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GABA agonists and omega conotoxin GVIA modulate responses to nerve activation of the perfused rat mesentery
Life Sciences, Vol. 48, pp. 2331-2339 Printed in the U.S.A.
Pergamon Press
GABA AGONISTS AND OMEGA CONOTOXIN GVIA M O D U L A T E RESPONSES TO NERVE ACTIVATION OF THE PERFUSED RAT MESENTERY Yuanjian Li and Sue Piper Duckies Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, CA 92717 (Received in final form April 9, 1991)
Summary The modulatory actions of T-aminobutyric acid (GABA) receptor agonists and co-conotoxin GVIA (CTX) on s y m p a t h e t i c and sensory nerves were e x a m i n e d on contractile responses of the perfused rat mesentery to transmural nerve stimulation (TNS). GABA and baclofen, a selective GABAB receptor agonist, significantly inhibited vasoconstrictor responses to TNS, while muscimol, a selective GABAA receptor agonist, had no effect. In the guanethidine treated and methoxamine-contracted mesentery, TNS caused a vasodilator response which was unaffected by GABA. CTX (10-8 M) markedly suppressed the vasoconstrictor response to TNS, but did not affect vasodilator responses. These findings suggest that in the rat mesentery: (1) GABA receptors modulate the activity of sympathetic nerves via prejunctional GABAB receptors, but do not influence sensory nerves, and (2) calcium channels which participate in sympathetic nerve activation have different properties than calcium channels in capsaicinsensitive sensory nerves. T-aminobutyric acid (GABA), an inhibitory neurotransmitter, is distributed in central and peripheral nerves and has been reported to have modulatory actions on sympathetic and parasympathetic nerves as well as capsaicin sensitive sensory nerves (1-4). Actions of the GABA receptor are mediated by at least two distinct receptor types, termed GABAA and GABAB (5). It is well known that calcium ions play a crucial role in d e p o l a r i z a t i o n - c o u p l e d transmitter release from nerve terminals. Recent investigations have suggested that voltage-sensitive calcium 0024-3205/91 $3.00 + .00 Copyright (e) 1991 Pergamon Press plc
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channels can be divided into L-, N- and T types and that N-channels play a major role in the control of neurotransmitter release (6-8). 0~Conotoxin GVIA (CTX), a very potent and selective blocker of L- and N type channels, has been shown to inhibit neurotransmitter release from sympathetic nerves and also from some sensory nerves (9-12). It has recently been demonstrated that rat mesenteric arteries are innervated both by adrenergic and capsaicin-sensitive sensory nerves which mediate vasoconstrictor and vasodilator responses, respectively (13,14). GABA receptors and N-type calcium channels have been found to affect the transmission of both types of nerves in some tissues. However, differential sensitivity to CTX has been shown for adrenergic and capsaicin-sensitive sensory nerves (9,12). Previous investigations concerning the effect of GABA agonists or CTX on the modulatory action of sympathetic or sensory nerves were separately performed in different tissues, and it is not clear whether GABA agonists and CTX modulate the activity of both sympathetic and sensory nerves in the rat mesentery. Therefore, the aim of this study was to explore the modulatory effect of GABA agonists and CTX on vasomotor responses to activation of vasoconstrictor (sympathetic) or vasodilator (sensory) nerves in the perfused rat mesentery. Materiab and Methods Tissue preparation and perfusion. Mesenteric vasculature of male Sprague Dawley rats (250 300 g) was isolated and prepared for perfusion as described by Kawasaki and Takasaki (15) with some modifications. Rats were decapitated, and the mesenteric artery was quickly cannulated at its origin at the aorta with PE 50 tubing and perfused with warm Krebs' solution, saturated with 95% 02 and 5% CO2. The mesentery was isolated under perfusion, and preparations were then placed in a water jacket maintained at 37°C. The system was perfused with Krebs' solution with the help of a peristaltic pump at a rate of 5 + 0.2 ml/min and superfused by gravity feed at a rate of 1 + 0.2 ml/min. The Krebs' solution had the following composition (in mM): NaCI, 118; KC1, 4.8; CaC12, 2.5; KH2PO4, 1.2; NaHCO3, 25; MgSO4, 1.2; EDTA.Na, 0.107; and dextrose, 11.5. The perfusion pressure was monitored and recorded by a pressure transducer, and resulting electrical signals were digitalized by Maclab analog to digital converter and recorded by Macintosh SE computer. Electrical stimulation. Two platinum electrodes, one placed around the superior mesenteric artery and the other resting on the vasculature in a lower part of the intestine, were used to create
