Central vasopressin pretreatment sensitizes phosphoinositol hydrolysis in the rat septum

Brain Research, 531 (1990) 167-172 Elsevier

167

BRES 15967

Central vasopressin pretreatment sensitizes phosphoinositol hydrolysis in the rat septum C.J. Lebrun, M.G. Gruber, M. Meister and Th. Unger Department of Pharmacology, University of Heidelberg and the German Institutefor High Blood Pressure Research, Heidelberg (E R. G.) (Accepted 24 April 1990)

Key words: Vasopressin; Vasopressin receptor; Brain; Septum; Phosphoinositol hydrolysis

Previous in vivo and in vitro studies have demonstrated that exposure of the brain to arginine vasopressin (AVP) can potentiate various responses to a second central challenge with AVE To determine whether this sensitization is mediated by changes at the receptor level, we investigated the effects of AVP on the phosphoinositide metabolism in septal slices prepared from rats centrally pretreated with saline or AVE Addition of vasopressin (10-7 M, 10-6 M) to septal slices from saline-pretreated rats failed to elicit a significant stimulation of inositol-l-phosphate (IP1). In contrast, AVP (10-7 M) significantly stimulated IP1 release in septal slices prepared from rats pretreated intracerebroventricularly (i.c.v.) 24 h earlier with 10 or 100 ng AVE Pretreatment with the same i.c.v, doses of AVP also induced a significant enhancement of the carbachol-induced stimulation of IP~ release, but i.c.v, pretreatment with carbachol did not stimulate the IP~ release in response to AVE Our results suggest that a novel facilitation of phosphoinositide metabolism can be induced by central AVP pretreatment.

INTRODUCTION

havior 6'18 and electrophysiological activity of cells in the h i p p o c a m p u s 9.

T h e n e u r o h y p o p h y s e a l h o r m o n e , arginine vasopressin ( A V P ) , a p e p t i d e with well-known p e r i p h e r a l e n d o c r i n e functions, has been p o s t u l a t e d to serve as a neurotransm i t t e r or n e u r o m o d u l a t o r in various regions of the brain including the septum 1'19'21'22'38-4°. A u t o r a d i o g r a p h i c a l

W h e t h e r this e n h a n c e d sensitivity to A V P in A V P p r e t r e a t e d rats occurs at the r e c e p t o r level is unknown. O n e a p p r o a c h to study this possibility is to e x a m i n e the second messenger system activated by brain AVP-V1 receptors, i.e. p h o s p h o i n o s i t i d e hydrolysis. Thus, in the present study we have a d d r e s s e d the question of w h e t h e r i.c.v, p r e t r e a t m e n t with A V P w o u l d enhance the septal release of inositol p h o s p h a t e in response to AVP.

and biochemical d a t a have d e m o n s t r a t e d the presence of A V P in rat s e p t u m and its release from this area TM 26,27,29,35. N e u r o n s in septal slices r e s p o n d e d to A V P with a m a r k e d increase in their firing rate 15'16"28. In addition, A V P exerts several n e u r o m o d u l a t o r y actions such as increasing catecholaminergic turnover and enhancing the efficacy of excitatory a m i n o acid transmission on lateral septal neurons 16"36. F u r t h e r m o r e , A V P exerts an antipyretic action when perfused into the septal a r e a 1°'t7, facilitates passive avoidance b e h a v i o r ~9'1° and can induce severe m o t o r disturbances such as barrel rotation and myoclonic/myotonic cramps when microinjected into the ventral septal a r e a 2'4'6-8'1s. Most of the central actions of A V P were shown to be m e d i a t e d by stimulation of A V P r e c e p t o r s of the V l - s u b t y p e sA4'28'33. With respect to the regulation of the central A V P system, it has been d e m o n s t r a t e d that intracerebroventricular (i.c.v.) p r e t r e a t m e n t with A V P e n h a n c e d the responses to a second challenge of AVP, including those of b l o o d pressure and splanchnic nerve activity22, be-

