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Antidiuretic action of tachykinin NK-3 receptor in the rat paraventricular nucleus
Brain Research 743 Ž1996. 49–55
Research report
Antidiuretic action of tachykinin NK-3 receptor in the rat paraventricular nucleus Takashi Eguchi a , Yukio Takano a, ) , Takashi Hatae a,1, Ryo Saito a , Yasuhisa Nakayama Yasufumi Shigeyoshi b , Hitoshi Okamura b , James E. Krause c , Hiro-o Kamiya a a
c
a,2
,
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka UniÕersity, Fukuoka 814-80, Japan b Department of Anatomy and Neurobiology, Kobe UniÕersity School of Medicine, Kobe 650, Japan Department of Anatomy and Neurobiology, Washington UniÕersity School of Medicine, St. Louis, MO 63110, USA Accepted 13 August 1996
Abstract Studies were performed on the central antidiuretic actions via the tachykinin NK-3 receptor in the rat hypothalamic paraventricular nucleus ŽPVN.. Microinjections of the selective tachykinin NK-3 receptor agonist senktide Ž2–200 pmol. into the PVN resulted in prolonged inhibition of urine output in water-loaded rats, its effect being dose-dependent. The antidiuretic action of senktide was blocked by pretreatment with the vasopressin V2 receptor antagonist OPC-31260 Ž1 mgrkg, i.v.., but not by microinjection of the angiotensin II AT-1 receptor antagonist losartan Ž1 nmol. into the PVN. NK-3 receptor mRNA was strongly detected in the magnocellular part of the PVN and the supraoptic nucleus ŽSON. of the hypothalamus as detected by in situ hybridization histochemistry. Moreover, w 3 Hxsenktide binding sites were also detected in the PVN and the SON by receptor autoradiography. These findings suggest that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and that vasopressin caused water reabsorbtion via the kidney V2 receptor. Keywords: Tachykinin; NK-3 receptor; Antidiuretic action; Paraventricular nucleus; Vasopressin V2 receptor; Senktide
1. Introduction Tachykinin peptides have a common C-terminal amino acid sequence ŽPhe-X-Gly-Leu-Met-NH 2 ., and have a variety of pharmacological effects. Several lines of evidence indicate the existence of three major tachykinin receptors, NK-1, NK-2 and NK-3, which have highest affinities for substance P ŽSP., neurokinin A ŽNKA. and neurokinin B ŽNKB., respectively w5,7,11,20x. The distributions of these receptor subtypes in the central nervous system ŽCNS. has been extensively studied by biochemical and autoradiographic analyses of binding sites. Binding studies demonstrated the presence of the NK-3 receptors in the paraventricular ŽPVN. and supraoptic nuclei ŽSON. of the hypo)
Corresponding author. Fax: q81 Ž92. 863-0389. Present address: Department of Anatomy, Saga Medical School, Saga 849, Japan. 2 Present address: Division of Pediatric Pharmacology, National Children’s Medical Resaerch Center, Tokyo 154, Japan. 1
thalamus w4x. In addition, we found previously that the PVN, the SON and nucleus tractus solitarius ŽNTS. have high contents of NKB-like immunoreactivity w10x. Several lines of evidence have indicated that the NKB agonist senktide has a potent effect in causing endothelium-dependent relaxation of mesenteric arteries w9x and that central administration of senktide increases the blood pressure w21x. However, little is known about the physiological role of NKB in the CNS. Previous studies have shown that intracerebroventricular Ži.c.v.. injection of the tachykinin NK-3 agonist senktide had an antidiuretic action by stimulating argininevasopressin ŽAVP. release in water-loaded rats w17x. In addition, eledoisin reduced urine output in Wistar rats, but not in Brattleboro rats w1x. Therefore, in the present study, we examined the site of action of the tachykinin NK-3 receptor in central water regulation after a water-load and spontaneous urine output. We used senktide rather than NKB as an NK 3 agonist because NKB is essentially insoluble in water w24x.