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transmural field stimulation. Transmural nerve stimulation (TNS) (amplitude of 70 V and pulse duration of 3 msec) was applied at various frequencies and train lengths using a Grass $48 stimulator. For constrictor responses to TNS, 3 min were allowed between each stimulation train. In the case of vasodilator responses to TNS, sufficient time was allowed between each stimulation train for the perfusion pressure to return to a stable level, usually 10 to 20 min. E x o e r i m e n t a l procedures. Tissues were equilibrated for 60 min before beginning each experiment. All drugs were administered by switching the perfusion solution to solution containing drug in the final concentrations indicated. Exposure to capsaicin was for 20 min, and capsaicin was then washed out. For measurement of vasodilator responses, the tissues were pretreated with guanethidine (5 x 10-6 M) for 20 min, and then contracted by methoxamine (5 x 10 -6 to 10-5 M). Both guanethidine and methoxamine then remained in the perfusate for the remainder of the study. Tissues were exposed to GABA agonists or CTX for 10 min, and TNS was applied at the seventh minute of drug administration. For all studies a paired design was used, that is the same tissue was studied both before and after treatment with GABA agonists or CTX. Drugs. The following drugs were used: ? - a m i n o b u t y r i c acid, baclofen, muscimol, tetrodotoxin and methoxamine HC1 (Sigma Chemical Co); 0~-conotoxin GVIA (Peninsula); capsaicin (ICN Biomedicals, Inc); and guanethidine (Ciba Pharmaceutical Co). All drugs were dissolved in Krebs' solution, except capsaicin which was initially dissolved in ethanol and further diluted in Krebs' solution to the proper final concentration. Statistics. Results are expressed as means + S.E.M. Student's paired t-test was used to determine statistical differences between two means. The level of significance was chosen as p<0.05. Results Effect of GABA agonists. TNS of the mesenteric vascular bed caused an increase in perfusion pressure. This response was completely blocked by guanethidine (5 x 10-6 M) and tetrodotoxin (10 -6 M), confirming that it is due to nerve stimulation. Vasoconstrictor responses to TNS were significantly depressed in the presence of GABA (10 -5 or 10-4 M) (Fig 1A). There is evidence in the literature suggesting that GABA agonists cause the release of substance P and calcitonin gene related peptide (CGRP) from isolated guinea-pig ilea and left atria
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(16,17). In order to rule out the possible contribution of vasodilator transmitter release from capsaicin-sensitive sensory nerves, the preparation was pretreated with capsaicin (3 x 10 -7 M ) f o r 20 min to d e s e n s i t i z e the tissue to sensory nerve stimulation. As we have p r e v i o u l y reported, in tissues pretreated with e a p s a i c i n v a s o d i l a t o r r e s p o n s e s to TNS are c o m p l e t e l y abolished (14). After capsaicin treatment, however, G A B A still inhibited vasoconstrictor responses to TNS (Fig 1B). I~ []
B. After Capsalcin
A. Before Capsalcln 40
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FIG. 1 Effect of various concentrations of G A B A on vasoconstrictor responses to TNS before (A) and after (B) treatment with capsaicin (3 x 10 -7 M). TNS was applied at 8 Hz for 10 sec trains. Each value is the mean + S.E.M (N=5). ** p<0.01 compared with control. A. Baclofen
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FIG. 2 Effect of baclofen (A) (N=6) and muscimol (B) (N=5) on v a s o c o n s t r i c t o r r e s p o n s e s to TNS (8 Hz, 10 see trains). Values are given as mean + S.E.M. *** p<0.001 compared with control.
