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Inhibitory effects of neuropeptide Y on cyclic AMP accumulation in slices of the nucleus tractus solitarius region of the rat
Neurosciem'e Letters. 76 (1987) 185 191) Elsevier Scientific Publishers Ireland Ltd.
185
NSL t)4531
Inhibitory effects of neuropeptide Y on cyclic AMP accumulation in slices of the nucleus tractus solitarius region of the rat Anders H~irfstrand l, Bertil Fredholm2 and K jell F u x e l Department O/'~Hi.~'tology and : Phurmacolo~. ', Karolinska Instituter, Stockholm ~£'weden ) (Rccei,,ed 17 November 1986: Reviscd version received and accepted 29 December 1986) Ker wm'd.w Neuropeptide Y: Clonidine: Nucleus lractus solitarius" :~,-Receptor: Ral brain: Adcnylate cyclase: Rat
The ellkects of neuropeptide Y (NPY) on cyclic AMP (cAMP) accumulation in slices of the dorsal midline area of the caudal part of the medulla oblongata containing the nucleus tractus solitarius (nTS) have been studied Neuropeplide Y (30 and 3t)0 nM) significantly reduced the [3H]cAMP accumulation mduced by forskolin and phorboldibutyrate. Similar results were obtained after incubalion with the ~-adrenoceptor agonisl clonidine (I ItM). These results indicate that stimulation of the NPY receptors and >-adrenoceptors in the nTS region may cause inhibition of the adenylate cyclase. Such a mechanism may at least partl,, underlie the ccnlrally mediatcd hypotensive effects of the coslored transmitters adrenaline and NPY.
Neuropeptide Y (NPY) is a newly isolated neuropeptide containing 36 amino acid residues [21, 22]. This peptide is costored with noradrenaline (NA) in several peripheral adrenergic neurons [17, 18] and also in catecholamine-containing neurons in the brain [2, 14. 15]. NPY immunoreactive (IR) terminals are particularly abundant in the nucleus tractus solitarius (nTS) region [2, 5, 13], where there is evidence that NPY is costored with adrenaline [5, 12]. The nTS also contains substantial amounts of NPY-binding sites as evidenced by quantitative receptor autoradiography [9]. There is evidence that a reciprocal receptor-receptor interaction between the :~2-adrenoceptor and NPY receptor takes place in the nTS [5]. Whereas NPY released from peripheral nerve endings may be an important vasoconstrictor [17] NPY in the nTS area probably plays a vasodepressor role [5, 6] even in the presence of ~2-adrenoceptot blockade [I I]. Hypotension and bradycardia and bradypnea are also observed in awake unrestrained rats [10]. Despite increasing evidence of a physiologically important role of NPY, particularly together with catecholamines, little is known about the mechanism of action
('orrespon&,nce: A. Hz'irfstrand. Karolinska lnstilutet, Box 64100. S-104 01 Stockholm, Sweden. 0304-3940,'87 S 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
186 of NPY. Recently it was reported that NPY inhibits the cyclic A M P (cAMP) accumulation in feline cerebral blood vessels [3]. These inhibitory effects were shared by the ~2-receptor agonist clonidine [3]. Based on these findings we have examined the effects of NPY on c A M P accumulation in the rat brain, particularly in the nTS area. A few experiments with clonidine were also carried out. Male specific pathogen-tYee Sprague-Dawley rats (150-200 g; A L A B Stockholm, Sweden) were used. After decapitation the hippocampus or the dorsal midline area of the caudal part of the medulla oblongata ( D C M O ) region containing inter alia the area postrema, the dorsal m o t o r nucleus of the vagus, and the hypoglossal nucleus was rapidly dissected. The D C M O was punched out from 1.0 mm thick coronal sections located at obex level ( -0.5 mm to +0.5 ram). Slices were cut with a McIIvain tissue chopper and put in a drop of oxygenated Krebs-Ringer solution and then dissected employing a rectangular (1 × 2 ram) punch. After collection the dissected slices were kept in a Krebs Ringer solution under continuous oxygenation throughout the whole experinaent. The preincubation with [~H]adenine and the accumulation of [~H]cAMP was studied as described by Fredholm et al. [3, 4]. In some experiments the brain slices were also treated with 100/tM N-ethylmaleimide (NEM) for 10 rain to inactivate the pathways leading to inhibition of adenylate cyclase (AC) activity [3]. A phorbolester was in the present experiments used to further increase the accumulation induced by forskolin. Phorbolesters do not directly activate AC but are able to raise c A M P levels in intact cells. Recent evidence strongly suggests that this effect is due to the activation of protein kinase C [7, 19]. The statistical analysis was carried out by means of the Mann Whitney U-test [20] and the Dunn test [8]. The Jonckheere-Terpstra test was used