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 2 0 - 7
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T. Eguchi et al.r Brain Research 743 (1996) 49–55
2. Materials and methods 2.1. Animals Male Wistar rats Ž200–350 g. were purchased from Kyudo ŽKumamoto, Japan.. The animals were kept in a room at 24 " 28C with a 12 h light–dark cycle Žlight on at 07.00 h. and were given free access to commercial food and tap water. 2.2. Antidiuretic responses For microinjections into the PVN, rats were anesthetized with 1–3% halothane and placed in a stereotaxic apparatus ŽNarishige, Japan.. A guide cannula Ž26-gauge stainless steel. was inserted into the right PVN ŽA: 7.2, L: 0.6, V: 2.1. from the interaural region and secured to the skull with dental acrylic cement. A silicone catheter ŽSilastic, MI, USA. was inserted into the right external jugular vein for intravenous injections. Rats were used at least 5 days after surgery. Experiments on antidiuretic responses were performed as described previously w17x. Rats were first given water orally Ž4.5% body weight. and placed in a metabolic cage. From l h later, urine was collected every 30 min for 2–2.5 h for measurement of its volume. All experiments were carried out between 09.00 and 14.00 h. In an experiment on the effects of injections of senktide and substance P into the PVN on spontaneous urine output, these peptides were injected into rats at 19:00 and the volume of urine and water intake was measured every 30 min for 24 h. The non-peptide angiotensin II AT-1 receptor antagonist losartan was injected 10 min before injection of senktide or angiotensin II. Senktide, substance P, angiotensin II and losartan were dissolved in artificial cerebrospinal fluid ŽACSF: NaCl 128.5 mM, KCl 3 mM, CaCl 2 1.2 mM, MgCl 2 0.8 mM, NaH 2 PO4 0.25 mM, NaHCO 3 21 mM, glucose 3.4 mM, pH 7.4. and 500 nl volumes of solution were injected into the PVN with a microinjection pump over a period of 150 s. The non-peptide vasopressin receptor antagonists, OPC21268 and OPC-31260, were dissolved in isotonic saline contained 10% dimethylsulphoxide ŽDMSO.. After each experiment, malachite-green was injected to confirm the site of microinjection. 2.3. [ 3 H]Senktide binding Receptor autoradiography was carried out according to the protocol developed by Dam et al. w4x. Male Wistar rats were sacrificed by decapitation and their brains were removed, rapidly frozen in powdered dry-ice, mounted on cryostat chuks and cut into 20-m m-thick sections at y228C ŽCryocut 1800, Reichert-Jung.. Sections were thawmounted on precleaned gelatin-coated slide glasses, dried
overnight in a desiccator at 48C and then incubated for 90 min at 258C in 50 mM Tris-HCl buffer ŽpH 7.4., containing 3 mM MnCl 2 , 0.02% Žwrv. bovine serum albumin, 40 m grml bacitracin, 2 m grml chymostatin and 4 m grml leupeptin in the presence of 3 nM w 3 Hxsenktide Ž2738.0 GBqrmmol; Dupont NEN.. Non-specific binding was determined by the addition of 1.0 m M unlabelled senktide to the incubation medium. At the end of the incubation period, slides were placed in racks and transferred sequentially through four rinses Ž1 min each. of ice-cold 50 mM Tris-HCl buffer ŽpH 7.4., followed by rapid dip in cold deionized water to remove ions. Incubated slides were then rapidly dried under a stream of cold air, juxaposed tightly against tritium-sensitive film ŽUltrofilm- 3 H, PharmaciaLKB. and stored at y808C for 6 months. Computerized densitometry ŽMCID, Imazing Research, Canada. was performed using 3 H-microscales as standards. Anatomical localization was determined using the atlas of Paxinos and Watson w12x. 2.4. In situ hybridization A rat NK-3 receptor cDNA fragments Žcoding region bases 1014–1359. was previously isolated and cloned into the plasmid Bluescript SK9-vector w6x. The cDNA-containing vector was linearized with restriction enzymes and were used as template for sense or antisense cRNA probes. Radiolabeled probes for NK-3 receptor were made by using T7 or T3 polymerase in the presence of w a- 32 PxCTP ŽNew England Nuclear, USA.. Male albino rats were anesthetized with pentobarbital and perfused intracardially with 100 ml of ice-cold saline and 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB., pH 7.4. Brains were removed, postfixed for 2 h at 48C, and placed in 0.1 M PB with 20% sucrose for 4 h. Then they were frozen in dry ice and stored at y808C until use. Tissues were cut at 25 m m thickness on a cryostat and slices were collected in 0.1 M PBS. In situ hybridization histochemistry was performed as described previously w19x. Briefly, tissue sections were placed in 4 = SSC, proteinase K Ž1 m grml, 0.1 M Tris buffer pH 8.0; 50 mM EDTA. for 15 