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T w o G A B A receptor agonists were selected to represent actions on two types of G A B A receptor, muscimol ( G A B A A ) and baclofen (GABAB). As shown in Fig 2A, vasoconstrictor responses to TNS were markedly inhibited by baclofen (10 -5 and 10 -4 M). However, muscimol had no effect on vasoconstrictor responses to TNS (Fig 2B). 60 50 v r#$
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FIG, 3 E f f e c t of G A B A on vasodilator r e s p o n s e s to TNS after treatment with guanethidine (5 x 10 -6 M) and in the presence of methoxamine (5 x 10 -6 to 10 -5 M) (N=6). TNS was applied at 8 Hz for 10 sec trains. Values are means + S.E.M. A. Vasoconstrictor response 25
B. Vasodllator response
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Ok) FIG. 4 Effect of CTX (10 -8 M) on vasoconstrictor responses (A) (N=5) and vasodilator responses (B) (N=6) to TNS (8 Hz, 10 see trains). Values are given as means 5: S.E.M. *** p<0.001 compared with control. To investigate the effect sensory nerve stimulation, guanethidine (5 x 10-6 M)
of G A B A on vasodilator responses to preparations were pretreated with to b l o c k s y m p a t h e t i c nerves. A f t e r
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methoxamine contraction, G A B A itself did not produce any relaxation (N=6) and TNS caused a vasodilator response as previously shown (13). However, the vasodilator response to TNS was unaffected by GABA at any of the concentrations tested (Fig 3). Effect~ of CTX. As shown in Fig 4A, vasoconstrictor responses to TNS were significantly suppressed in the presence of CTX (10 -8 M). In the presence of guanethidine (5 x 10-6 M), CTX did not significantly a f f e c t the m e t h o x a m i n e - i n d u c e d vasoconstrictor response of the perfused rat mesentery. Contractile responses to methoxamine were 75 _+ 9 and 78 + 12 mmHg for control and CTX treated tissues, respectively (p>0.3, N=6). In contrast, CTX had no significant effect on vasodilator responses to TNS (Fig 4B). Discussion This study shows that GABA significantly suppresses vasoconstrictor responses of the perfused rat mesentery to TNS. Several p o s s i b l e mechanisms have been considered. Others have shown that G A B A A agonists can stimulate capsaicin-sensitive sensory nerves in the guinea-pig ileum and left atrium (16,17). Therefore, the possibility that inhibition of the pressor response of sympathetic nerves to TNS was due to stimulation of sensory nerves was considered. H o w e v e r , in the p r e s e n t study, after pretreatment with capsaicin to d e s e n s i t i z e the tissue to s e n s o r y nerve stimulation, GABA still inhibited the vasoconstrictor response to TNS. A n o t h e r p o s s i b i l i t y we c o n s i d e r e d was that G A B A r e c e p t o r s mediate a direct vasodilatory effect via a postjunctional site of action. It has been demonstrated that G A B A , via a GABAA receptor, causes vasodilatation in isolated cerebral blood vessels from cat, dog and rabbit (18-20). However, in the present study, G A B A did not produce any relaxation in the methoxamine-contracted mesentery. Similar results were reported in K+-contracted rat mesenteric arteries where G A B A did not produce any relaxation (21). Thus, we conclude that it is most possible that G A B A inhibits vasoconstrictor responses to TNS by an action on p r e j u n c t i o n a l r e c e p t o r s to inhibit adrenergic transmitter release. Others have shown that GABAB receptors inhibit stimulationinduced contraction and [3H] norepinephrine release in the isolated goat cerebrovascular bed (1), in further support of this conclusion. GABA receptors are subdivided into GABAA and GABAB types. The former is sensitive to muscimol, while the latter is sensitive to baclofen (5). In order to explore the type of G A B A receptor mediating the