MATERIALS AND METHODS

Animals Experiments were performed in male Wistar rats weighing 300-350 g (Thomae, Biberach, ER.G.). Animals were kept under controlled conditions with respect to temperature, humidity, and light periodicity and were allowed free access to food and water. Labelling of brain slices with f H]inositol, extraction of water-soluble inositol phosphates and their separation by anion exchange chromatography Measurement of inositol phosphates was performed as described previously using the method of Bone et al. 5 with slight modifications. Following decapitation, the brains were rapidly removed and dissected on ice. Septal slices were cross-chopped using a McIiwain tissue chopper and incubated for 2.5 h at 37 °C in a shaking incubator in Krebs-Ringer bicarbonate medium (composition in raM: NaCI 125; KCI 3.5; KH2PO 4 1.25; MgSO4 1.2; CaCI2 0.75; NaHCO 3 25; glucose 10) containing 7.5 /~Ci myo-2[aH]inositol. Incubations were gassed every 30 min with an atmosphere of 95%

Correspondence: C. Lebrun, Dept. of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G. 0006-8993/90/$03.50 t~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

168 O 2 and 5% CO 2, The slices (200-300/~g) were then rinsed with the same medium devoid of inositol. They were transferred to a medium containing 160/~1 of prewarmed oxygenated Krebs-Ringer bicarbonate. After 20 rain, 10 mM LiCI was added to inhibit the hydrolysis of any inositol phosphates formed during stimulation 3. Twenty minutes later, the agonist to be tested (AVP or carbachol as control) was added and incubation continued for the appropriate time. Incubation was terminated by addition of perchloric acid (HCIO4). The structures were homogenized and the organic and the aqueous phase were separated by centrifugation. The aqueous phase was neutralized with 1,5 M KOH, and buffered with 75 mM HEPES. The neutralized extracts were diluted to 15 ml with 5 mM sodium tetraborate/0.5 mM EDTA and applied to ion exchange chromatography 1 x 4(200-400). Free inositol was eluted with 28 ml H20; glycero-phosphoinositol (GroPIns) with 28 ml of 5 mM sodium tetraborate/60 mM ammonium formate; inositol monophosphate (IP 0 with 28 ml of 5 mM sodium tetraborate/150 mM ammonium formate; inositol biphosphate (IP2) with 28 ml of 0.1 M formic acid/0.4 M ammonium formate and inositol triphosphate (IP3) with 28 ml of 0.1 M formic acid/1 M ammonium formate. The radioactivity of each fraction was determined by liquid scintillation counting.

Reagents' Arginine vasopressin was purchased from Boehringer/Mannheim, ER.G. Carbachol was purchased from Aldrich Chemie, Steinheim, ER.G. Vasopressin was dissolved in isotonic NaCl and stored in 0.1% albumin-coated vials at -20 °C until use. Aliquots were diluted as required on the day of the experiment. Myo-2[3H]inositol (16 Ci/mmoi) was purchased from Amersham.

Statistical analysis Results are reported as means + S.E.M. The data were subjected to one-way analysis of variance (ANOVA). Student's t-test was used as follow-up test to analyse differences between groups. A significance level of P < 0.05 was accepted. RESULTS

Effects o f A V P and carbachol on the accumulation of labelled inositol phosphates in brain slices I n c u b a t i o n o f s e p t a l slices w i t h A V P a t c o n c e n t r a t i o n s o f 10 -7 M a n d 10 -6 M f a i l e d t o elicit a s i g n i f i c a n t i n c r e a s e

Extraction of lipids

in I P 1 levels c o m p a r e d t o t h o s e o b t a i n e d w i t h c o n t r o l s

Lipids were extracted from the organic phase using chloroform/ methanol/HCl (200:100:1, by volume). The washed lipid extracts were evaporated to dryness in counting vials and the incorporation of [3H]inositol into the lipids was determined.

significant time-dependent stimulation of the breakdown

Expression of results To eliminate the variations caused by differences in the size of slices or in [3H]inositol incorporation, we expressed [3H]IP1 release as a percentage of the total radioactivity found in inositol lipids in each sample.

