3 a

nd 5

2.5

k0.5

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on t

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at

the

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1.4k

0.5

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.9

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and

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.8k

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J .-L. BISTER, M. HEINDRICHS and R. PAQUAY (Laboratoire de Physiologie Animale, Facultb Universitaires de Namur, rue de Bruxelles 61, 8-5000 Namur).

Twin pregnancy diagnosis by progesterone measurement in the ewe (1 figure).

The knowledge of the number of foetuses carried by an ewe is of great interest in sheep-farming, namely for the food’s measurement during the second half of the pregnancy.

In order to establish a twin-pregnancy diagnosis based on plasmatic progesterone level, we randomly take out of a collection of venipuncture realized during several years 10 tubes of plasma for each 10 days’ period throughout the whole pregnancy and for each category of ewes (those which will lamb one or two lambs).

Results During the 70 first days, the progesterone levels seem to be constant. This is cor-

roborated by a two-way analysis of variance which gives a significant effect for the number of foetuses (P < 0.001) but no effect for time or interaction between time and foetuses.

Discriminant analysis between the results yielded by ewes carrying one or two lambs during this first half of pregnancy period gives the discriminant value of 2.09 ng/ml with a probability of misclassification of 0.15.

5 0 -

4 5

4 0 ~. 3 5 - -

3 0

.-

-.

* Ewes mlh 2 lceluses

10..

0 5 -.

25 35 4 5 55 65 75 85 95 105 115 125 135

Day; d vg~vry

FIG. 1. Evolution of P, levels throughout the pregnancy in ewes carrying one or two lambs.

Conclusion We may safety conclude that the R.I.A. measurement of plasmatic progesterone

during the 70 first days of pregnancy is able to diagnose with a good probability (85 chances Yo) that an ewe is carrying one or two lambs, if her P, level is respectively below or above 2.09 ng/ml.


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B. VANHEEL and A. DE HEMPTINNE (Laboratoriurn voor Norrnale en Pathologische Fysiologie, Universiteit Gent, B-9000 Gent).

Facilitated diffusion of L-lactate and pyruvate across red and white skeletal muscle cell membranes of the mouse.

In several cell types a specific monocarboxylate carrier has been shown to be involved in the transmembrane movement of lactate and pyruvate. The transport of these organic acids can be blocked by a-cyano-4-hydroxycinnamate (HALESTRAP, 1976; SPENCER & LEHNINGER, 1976). Kinetic analysis of the effect of medium pH on lactate fluxes suggested a lactate-H+ symport (or lactate-OH- antiport) carrier system (DUBINSKY & RACKER, 1978; MOLL et al., 1980; MONSON et al., 1982; DEUTICKE et al., 1982; DE BRUUNE et al., 1983). Measurements of the rate of decrease of the intracellular pH (PHI) caused by monocarboxylate influx in the absence and presence of cinnamate also indicate the presence of a L-lactate carrier in sheep cardiac Purkyni? strands (DE HEMPTINNE el al., 1983) and frog sartorius muscle fibres (MASON & THOMAS, 1985). In the present study we investigated the possible existence of a similar transport mechanism in mammalian muscles, a typical fast twitch muscle (the m. extensor digitorum longus) and a typical slow twitch muscle (m. soleus).

The rate of H+-associated lactate influx (the sum of passive diffusion of the undissociated form and lactate-H' symport or -OH- antiport) was assessed from the initial rate of change in pHi recorded when the organic acid was added to the superfusion solution. pHi was measured with a double barreled pH-sensitive glass microelectrodes in superficial cells of isolated m. soleus (SOL) and m. extensor digitorum longus (EDL) of mice. The muscles were superfused at 37" C with CO,/HCO, buffered Ringer solutions (pH 7.45). Na-propionate, -lactate or -pyruvate were added to the superfusate, at constant pH, in a concentration range of 2.5-20 mM.

Addition of 20 mM propionate, lactate or pyruvate caused acidification of the muscle fibres, as shown for rat soleus (DE HEMPTINNE & MARRANNES, 1979), crab extensor propodite (SHARP & THOMAS, 1981) and frog sartorius muscle (MASON & THOMAS, 1985). With L-lactate, the mean initial intracellular acidification rate was 0.059 pH unit/min in SOL (n = 1 I), and 0.053 pH unit/min in EDL (n = 18). Due to differences in cellular buffering capacity and surface to volume ratio between SOL and EDL fibres, direct comparison of these absolute values in the two muscle types is difficult. However, the ratio of acidification rate caused by L-lactate and pyruvate to that caused by propionate in the same cell was considerably larger in SOL fibres than in EDL fibres (for L-lactate 84.3 070 and 54.6 070 respectively). The presence of 4 mM a- cyano-4-hydroxycinnamate substantially lowered the rate of pHi decrease induced by 20 mM L-lactate and pyruvate both in SOL and EDL fibres, and had little effect on that produced by 20 mM propionate. Inhibition was larger in SOL than in EDL (for L-lactate 55.1 070 and 3 1 .O 070 respectively). Preliminary experiments indicate differences in concentration dependence of the effect of L-lactate and pyruvate in SOL and EDL fibres. At higher concentration, acidification rates levelled off in both muscle types, but saturation was much more pronounced in EDL.

These experiments suggest that both in SOL and EDL muscle of mice, transmembrane movement of L-lactate and pyruvate is mediated at least partly by an organic anion-Hi symport (or -OH- antiport) carrier system. Differences in modalities of transport of these monocar- boxylates could exist between both muscle types.

References DE BRUIJNE. A. W., VREEBURG, H. & VAN STEVENINCK, J . (1983) Eiochim. Eiophys. Acfa 732. 562-568. DE HEMPTINNE, A. & MARWNES. R . (1979) J . Physiol. (London) 295, 22P-23P. DE HEMPTINNE, A,, MARRANNES. R . & VANHEEL, B. (1983) Amer. J. Physiol. 245, C178-CI83. DEUTICKE, B., BEYER. E. & FORST, B. (1982) Biochim. Biophys. Acta 684, 96-110. DUBINSKY, W. P. & RACKER, E. (1978) J. Membrane Biol. 44, 25-36. HALESTRAP, A. P. (1976) Biochem. J. 156, 193-207. MASON, M. J . & THOMAS, R. C. (1985) J . Physiol. (London) 361, 23P. MOLL, W., GIRARD, H. & GROS, G . (1980) Pfliigers Arch. Eur. J . Physiol. 385, 229-238. MONSON, J . P. , COHEN, R . D., SMITH, J . A. & ILES, R . A. (1982) Clin. Sci. 62. 411-420. SHARP, A. P. & THOMAS, R. C. (1981) J. Physiol. (London) 312, 71-80. SPENCER, T. L. & LEHNINCER. A. L. (1976) Biochem. J . 154, 405-414.

Arch. int. Physiol. Biochirn. 1986, 94 ( 5 )

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