to evaluate the concentration response relationship [8]. [~H]Adenine (27 Ci/mmol) was obtained from the Radiochemical Center (Amersham, U.K.). Dithiothreitol and N E M were obtained from Sigma (St. Louis, MO, U.S.A.). Forskolin was purchased from Calbiochem. Boehring, (La Jolla, CA, U.S.A.) and porcine NPY from Bachem (Bubendorf, Switzerland). Clonidine was a generous gift from Boehringer-lngelheim, F.R.G. In slices of the nTS area, incubation with forskolin had a stimulatory effect on c A M P accumulation. (Fig. 1). A clear-cut effect was seen already with 0.3/iM forskolin and a more than 10-fold stimulation was obtained with l 0 / t M forskolin. This stimulation was further enhanced by phorbol dibutyrate (PBB) (1 10/~M). In the rat hippocampal slices (used as reference tissue) forskolin (0.3 /~M) also increased the c A M P accumulation from a control value of 0.70% of total radioactivity to 2.88% in agreement with earlier findings [3, 4]. Clonidine over a range of concentrations from O. 1 to 1000 nM and NPY from 0.1 100 nM did not modify the c A M P accumulation afforded by forskolin in the hippocampal slices. In the presence of 3 /~M forskolin NPY tended to reduce the accumulation of c A M P in nTS slices at a concentration of 300 riM, whereas lower concentrations were ineffective (Fig. 2). Addition of the phorbolester led to a significant increase in the accumulation of cAMP. After combined stimulation with forskolin (1/~M) and PBB
187
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FORSKOLIN ~JM PBB ~JM
(pM FORSKOLIN)
Fig. 1. Effects of increasing concentrations of forskolin (A) used in the absence or presence of PBB (B) on the % conversion in to cAMP. Means_-1-S.D. are shown (n - 7 rats in each group). The Joncheere Terpstra test was used for the calculation of the concentration response relationship. In (B) the Mann-Whitney U-test was used.
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IlJM FORSKOLIN + INDOMETHACIN 3#M 1
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30
300
NPY(nM) Fig. 2. Efl'ects of two concentrations of NPY (30 and 300 nM) on the cAMP accumulation induced by t'orskolin in the absence or presence of phorbol dibutyrale (PBB, I HM) in slices of the nTS area. The accumulation of tritiated cAMP is given in percent of total radioactivity in the slice. Number of experiments (each representing one rat) are indicated in parentheses. Means_+S.D. are shown. In the statistical analysis the Dunn test (see ref. 8) was used.
188 FORSKOLIN lpM
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Fig. 3. A: inhibitory effects of c]onidine (l pM) on the accumulation of [3HJcAMPin slices of the nYS area followingincubation with forskolin (1 t~M) and their prevention by the presence of NEM (100 pM). B: the counteractive effect of DTT on the increase in [3H]cAMPconversion seen after NPY/NEM cotreatment is shown in the presence of forskolin ( 1~M). Means_ S.D. (n = 6 rats in each group). Mann Whitney U-tesl was used.
(] /zM) NPY (0.03-0.3 /~M) caused a significant inhibition of c A M P accumulation (Fig. 2). In the presence of indomethacin (3 ItM) and enprofylline (100/tM), which block the influence of the two major endogenous stimulating compounds i.e. prostaglandins and adenosine [1] respectively, NPY (0.03-0.3/~M) caused no change (Fig. 2). N E M ( 100/~M) is shown to counteract the inhibitory effects of clonidine and NPY and c A M P accumulation (Fig. 3). Thus, in the experiments with NPY the N E M effect was counteracted by dithiothreitol (100/~M) unmasking the inhibitory role of NPY (Fig. 3). After combined stimulation with forskolin and the phorbolester clear-cut and significant inhibitory effects of NPY were observed. Thus, the relative absence of gross changes in c A M P accumulation under most conditions may be due to the fact that the transmission systems operate to maintain a certain homeostasis and that maximal stimulation does not take place under physiological in vivo conditions. Also clonidine exerts little effect on c A M P accumulation in slices of the nTS area under normal circumstances but when stimulated with forskolin an inhibitory effect of clonidine on c A M P accumulation could be observed. This effect was blocked by NEM pretreatment. Furthermore, NPY in combination with N E M induced an increase in c A M P accumulation which was counteracted by dithiothreitol (DTT). The present results are in good agreement with other findings suggesting interactions of the ~2-adrenoceptors and NPY receptors with G T P binding proteins [16, 23]. In conclusion, the present results are compatible with an interaction of N P Y and ~2receptors in the inhibitory regulation of AC in the nTS area probably taking place at the level of the Ni protein.