Experimental protocols Effects of A VP and carbachol on septal phosphoinositide metabolism. Labelled inositol phosphate and lipids were determined in septal slices from untreated rats (n = 7) in the presence of AVP (10 -7 M, 10-6 M), carbachol (10 -4 M) and buffer (control). Sensitization study. Rats were anaesthetized with chloralhydrate (400 mg/kg). An i.c.v, cannula (PP20, Portex, Hythe, Kent, U.K.) was implanted into the right lateral ventricle. The animals were allowed one week to recover from surgery; they were then treated with i.c.v, injections of either isotonic saline (5 /A), AVP (1 ,ul peptide solution plus 4/~1 isotonic saline) at doses of I ng, I0 ng or 100 ng, or carbachol (10 4 M). Twenty-four hours after the i.c.v. injection the rats were killed, their brains removed, and labelled inositol phosphates and lipids were determined in the septum in the absence or in the presence of AVP (10 -7 M) or carbachol (10 -4 M) in the incubation. Rats were divided into 5 groups as shown in Table I. In group 1, the effect of central pretreatment with 1 ng AVP on phosphoinositide metabolism in the septum was investigated. In group IA, septal slices of saline-pretreated rats were incubated in the absence of AVP to determine the basal IP 1 release. In group 1B, IP 1 levels were measured in septal slices of rats pretreated with 1 ng AVP without adding AVP to the incubation. In group 1C, septal slices from saline-pretreated rats were incubated with AVP. In group 1D, septal slices from AVP-pretreated rats were incubated with AVP. Group 1E served as validation of the assay with septai slices from saline-pretreated rats incubated with carbachol. In groups 2 and 3 the effects of central pretreatment with AVP at doses of 10 ng and 100 ng, respectively, were investigated with the same protocol as described for group 1. Groups 4 and 5 served as control for the specificity of the effects obtained with AVP pretreatment: in group 4, the effects of central pretreatment with AVP (1, 10, 100 ng) on the IPl release in response to incubation with carbachol were determined. In group 5, rats were pretreated i.c.v, with 18.30 ng carbachol (corresponding to an equimolar dose of 100 ng AVP), and the IP1 release in response to incubation with AVP was measured.

(Fig.

1). I n c o n t r a s t ,

carbachol

(10 -4 M )

induced

a

o f p h o s p h o i n o s i t i d e s (Fig. 2).

Effects of central pretreatment with A VP on the phosphoinositide metabolism in septal slices C e n t r a l p r e t r e a t m e n t w i t h A V P (1 n g , 10 n g , 100 n g )

TABLE I

Experimental design of the sensitization study SAL, isotonic saline; CARB, carbachol; AVP, a r g i n i ~ vasopressin,

Group 1

A B

C D E Group 2

Group 3

Group 4

Group 5

In vivo

In vitro

SAL AVP 1 ng SAL AVP 1 ng SAL SAL AVP 10 ng SAL AVP 10 ng SAL SAL AVP 100 rig SAL AVP 100 ng SAL SAL SAL AVP 1 ng AVP 10 ng AVP 100 ng SAL CARB SALT CARB SAL CARB

BUFFER BUFFER AVP AVP CARB BUFFER BUFFER AVP AVP CARB BUFFER BUFFER AVP AVP CARB BUFFER CARB CARB CARB CARB BUFFER BUFFER AVP AVP

CARB CARB

169 30

A

1 ng AVP

25 "O

i

20 15 0 o

10 5

Control

C o n t r o l Carb AVP AVP 10-4 M10-7 M10-6 M Fig. 1. Effects of AVP (10-7 M, 10-6 M) on IP 1 accumulation in septM slices. Septal slices were preincubated with [3H]inositol and the accumulation of IP 1 was measured in the presence of Li + as described in the text. Results are expressed as mean + S.E.M., n = 7-11 determinations. **P < 0.01 when compared to control (ANOVA followed by Student's t-test. Carb = carbachol.

did not affect the unstimulated phosphoinositide b r e a k down in the absence of A V P (Fig. 3). In septal slices from rats p r e t r e a t e d with the lowest dose of 1 ng, A V P ( A V P 10 -7 M ) failed to p r o d u c e a significant stimulation of IP 1 release (Fig. 3A). H o w e v e r , in septal slices from rats p r e t r e a t e d with 10 ng or 100 ng AVP, A V P (10 -7 M) p r o d u c e d a significant stimulation of IP 1 release when c o m p a r e d to s a l i n e - p r e t r e a t e d rats (Fig. 3B,C). Central p r e t r e a t m e n t with A V P (10 ng, 100 ng) also e n h a n c e d the septal IP 1 release in response to carbachol (Fig. 4). O n the o t h e r hand, in septal slices from rats p r e t r e a t e d with carbachol, A V P did not significantly stimulate IP 1 release, and the IP 1 release in response to carbachol r e m a i n e d u n a l t e r e d (Fig. 5).