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modulation of vasoconstrictor nerve effects, muscimol and baclofen were used. Vasoconstrictor responses to TNS of the perfused rat mesentery were markedly inhibited in the presence of baclofen, while muscimol had no effect. These results suggest that this inhibitory effect of GABA is due to an action at GABAB receptors. The mechanism of the inhibitory effect of GABAB on neurotransmitter release from sympathetic nerves is still unclear, but recent evidence suggests that the GABAB receptor is functionally coupled to the calcium ion channel via a GTP-binding protein (4). It has also been demonstrated that G A B A receptors can modulate the actions of s e n s o r y nerves. G A B A and b a c l o f e n inhibited the b r o n c h o c o n s t r i c t i o n p r o d u c e d by stimulation of non-adrenergic, noncholinergic nerves in the guinea-pig, and GABAB receptors have been shown to inhibit electrical stimulation-induced release of substance P from e m b r y o n i c chick dorsal root ganglia (2,3). In contrast, as mentioned p r e v i o u s l y GABAA receptors stimulate neurotransmitter release from sensory nerves in the guinea-pig isolated ileum and left atrium (16,17). However, in the present study, TNS-induced vasodilator responses were not affected by GABA, suggesting that GABA receptors do not modulate sensory nerve activation in the rat mesentery. CTX, a selective blocker of L- and N type calcium channels, has been shown to inhibit neurotransmitter release from sympathetic and parasympathetic nerves as well as sensory nerves in the central nervous system and periphery (9-11). In the present study, CTX markedly inhibited v a s o c o n s t r i c t o r r e s p o n s e s to TNS, but had no effect on vasodilator responses, suggesting that N-type calcium channels are only i n v o l v e d in activation of s y m p a t h e t i c nerves and do not mediate activation of sensory nerves in the rat mesentery. It was previously reported that CTX did not affect the release of substance P from sensory nerves in the guinea-pig bladder or the actions of non-adrenergic nonc h o l i n e r g i c nerves in the rat a n o c o c c y g e u s , d u o d e n u m or urinary b l a d d e r (9,22,23). More recently, differential sensitivities to CTX between different transmission processes have been shown in a single tissue, for example, in rat gastric fundus strips and in gunea-pig taenia cacti (12). It was postulated that this discrepancy may be due to the e x i s t e n c e of a h e t e r o g e n e o u s population of v o l t a g e - s e n s i t i v e calcium channels or the lack of involvement of N-type channels in controlling Ca 2+ influx during excitation-secretion coupling (9,12). Using guanethidine and capsaicin as tools to reveal distinct r e s p o n s e s to v a s o c o n s t r i c t o r and v a s o d i l a t o r nerves, we and other authors have demonstrated that the rat mesentery is innervated both by
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adrenergic and capsaicin-sensitive sensory nerves (13). The present study demonstrates that sensitivity to GABA agonists and CTX differs between these two types of nerves. Thus, prejunctional GABA receptors appear to modulate transmitter release from sympathetic nerves but are not active in sensory nerves. Similarly, the calcium channel blocker CTX effectively blocks activation of sympathetic nerves, but is ineffective in the case of sensory nerves. This study, along with previous findings (12), underscores the differential sensitivity to n e u r o m o d u l a t o r y influences of sympathetic and capsaicin-sensitive sensory nerve transmission in a single tissue.
Acknowledgements This work was supported by Grant #PO1 DK36289 from the National Institutes of Health and by a postdoctoral fellowship from the California Affiliate of the American Heart Association. References 1.
F.J. MIRANDA, G. TORREGROSA, J.B. SALOM, V. CAMPOS, J.A. ALABADI AND E. ALBORCH, Brain Res. 492 45-52 (1989). 2. M.G. BELVISI, M. ICHINOSE AND P.J. BARNES, Br. J. Pharmacol. 9._27 1225-1231 (1989). 3. D.W.Y. SAH, J. Neurosci. 10 136-141 (1990). 4. Y. OHMORI, M. HIROUCHI, J.I. TAGUCHI AND K. KURIYAMA, J. Neurochem. 54 80-85 (1990). 5. D.R. HILL AND N.G. BOWERY, Nature 290 149-152 (1981). 6. R.J. MILLER, Science 235 46-52. (1987). 7. R.W. TSIEN, D. LIPSCOMBE, D.V. MADISON, K.R. BLEY AND A.P. FOX, Trends. Neurosci. 11 431-438 (1988). 8. R.A. KEITH, T.J. MANGANO, M.A. PACHECO AND A.I. SALAMA, J. AUTON. Pharmacol. 9 243-252 (1989). 9. C.A. MAGGI, R. PATACCHINI, P. SANTICIOLI, I. TH. LIPPE, S. GIULIANI, P. GEPPETTI, E.D. BIANCO, S. SELLERI AND A. MELI, Naunyn-Schmiedeberg's. Arch. Pharmacol. 338 107-113 (1988). 10. H. KASAI T. AOSAKI AND J. FUKUDA, Neurosci. Res. 4 228-235 (1987). 11. J.A. BROCK, T.C. CUNNANE, R.J. EVANS AND J. ZIOGAS, Clin. Exp. Pharmacol. Physiol. 16 333-339 (1989). 12. A. DE LUCA, C.G. LI, M.J. RAND, J.J. REID, P. THAINA AND H.K. WONG-DUSTING, Br. J. Pharmacol. 101 437-447 (1990).