189
This work has been supported by Grants 02553 and 04X-715 from the Swedish Medical Research Council, fi'om the L. Ostermans Foundation and from the Karolinska lnstitutet. We are grateful to Karin Lindstr6m and Agneta Wallman for skillful technical assistance.
I Daly, I.W_ ('yclic Nuclcotides in Ihe Nervous System, Plenum. New York, 1977. 2 Everitt. B., H(ikfell, T., Terenius, L., Tatemolo, K., M u n , V. and Ooldsteiu, M., Differential co-cxis tence of neuropeptide Y (NPY)-like immunoreaclivity with catecholamines in the central nervous s,,stcm of tile rat, Neuroscience, I 1 (1984) 443 462. 3 Fredholm, B.B., Jansen, I. and Edvinsson, L., Neuropeptide Y is a potent inhibitor of c A M P accumulation in feline cerebral vessels, Acta Physiol. Stand.. 124 (1985i 467 469. 4 Fredholm. B.B., Lindgren. E. and Lindslr6m, K., Treatment with N-ethylmaleimide selectively reduces adcnosine receptor-mediated decreases in c A M P accumulation in rat hippocampus slices. Br. J. Pharmacol., 86 (1985) 509 513. 5 Fuxc. K., Agnati, L.F., Hiirfstrand, A., Janson, A.M., Neumeycr, A., Andersson, K.. Ruggeri. M.. Zoli, M. and Goldsiein, M., Morphofunctional studies on the ncuropeptide Y/adrenaline costoring tcrnfinal systems in the dorsal cardiovascular region of the medulla oblongala. Focus on Receptor Receptor Interactions in Cotransmission. In T. tt6kfelt, K. Fuxe and B. Pernow (Eds.), Progress in Brain Research, Vol. 68, Elsevier, Amsterdanl, 1986, pp. 303 320. 6 Fuxc, K., Agnali. L.F., ltfirfstrand, A., Zini, l., Yatemolo, K., Merlo Pick, E., H6kfelt, T., Mutt. V. and Tcrenius. L., Central administration of neuropeptide Y induces hypotcnsion bradypnea and El!(; s ~ n c h r o n i z a t i o n i u l h e r a t , AclaPhysiol. Scand., 118(1983) 189 19(I. 7 1|ollingsworth. E.B. and Daly, J.W., Accumulation ofinosilol phosphates and cyclic A M P in guineapig cerebral cortex preparations. Effects of norcpinephrine, histamine, carbamycholinc and 2-chloroadcnosme. Biochim. Biophys. Acta, 847 (I985) 21)7 216. 8 Hollandcr, M. and Wolfe. D.A., Nonparametrical statistical methods. Wiley, New York, 1973. 9 t l/.irfslrand, A., Fuxe, K., Agnali, L.F., Benfenati, F. and Goldstein, F., Reccptor autoradiographical evidence for high densities of 125-1 neuropeptide Y binding sites in the nucleus tractus solilafius of normal male rat. Acta Physiol. Scand., 128 (1986) 195 2t)(I. 10 llLirfslrand, A., Fuxc, K., Agnati. L.F., Eneroth, P., zmi, I., Zoli, M., Andersson, K., yon [iuler, (i.. Terenius. L., Mull, V. and Goldstein, M., Studies oil neuropepiide Y-catecholamine interactions in the h5 poihalamus and in the forebrain of Ihe male rat. Relationship to ncuroendocrinc function, Ncurochem. Int.. g (1986) 355 376. I I ttfirfstrand, A., Fuxe. K., Agnati. L.V., Ganten, D.. Encroth, P., Tatemolo, K. and Mutt, V.. Studies on neuropcptide-Y catecholamine interactions in central cardiovascular regulation in the :~-chloralosc anaesthetized rat. Evidence l\~r a possible new way of activating tile :~-2 adrenergic transmission line, ('lin. Exp. liypcrtcnsionTheory P r a c t . , A 6 ( 1 0 a n d II)(I984) 1947 195(t. 12 ltzliffstrand, A., Fuxc, K., Agnati, L.F., Kitayama, l., Cintra. A.. Janson, A.-M., Kalia. M., Vanderhaegen, J.-J.. Goldstein, M. and Tercnius. L., Intracisternal administration of cholecvstokinin-S counteracts the central cardiovascular effects of adremdinc and NPY. A study based o n the existcncc of cholccysiokinm, phenylcthal~olamine N-mcthyltransferase and neuropepiide Y immunoreactivil\ in lhc salne neurons of the nucleus tractus solilarius. Neurochem. Int., (1987) in press. 