251 B

AVP 10-7 M

Carb 10-4 M

10 n g A V P

200J~l L

~o

15o

~.~

105-

N Control

16

C

AVP 10-7 M

1 0 0 n g AVP"

Carb 10-4 M

[

14 12 10 8 6

4 2 0

I

Control

AVP 10-7 M

Carb 10-4 M

Fig. 3. Effect of HAVP (10-7 M) on IP 1 release in scptal slices from rats sacrificed 24 h after i.c.v, injection of saline or 1, 10, 100 ng AVP. Clear columns depict saline-pretreated rats, and hatched columns depict AVP-pretreated rats. Results are expressed as means + S.E.M., n = 4-8 determinations. *P < 0.05, **P < 0.01 when compared to control (incubation with buffer in the absence of AVP) (ANOVA followed by Studentes t-test).

500

400 Z

o -C

300

DISCUSSION 200 09

100'

10

i

i

,

i

60

90

TIME (min)

Fig. 2. Percentage of stimulation (% of control) induced by carbachol (10" M) on IP~ release in septal slices.

T h e results of o u r study d e m o n s t r a t e that central p r e t r e a t m e n t with vasopressin can sensitize the p h o s p h o inositide m e t a b o l i s m in response to A V P in the rat septum. O u r results t o g e t h e r with a u t o r a d i o g r a p h i c a l and electrophysiological results 1'z'13'15 a n d the biochemical evidence that e n d o g e n o u s A V P is p r e s e n t in the septum and can be r e l e a s e d from this a r e a 11"26, all s u p p o r t the

170 30-

• Carb

~.~ l s ~=,~ lO5 0 Control

il

AVP 10-7 M

pretreated

E3 Saline pretreated

Carb

10-4 M

Fig. 4. Effect of carbachol (10 -4 M) on IP 1 release in septal slices from rats after i.c.v, injection of 1, 10, 100 ng A V E Results are expressed as means + S.E.M., n = 3-7 determinations. Clear columns depict saline-pretreated rats, and hatched columns depict AVP-pretreated rats. *P < 0.05, **P < 0.01 when compared to control; +P < 0.05 when compared to carbachol (10 -4 M) (from saline-pretreated rats).

view that AVP acts as a neurotransmitter in the lateral septum by acting on AVP-V1 receptors. In our experiments, AVP failed to stimulate IP 1 release in septal slices of control rats that were not pretreated with AVE In contrast to our findings, Shewey and Dorsa z9'31 showed a dose-dependent increase in IP~ accumulation in response to AVP in the septum. The discrepancy between these and our results could be explained by the low concentration of protein per tube (200-300/~g) present in our experiment. According to Shewey and Dorsa 29, a tissue concentration of approximately 600/A/40/A is required for a maximal response to AVE Concentrations both higher and lower result in submaximal responses 29. It should also be kept in mind

Pretreatment AVP lng

iOng

lOOng

M

~0

18'~' E--~ _

Control

Carbachol

10-4

M

Fig. 5. Effect of AVP (10 -7 M) on IP 1 release in septal slices sacrificed 24 h after the i.c.v, injection of carbachol (18.30 ng). Results are expressed as means + S.E.M., n = 3-5 determinations. **P < 0.01 when compared to control or AVP (10 -7 M).