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13. H. KAWASAKI, K. TAKASAKI, A. SAITO AND K. GOTO, Nature 335 164-167 (1988). 14. YJ. LI AND S.P. DUCKLES, European J. Pharmacol. (in press) (1991). 15. H. KAWASAKI AND K. TAKASAKI, J. Pharmacol. Exp. Ther. 229 816-822 (1984). 16. M. TONINI, L. ONORI, C.A. RIZZI, E. PERUCCA, L. MANZO AND A. CREMA, Naunyn-Schmiedeberg's. Arch. Pharmacol. 3 3 5 6 2 9 - 6 3 5 (1987). 17. C.A. MAGGI, S. GIULIANI, S. MANZINI AND A. MELI, Br. J. Pharmacol. 9__7_7103-110 (1989). 18. L. EDVINSSON AND D.N. KRAUSE, Brain Res. 17389-97 (1979). 19. M. FUJIWARA AND I. MURAMATSU, Br. J. Pharmacol. 5__55561-562 (1975). 20. N. ANWAR AND D.F.J. MASON, Br. J. Pharmacol. 7__55177-181 (1982). 21. F.M. LAI, T. TANIKELLA AND P. CERVONI, J. Cardiovasc. Pharmacol. 1 2 3 7 2 - 3 7 6 (1988). 22. A.T. MCKNIGHT, J.J. MAGUIRE AND G.N. WOODRUFF, J. Physiol. 4 0 9 5 0 P (1989). 23. C.A. MAGGI, S. GIULIANI, P. SANTICIOLI, M. TRAMONTANA AND A. MELI, Neuroscience. 3 4 2 4 3 - 2 5 0 (1990).
Pergamon Press
GABA AGONISTS AND OMEGA CONOTOXIN GVIA M O D U L A T E RESPONSES TO NERVE ACTIVATION OF THE PERFUSED RAT MESENTERY Yuanjian Li and Sue Piper Duckies Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, CA 92717 (Received in final form April 9, 1991)
Summary The modulatory actions of T-aminobutyric acid (GABA) receptor agonists and co-conotoxin GVIA (CTX) on s y m p a t h e t i c and sensory nerves were e x a m i n e d on contractile responses of the perfused rat mesentery to transmural nerve stimulation (TNS). GABA and baclofen, a selective GABAB receptor agonist, significantly inhibited vasoconstrictor responses to TNS, while muscimol, a selective GABAA receptor agonist, had no effect. In the guanethidine treated and methoxamine-contracted mesentery, TNS caused a vasodilator response which was unaffected by GABA. CTX (10-8 M) markedly suppressed the vasoconstrictor response to TNS, but did not affect vasodilator responses. These findings suggest that in the rat mesentery: (1) GABA receptors modulate the activity of sympathetic nerves via prejunctional GABAB receptors, but do not influence sensory nerves, and (2) calcium channels which participate in sympathetic nerve activation have different properties than calcium channels in capsaicinsensitive sensory nerves. T-aminobutyric acid (GABA), an inhibitory neurotransmitter, is distributed in central and peripheral nerves and has been reported to have modulatory actions on sympathetic and parasympathetic nerves as well as capsaicin sensitive sensory nerves (1-4). Actions of the GABA receptor are mediated by at least two distinct receptor types, termed GABAA and GABAB (5). It is well known that calcium ions play a crucial role in d e p o l a r i z a t i o n - c o u p l e d transmitter release from nerve terminals. Recent investigations have suggested that voltage-sensitive calcium 0024-3205/91 $3.00 + .00 Copyright (e) 1991 Pergamon Press plc
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channels can be divided into L-, N- and T types and that N-channels play a major role in the control of neurotransmitter release (6-8). 0~Conotoxin GVIA (CTX), a very potent and selective blocker of L- and N type channels, has been shown to inhibit neurotransmitter release from sympathetic nerves and also from some sensory nerves (9-12). It has recently been demonstrated that rat mesenteric arteries are innervated both by adrenergic and capsaicin-sensitive sensory nerves which mediate vasoconstrictor and vasodilator responses, respectively (13,14). GABA receptors and N-type calcium channels have been found to affect the transmission of both types of nerves in some tissues. However, differential sensitivity to CTX has been shown for adrenergic and capsaicin-sensitive sensory nerves (9,12). Previous investigations concerning the effect of GABA agonists or CTX on the modulatory action of sympathetic or sensory nerves were separately performed in different tissues, and it is not clear whether GABA agonists and CTX modulate the activity of both sympathetic and sensory nerves in the rat mesentery. Therefore, the aim of this study was to explore the modulatory effect of GABA agonists and CTX on vasomotor responses to activation of vasoconstrictor (sympathetic) or vasodilator (sensory) nerves in the perfused rat mesentery. Materiab and Methods Tissue preparation