13 ttiirl~trand. A., Kalia, M., Terenius, L. and Fuxe, K., Neuropeptide Y immunoreacti,,e perikarya and ncrxc terminals in the rat medulla oblongala. Relationship to cytoarchilecture and catecholaminergic cell groups. J. Comp. Neurol., (1987) in press. 14 HSkfelt. T.. Lundberg, J.M., Lagercranlz, H., Talemoto, K., Mutt, V.. Lindberg, J.. Terenius. L., Everitl. B.M.. Fuxe. K., Agnali, L.t:. and Goldstein, M.. Occurrence of neuropeptide Y (NPY)-Iike immunoreactivity in catecholamine neurons in the h u m a n medulla oblongata, Ncurosci. Left.. 36 (1983) 217 222. 15 ll,3kfclt, T., Lundberg. J.M., Tatemoto, K., Mutt. V., Terenius, L., Polak, J., Bloom, A., Sasek, ('., Eldc. R. and Goldstein, M., Neuropeptide Y (NPY)-like and F R M F-amide neuropeptide-likc immu-
190
16
17
18
[9
20 21 22 23
noreactivities ill catecholamine ncurons of the rat medulla oblongata, Acta Physiol. Scand., 117 (I 9 ~ 315 318. Jakobs. K.H., Aktories, K., Minuth, M. and Schultz, G.. Inhibition of adenylate cyclase. In D.M.(' Cooper and K.B. Seamon (Eds.), Advances m Cyclic Nucleotide and Protein Phosphorylation Rcsearch, Vol. 19. Raven, NewYork, 1985,137 150, Lundberg, J.M. and Tatemolo, K., Pancreatic polypeptide family (APP, BPP NPY and PYY) in relation to sympathetic vasoconstriction resistant to ~-adrenoceptor blockade, Acta Physiol. Stand., 116 (1982) 393 4(12, Lundberg, J.M., Terenius, L., H6kfelt, T. and Tatemoto, K., Comparative immunohistochemical and biochemical analysis of pancreatic polypeptide-tike peptides with special reference to presence of neuropeptide Y in central and peripheral neurons, J. Neurosci., 49 0984) 237(>2386. Nordstedt, C. and Fredholm, B., Phorbol- [ 2,13-dibutyrate enhances the cyclic AMP accumulation in rat hippocampal slices induced by adenosine analogues, Naunyn-Schmiedeberg's Arch. Pharmacol., in press. Snedecor, G.W. and Cochran, W.G., Statistical Methods, 7th edn., The Iowa State University Press, Ames, IA, U.S.A., 1981). Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. USA, 79 (1982) 5485 5490. Talemoto, K., Karlqvist, M. and Mutt, V., Neuropeptide Y: a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London), 296 (1982) 659 660. Unden, A. and Bartfai, T., Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex, FEBS Left,, 177 (1984) 125 128.
185
NSL t)4531
Inhibitory effects of neuropeptide Y on cyclic AMP accumulation in slices of the nucleus tractus solitarius region of the rat Anders H~irfstrand l, Bertil Fredholm2 and K jell F u x e l Department O/'~Hi.~'tology and : Phurmacolo~. ', Karolinska Instituter, Stockholm ~£'weden ) (Rccei,,ed 17 November 1986: Reviscd version received and accepted 29 December 1986) Ker wm'd.w Neuropeptide Y: Clonidine: Nucleus lractus solitarius" :~,-Receptor: Ral brain: Adcnylate cyclase: Rat
The ellkects of neuropeptide Y (NPY) on cyclic AMP (cAMP) accumulation in slices of the dorsal midline area of the caudal part of the medulla oblongata containing the nucleus tractus solitarius (nTS) have been studied Neuropeplide Y (30 and 3t)0 nM) significantly reduced the [3H]cAMP accumulation mduced by forskolin and phorboldibutyrate. Similar results were obtained after incubalion with the ~-adrenoceptor agonisl clonidine (I ItM). These results indicate that stimulation of the NPY receptors and >-adrenoceptors in the nTS region may cause inhibition of the adenylate cyclase. Such a mechanism may at least partl,, underlie the ccnlrally mediatcd hypotensive effects of the coslored transmitters adrenaline and NPY.