that the septal area contains a number of anatomically and functionally different nuclei and that probably only some of these nuclei respond to AVP a2'25,27"2s'34. Since septal slices were used in our experiments, we cannot rule out that such preparation may have masked a discretely localized stimulation of phosphoinositide metabolism. However, when rats were centrally pretreated with AVP at doses of 10 and 100 ng, a significant stimulation of phosphoinositide metabolism in response to AVP was observed. This effect may underlie the altered sensitivity to AVP observed previously in various experimental models. For instance, we have shown that i.c.v, pretreatment with AVP can sensitize animals to the pressor effects and stimulation of sympathetic nerve activity induced by central AVP receptor stimulation 22. Other investigators observed that an increase in central vasopressin levels following AVP pretreatment increased the sensitivity of the animals to the convulsive effects of A M P 6"18. Moreover, hippocampal slices from rats pretreated with AVP exhibited an increased sensitivity to the depressant effect of population spike amplitude in response to AVP 9. Evidence for the presence of AVP-V1 receptors in the central nervous system has already been presented. For instance, vasopressin activated phosphoinositide metabolism in the superior cervical ganglion and in the hippocampus 5'33. In addition, using in vivo labelling with i.c.v, injections of [3H]inositol, AVP stimulated phosphoinositide metabolism in the medulla oblongata 24. So far, three different subtypes of vasopressin receptors have been described in the brain, namely a high affinity AVP binding site, a high affinity oxytocin binding site and a low affinity AVP binding site 27. Our finding that only the higher i.c.v, doses of AVP showed a sensitizing effect in the septum could reflect the fact that only one class of vasopressin receptors is involved in this action. Pretreatment with AVP also induced a sensitization of septal slices to the IP 1 generating effects of carbachol. It seems unlikely that AVP directly interfered with muscarinic receptors. However, it is possible that part of the carbachol response is due to release of endogenous AVE Cholinergic agonists are known to be potent stimulators for AVP release 32, and preliminary experiments from Shewey and Dorsa 29 suggest that carbachol may release vasopressin from septal slices. Furthermore, an AVP-V1 antagonist produced a partial but significant inhibition of the septal IPa release response to carbachoi 29. Thus, a sensitization to carbachol in septal slices from AVPpretreated rats could be taken as another indirect piece of evidence for a sensitization of AVP receptors. On the other hand, when rats were pretreated i.c.v. with carbachol, AVP failed to stimulate septal IP 1 release. Furthermore, the carbachol-induced stimulation

171 of IP 1 release was not e n h a n c e d by central p r e t r e a t m e n t with carbachol. These findings suggest that the o b s e r v e d sensitization is specifically induced by A V P and not due to previous stimulation of phosphoinositide metabolism p e r se. In contrast to o u r results, central p r e t r e a t m e n t with A V P has b e e n shown to induce a decrease in IP 1 release in response to A V P in the homozygous B r a t t l e b o r o rat 3°. These e x p e r i m e n t s differ from ours in that the animals were chronically p r e t r e a t e d with high doses of A V P (20 /~g/rat). F u r t h e r m o r e , h o m o z y g o u s B r a t t l e b o r o rats carry as a semirecessive trait the inability to synthetize A V P and exhibit severe behavioral and physiological deficiencies including diabetes insipidus 37. It is possible that in this animal m o d e l the regulation of A V P receptors is different from rats with an intact A V P system. Similar to o u r findings, an e n h a n c e d inositol phosp h a t e p r o d u c t i o n was o b s e r v e d after central p r e t r e a t m e n t with the p e p t i d e luteinizing hormone-releasing h o r m o n e ( L H - R H ) 23. Prior exposure to L H - R H caused a charac-

teristic p o t e n t i a t i o n of subsequent s e c r e t o r y responses and specifically e n h a n c e d L H - R H - i n d u c e d inositol phosp h a t e p r o d u c t i o n and mobilization of intracellular Ca 2+ stores 23. Since no c o r r e s p o n d i n g changes in r e c e p t o r p r o p e r t i e s were o b s e r v e d , the authors h y p o t h e s i z e d that a novel p h e n o m e n o n , i.e. a facilitation of the coupling of the L H - R H r e c e p t o r to its effector, was responsible for the e n h a n c e d response. Such a m e c h a n i s m could also be instrumental in the A V P - i n d u c e d sensitization of the phosphoinositide m e t a b o l i s m r e p o r t e d here. In conclusion, we have d e m o n s t r a t e d that central p r e t r e a t m e n t of rats with A V P enhances IP 1 release in response to A V P in septal slices, suggesting an increased sensitivity to A V P at the r e c e p t o r level. This receptorrelated sensitization m a y be a critical r e g u l a t o r y mechanism for the central actions of A V P in the septum and in o t h e r brain areas. Acknowledgement. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Un 47/1-2) to Th. U.

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