and perfusion. Mesenteric vasculature of male Sprague Dawley rats (250 300 g) was isolated and prepared for perfusion as described by Kawasaki and Takasaki (15) with some modifications. Rats were decapitated, and the mesenteric artery was quickly cannulated at its origin at the aorta with PE 50 tubing and perfused with warm Krebs' solution, saturated with 95% 02 and 5% CO2. The mesentery was isolated under perfusion, and preparations were then placed in a water jacket maintained at 37°C. The system was perfused with Krebs' solution with the help of a peristaltic pump at a rate of 5 + 0.2 ml/min and superfused by gravity feed at a rate of 1 + 0.2 ml/min. The Krebs' solution had the following composition (in mM): NaCI, 118; KC1, 4.8; CaC12, 2.5; KH2PO4, 1.2; NaHCO3, 25; MgSO4, 1.2; EDTA.Na, 0.107; and dextrose, 11.5. The perfusion pressure was monitored and recorded by a pressure transducer, and resulting electrical signals were digitalized by Maclab analog to digital converter and recorded by Macintosh SE computer. Electrical stimulation. Two platinum electrodes, one placed around the superior mesenteric artery and the other resting on the vasculature in a lower part of the intestine, were used to create
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transmural field stimulation. Transmural nerve stimulation (TNS) (amplitude of 70 V and pulse duration of 3 msec) was applied at various frequencies and train lengths using a Grass $48 stimulator. For constrictor responses to TNS, 3 min were allowed between each stimulation train. In the case of vasodilator responses to TNS, sufficient time was allowed between each stimulation train for the perfusion pressure to return to a stable level, usually 10 to 20 min. E x o e r i m e n t a l procedures. Tissues were equilibrated for 60 min before beginning each experiment. All drugs were administered by switching the perfusion solution to solution containing drug in the final concentrations indicated. Exposure to capsaicin was for 20 min, and capsaicin was then washed out. For measurement of vasodilator responses, the tissues were pretreated with guanethidine (5 x 10-6 M) for 20 min, and then contracted by methoxamine (5 x 10 -6 to 10-5 M). Both guanethidine and methoxamine then remained in the perfusate for the remainder of the study. Tissues were exposed to GABA agonists or CTX for 10 min, and TNS was applied at the seventh minute of drug administration. For all studies a paired design was used, that is the same tissue was studied both before and after treatment with GABA agonists or CTX. Drugs. The following drugs were used: ? - a m i n o b u t y r i c acid, baclofen, muscimol, tetrodotoxin and methoxamine HC1 (Sigma Chemical Co); 0~-conotoxin GVIA (Peninsula); capsaicin (ICN Biomedicals, Inc); and guanethidine (Ciba Pharmaceutical Co). All drugs were dissolved in Krebs' solution, except capsaicin which was initially dissolved in ethanol and further diluted in Krebs' solution to the proper final concentration. Statistics. Results are expressed as means + S.E.M. Student's paired t-test was used to determine statistical differences between two means. The level of significance was chosen as p<0.05. Results Effect of GABA agonists. TNS of the mesenteric vascular bed caused an increase in perfusion pressure. This response was completely blocked by guanethidine (5 x 10-6 M) and tetrodotoxin (10 -6 M), confirming that it is due to nerve stimulation. Vasoconstrictor responses to TNS were significantly depressed in the presence of GABA (10 -5 or 10-4 M) (Fig 1A). There is evidence in the literature suggesting that GABA agonists cause the release of substance P and calcitonin gene related peptide (CGRP) from isolated guinea-pig ilea and left atria
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(16,17). In order to rule out the possible contribution of vasodilator transmitter release from capsaicin-sensitive sensory nerves, the preparation was pretreated with capsaicin (3 x 10 -7 M ) f o r 20 min to d e s e n s i t i z e the tissue to sensory nerve stimulation. As we have p r e v i o u l y reported, in tissues pretreated with e a p s a i c i n v a s o d i l a t o r r e s p o n s e s to TNS are c o m p l e t e l y abolished (14). After capsaicin treatment, however, G A B A still inhibited vasoconstrictor responses to TNS (Fig 1B). I~ []
B. After Capsalcin