Neuropeptide Y (NPY) is a newly isolated neuropeptide containing 36 amino acid residues [21, 22]. This peptide is costored with noradrenaline (NA) in several peripheral adrenergic neurons [17, 18] and also in catecholamine-containing neurons in the brain [2, 14. 15]. NPY immunoreactive (IR) terminals are particularly abundant in the nucleus tractus solitarius (nTS) region [2, 5, 13], where there is evidence that NPY is costored with adrenaline [5, 12]. The nTS also contains substantial amounts of NPY-binding sites as evidenced by quantitative receptor autoradiography [9]. There is evidence that a reciprocal receptor-receptor interaction between the :~2-adrenoceptor and NPY receptor takes place in the nTS [5]. Whereas NPY released from peripheral nerve endings may be an important vasoconstrictor [17] NPY in the nTS area probably plays a vasodepressor role [5, 6] even in the presence of ~2-adrenoceptot blockade [I I]. Hypotension and bradycardia and bradypnea are also observed in awake unrestrained rats [10]. Despite increasing evidence of a physiologically important role of NPY, particularly together with catecholamines, little is known about the mechanism of action
('orrespon&,nce: A. Hz'irfstrand. Karolinska lnstilutet, Box 64100. S-104 01 Stockholm, Sweden. 0304-3940,'87 S 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
186 of NPY. Recently it was reported that NPY inhibits the cyclic A M P (cAMP) accumulation in feline cerebral blood vessels [3]. These inhibitory effects were shared by the ~2-receptor agonist clonidine [3]. Based on these findings we have examined the effects of NPY on c A M P accumulation in the rat brain, particularly in the nTS area. A few experiments with clonidine were also carried out. Male specific pathogen-tYee Sprague-Dawley rats (150-200 g; A L A B Stockholm, Sweden) were used. After decapitation the hippocampus or the dorsal midline area of the caudal part of the medulla oblongata ( D C M O ) region containing inter alia the area postrema, the dorsal m o t o r nucleus of the vagus, and the hypoglossal nucleus was rapidly dissected. The D C M O was punched out from 1.0 mm thick coronal sections located at obex level ( -0.5 mm to +0.5 ram). Slices were cut with a McIIvain tissue chopper and put in a drop of oxygenated Krebs-Ringer solution and then dissected employing a rectangular (1 × 2 ram) punch. After collection the dissected slices were kept in a Krebs Ringer solution under continuous oxygenation throughout the whole experinaent. The preincubation with [~H]adenine and the accumulation of [~H]cAMP was studied as described by Fredholm et al. [3, 4]. In some experiments the brain slices were also treated with 100/tM N-ethylmaleimide (NEM) for 10 rain to inactivate the pathways leading to inhibition of adenylate cyclase (AC) activity [3]. A phorbolester was in the present experiments used to further increase the accumulation induced by forskolin. Phorbolesters do not directly activate AC but are able to raise c A M P levels in intact cells. Recent evidence strongly suggests that this effect is due to the activation of protein kinase C [7, 19]. The statistical analysis was carried out by means of the Mann Whitney U-test [20] and the Dunn test [8]. The Jonckheere-Terpstra test was used to evaluate the concentration response relationship [8]. [~H]Adenine (27 Ci/mmol) was obtained from the Radiochemical Center (Amersham, U.K.). Dithiothreitol and N E M were obtained from Sigma (St. Louis, MO, U.S.A.). Forskolin was purchased from Calbiochem. Boehring, (La Jolla, CA, U.S.A.) and porcine NPY from Bachem (Bubendorf, Switzerland). Clonidine was a generous gift from Boehringer-lngelheim, F.R.G. In slices of the nTS area, incubation with forskolin had a stimulatory effect on c A M P accumulation. (Fig. 1). A clear-cut effect was seen already with 0.3/iM forskolin and a more than 10-fold stimulation was obtained with l 0 / t M forskolin. This stimulation was further enhanced by phorbol dibutyrate (PBB) (1 10/~M). In the rat hippocampal slices (used as reference tissue) forskolin (0.3 /~M) also increased the c A M P accumulation from a control value of 0.70% of total radioactivity to 2.88% in agreement with earlier findings [3, 4]. Clonidine over a range of concentrations from O. 1 to 1000 nM and NPY from 0.1 100 nM did not modify the c A M P accumulation afforded by forskolin in the hippocampal slices. In the presence of 3 /~M forskolin NPY tended to reduce the accumulation of c A M P in nTS slices at a concentration of 300 riM, whereas lower concentrations were ineffective (Fig. 2). Addition of the phorbolester led to a significant increase in the accumulation of cAMP. After combined stimulation with forskolin (1/~M) and PBB
187
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0.1
0.3
1
3
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4 ¸
2.