A. Before Capsalcln 40
CONT GABA
30
i ¢
i
,
10-
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6 5 4 G A B A concentration (-log M)
6 5 GABA concentration (-log M)
FIG. 1 Effect of various concentrations of G A B A on vasoconstrictor responses to TNS before (A) and after (B) treatment with capsaicin (3 x 10 -7 M). TNS was applied at 8 Hz for 10 sec trains. Each value is the mean + S.E.M (N=5). ** p<0.01 compared with control. A. Baclofen
B. Muscimol
[] []
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25
Control +GABA agonist
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5
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FIG. 2 Effect of baclofen (A) (N=6) and muscimol (B) (N=5) on v a s o c o n s t r i c t o r r e s p o n s e s to TNS (8 Hz, 10 see trains). Values are given as mean + S.E.M. *** p<0.001 compared with control.
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T w o G A B A receptor agonists were selected to represent actions on two types of G A B A receptor, muscimol ( G A B A A ) and baclofen (GABAB). As shown in Fig 2A, vasoconstrictor responses to TNS were markedly inhibited by baclofen (10 -5 and 10 -4 M). However, muscimol had no effect on vasoconstrictor responses to TNS (Fig 2B). 60 50 v r#$
4o 30
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FIG, 3 E f f e c t of G A B A on vasodilator r e s p o n s e s to TNS after treatment with guanethidine (5 x 10 -6 M) and in the presence of methoxamine (5 x 10 -6 to 10 -5 M) (N=6). TNS was applied at 8 Hz for 10 sec trains. Values are means + S.E.M. A. Vasoconstrictor response 25
B. Vasodllator response
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4
8
Ok) FIG. 4 Effect of CTX (10 -8 M) on vasoconstrictor responses (A) (N=5) and vasodilator responses (B) (N=6) to TNS (8 Hz, 10 see trains). Values are given as means 5: S.E.M. *** p<0.001 compared with control. To investigate the effect sensory nerve stimulation, guanethidine (5 x 10-6 M)
of G A B A on vasodilator responses to preparations were pretreated with to b l o c k s y m p a t h e t i c nerves. A f t e r
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methoxamine contraction, G A B A itself did not produce any relaxation (N=6) and TNS caused a vasodilator response as previously shown (13). However, the vasodilator response to TNS was unaffected by GABA at any of the concentrations tested (Fig 3). Effect~ of CTX. As shown in Fig 4A, vasoconstrictor responses to TNS were significantly suppressed in the presence of CTX (10 -8 M). In the presence of guanethidine (5 x 10-6 M), CTX did not significantly a f f e c t the m e t h o x a m i n e - i n d u c e d vasoconstrictor response of the perfused rat mesentery. Contractile responses to methoxamine were 75 _+ 9 and 78 + 12 mmHg for control and CTX treated tissues, respectively (p>0.3, N=6). In contrast, CTX had no significant effect on vasodilator responses to TNS (Fig 4B). Discussion This study shows that GABA significantly suppresses vasoconstrictor responses of the perfused rat mesentery to TNS. Several p o s s i b l e mechanisms have been considered. Others have shown that G A B A A agonists can stimulate capsaicin-sensitive sensory nerves in the guinea-pig ileum and left atrium (16,17). Therefore, the possibility that inhibition of the pressor response of sympathetic nerves to TNS was due to stimulation of sensory nerves was considered. H o w e v e r , in the p r e s e n t study, after pretreatment with capsaicin to d e s e n s i t i z e the tissue to s e n s o r y nerve stimulation, GABA still inhibited the vasoconstrictor response to TNS. A n o t h e r p o s s i b i l i t y we c o n s i d e r e d was that G A B A r e c e p t o r s mediate a direct vasodilatory effect via a postjunctional site of action. It has been demonstrated that G A B A , via a GABAA receptor, causes vasodilatation in isolated cerebral blood vessels from cat, dog and rabbit (18-20). However, in the present study, G A B A did not produce any relaxation in the methoxamine-contracted mesentery. Similar results were reported in K+-contracted rat mesenteric arteries where G A B A did not produce any relaxation (21). Thus, we conclude that it is most possible that G A B A inhibits vasoconstrictor responses to TNS by an action on p r e j u n c t i o n a l r e c e p t o r s to inhibit adrenergic transmitter release. Others have shown that GABAB receptors inhibit stimulationinduced contraction and [3H] norepinephrine release in the isolated goat cerebrovascular bed (1), in further support of this conclusion. GABA receptors are subdivided into GABAA and GABAB types. The former is sensitive to muscimol, while the latter is sensitive to baclofen (5). In order to explore the type of G A B A receptor mediating the
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modulation of vasoconstrictor nerve effects, muscimol and baclofen were used. Vasoconstrictor responses to TNS of the perfused rat mesentery were markedly inhibited in the presence of baclofen, while muscimol had no effect. These results suggest that this inhibitory effect of GABA is due to an action at GABAB receptors. The mechanism of the inhibitory effect of GABAB on neurotransmitter release from sympathetic nerves is still unclear, but recent evidence suggests that the GABAB receptor is functionally coupled to the calcium ion channel via a GTP-binding protein (4). It has also been demonstrated that G A B A receptors can modulate the actions of s e n s o r y nerves. G A B A and b a c l o f e n inhibited the b r o n c h o c o n s t r i c t i o n p r o d u c e d by stimulation of non-adrenergic, noncholinergic nerves in the guinea-pig, and GABAB receptors have been shown to inhibit electrical stimulation-induced release of substance P from e m b r y o n i c chick dorsal root ganglia (2,3). In contrast, as mentioned p r e v i o u s l y GABAA receptors stimulate neurotransmitter release from sensory nerves in the guinea-pig isolated ileum and left atrium (16,17). However, in the present study, TNS-induced vasodilator responses were not affected by GABA, suggesting that GABA receptors do not modulate sensory nerve activation in the rat mesentery. CTX, a selective blocker of L- and N type calcium channels, has been shown to inhibit neurotransmitter release from sympathetic and parasympathetic nerves as well as sensory nerves in the central nervous system and periphery (9-11). In the present study, CTX markedly inhibited v a s o c o n s t r i c t o r r e s p o n s e s to TNS, but had no effect on vasodilator responses, suggesting that N-type calcium channels are only i n v o l v e d in activation of s y m p a t h e t i c nerves and do not mediate activation of sensory nerves in the rat mesentery. It was previously reported that CTX did not affect the release of substance P from sensory nerves in the guinea-pig bladder or the actions of non-adrenergic nonc h o l i n e r g i c nerves in the rat a n o c o c c y g e u s , d u o d e n u m or urinary b l a d d e r (9,22,23). More recently, differential sensitivities to CTX between different transmission processes have been shown in a single tissue, for example, in rat gastric fundus strips and in gunea-pig taenia cacti (12). It was postulated that this discrepancy may be due to the e x i s t e n c e of a h e t e r o g e n e o u s population of v o l t a g e - s e n s i t i v e calcium channels or the lack of involvement of N-type channels in controlling Ca 2+ influx during excitation-secretion coupling (9,12). Using guanethidine and capsaicin as tools to reveal distinct r e s p o n s e s to v a s o c o n s t r i c t o r and v a s o d i l a t o r nerves, we and other authors have demonstrated that the rat mesentery is innervated both by
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adrenergic and capsaicin-sensitive sensory nerves (13). The present study demonstrates that sensitivity to GABA agonists and CTX differs between these two types of nerves. Thus, prejunctional GABA receptors appear to modulate transmitter release from sympathetic nerves but are not active in sensory nerves. Similarly, the calcium channel blocker CTX effectively blocks activation of sympathetic nerves, but is ineffective in the case of sensory nerves. This study, along with previous findings (12), underscores the differential sensitivity to n e u r o m o d u l a t o r y influences of sympathetic and capsaicin-sensitive sensory nerve transmission in a single tissue.
Acknowledgements This work was supported by Grant #PO1 DK36289 from the National Institutes of Health and by a postdoctoral fellowship from the California Affiliate of the American Heart Association. References 1.
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