0 0
0
0 10 10
FORSKOLIN ~JM PBB ~JM
(pM FORSKOLIN)
Fig. 1. Effects of increasing concentrations of forskolin (A) used in the absence or presence of PBB (B) on the % conversion in to cAMP. Means_-1-S.D. are shown (n - 7 rats in each group). The Joncheere Terpstra test was used for the calculation of the concentration response relationship. In (B) the Mann-Whitney U-test was used.
I
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IuM FORSKOLIN (11/
< ,o -G
IlJM FORSKOLIN + INDOMETHACIN 3#M 1
+ ENPROFYLLINE 100#M // O
30
300
NPY(nM) Fig. 2. Efl'ects of two concentrations of NPY (30 and 300 nM) on the cAMP accumulation induced by t'orskolin in the absence or presence of phorbol dibutyrale (PBB, I HM) in slices of the nTS area. The accumulation of tritiated cAMP is given in percent of total radioactivity in the slice. Number of experiments (each representing one rat) are indicated in parentheses. Means_+S.D. are shown. In the statistical analysis the Dunn test (see ref. 8) was used.
188 FORSKOLIN lpM
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Fig. 3. A: inhibitory effects of c]onidine (l pM) on the accumulation of [3HJcAMPin slices of the nYS area followingincubation with forskolin (1 t~M) and their prevention by the presence of NEM (100 pM). B: the counteractive effect of DTT on the increase in [3H]cAMPconversion seen after NPY/NEM cotreatment is shown in the presence of forskolin ( 1~M). Means_ S.D. (n = 6 rats in each group). Mann Whitney U-tesl was used.
(] /zM) NPY (0.03-0.3 /~M) caused a significant inhibition of c A M P accumulation (Fig. 2). In the presence of indomethacin (3 ItM) and enprofylline (100/tM), which block the influence of the two major endogenous stimulating compounds i.e. prostaglandins and adenosine [1] respectively, NPY (0.03-0.3/~M) caused no change (Fig. 2). N E M ( 100/~M) is shown to counteract the inhibitory effects of clonidine and NPY and c A M P accumulation (Fig. 3). Thus, in the experiments with NPY the N E M effect was counteracted by dithiothreitol (100/~M) unmasking the inhibitory role of NPY (Fig. 3). After combined stimulation with forskolin and the phorbolester clear-cut and significant inhibitory effects of NPY were observed. Thus, the relative absence of gross changes in c A M P accumulation under most conditions may be due to the fact that the transmission systems operate to maintain a certain homeostasis and that maximal stimulation does not take place under physiological in vivo conditions. Also clonidine exerts little effect on c A M P accumulation in slices of the nTS area under normal circumstances but when stimulated with forskolin an inhibitory effect of clonidine on c A M P accumulation could be observed. This effect was blocked by NEM pretreatment. Furthermore, NPY in combination with N E M induced an increase in c A M P accumulation which was counteracted by dithiothreitol (DTT). The present results are in good agreement with other findings suggesting interactions of the ~2-adrenoceptors and NPY receptors with G T P binding proteins [16, 23]. In conclusion, the present results are compatible with an interaction of N P Y and ~2receptors in the inhibitory regulation of AC in the nTS area probably taking place at the level of the Ni protein.
189
This work has been supported by Grants 02553 and 04X-715 from the Swedish Medical Research Council, fi'om the L. Ostermans Foundation and from the Karolinska lnstitutet. We are grateful to Karin Lindstr6m and Agneta Wallman for skillful technical assistance.
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