min at 378C, 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, and 4 = SSC for 10 min. The sections were then incubated in a hybridization buffer w55% formamide, 10% dextran sulfate, 10 mM Tris-HCl ŽpH 8.0., 1 mM EDTA ŽpH 8.0., 0.6 M NaCl, 0.2% N-laurylsarcosine, 500 mgrml, 200 m grml tRNA, 1 = Denhardt’s, 0.25% SDS, 10 mM dithiothreitol ŽDTT.x containing radio-labeled cRNA for 16 h at 608C. After 2 rinses in 2 = SSC 50% formamide at 608C for 15 min, the sections were treated with RNase solution Ž10 m grml RNase A., 10 mM Tris-HCl ŽpH 8.0., 1 mM EDTA, 0.5 M NaCl for 30 min at 378C. The sections were further washed two times each in 2 = SSC 50% formamide at 608C for 15 min, and 0.4 = SSC Ž608C, 15 min.. Air-dried
T. Eguchi et al.r Brain Research 743 (1996) 49–55
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sections were exposed to b-max film ŽAmersham, UK. for 7 days. They were then dipped in nuclear track emulsion ŽIllford K5. and exposed for 4 weeks at 48C. Following development of the tissue autoradiograms, the sections were stained with cresyl violet and analyzed under brightfield and darkfield microscopy. For confirmation of the specificity of the NK-3 receptor cRNA probes for in situ hybridization method, in situ hybridization was performed using message sense probe and using antisense probe on RNase pretreated sections. 2.5. Drugs Senktide was obtained from Peninsula ŽCA, USA.. Substance P and angiotensin II were obtained from the Peptide Institute ŽOsaka, Japan.. OPC-21268 and OPC31260 were gifts from Otsuka Pharmaceutical Co. ŽTokushima, Japan.. Losartan was a gift from Du Pont Merk Pharnaceutical Co. w 3 Hxsenktide was obtained from
Fig. 2. Effects of senkide ŽA. and substance P ŽB. on diuresis in water-loaded rats. Senktide Ž2–200 pmol. and Substance P Ž0.2–2 nmol. were injected into the PVN 1 h after water loading. Values are means" S.E.M. for 6 rats. ) P - 0.01 and † P - 0.05 vs. ACSF control by Dunnett’s test.
Fig. 1. Effects of senkide ŽA. and substance P ŽB. on spontaneous urine output in rats. Senktide and substance P were injected into the PVN at 19:00h. Values are means"S.E.M. for 6 rats. ) P - 0.01 vs ACSF control by Student’s t-test.
Fig. 3. Effects of the vasopressin receptor antagonists OPC-21268 and OPC-31260 on the antidiuretic action of senktide in water-loaded rats. OPC-21268 and OPC-31260 were injected intravenously 10 min before senktide injection. Values are means"S.E.Ms. for 6 rats. ) P - 0.01 and † P - 0.05 vs. ACSF control by Dunnett’s test.
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3. Results
Fig. 4. Effects of the angiotensin II AT-1 receptor antagonist losartan on the antidiuretic actions of senktide and angiotensin II in water-loaded rats. Values are means"S.E.M. for 6 rats. ) P - 0.01 vs. ACSF control by Dunnett’s test.
Du pont NEN ŽBoston, MA.. Data are expressed as means " S.E.M. Statistical anaysis of data were performed by Dunnett’s test or Student’s t-test.
Fig. 1 shows the effect of the NK-3 agonist senktide on spontaneous urine output in rats. The volume of urine and water intake was obviously increased in the dark period Ž19.00–07.00 h., because rats are nocturnal animals. Microinjection of the NK-3 agonist senktide Ž200 pmol. into the PVN at 19.00 h completely inhibited spontaneous urine output and water intake for 5 h ŽFig. 1A.. The endogenous NK-1 agonist substance P Ž200 pmol., however, did not inhibit spontaneous urine output or water intake ŽFig. 1B.. In water-loaded rats, injection of senktide Ž2–200 pmol. caused a dose-dependent prolonged inhibition of urine output ŽFig. 2A.. But substance P Ž200 pmol. did not affect urine output, although at a high dose Ž2 nmol., it had a slight antidiuretic effect ŽFig. 2B.. To determine whether vasopressin receptors in the kidney are involved in the antidiuretic response to senktide, we injected vasopressin receptor antagonists Ži.v.. 10 min before the injection of senktide into water-loaded rats. Pretreatment with the selective vasopressin V2 receptor antagonist OPC-31260 Ž1 mgrkg, i.v.. significantly inhibited the antidiuretic effect of senktide Ž0.2 nmol, PVN. in water-loaded rats. However, pretreatment with a vasopressin V1 receptor antagonist OPC-21268 Ž1 mgrkg, i.v.. did not prevent senktide’s antidiuretic effect ŽFig. 3.. And the vasopressin antagonist, OPC-31260 or OPC-21268 itself did not cause diuretic response. In the brain angiotensin II stimulates the releases of vasopressin, oxytocin and adrenocorticotropic hormone, and has an antidiuretic action. To determine the contribution of the central angiotensin II system on the antidiuretic action of senktide, we examined the effect of pretreatment with the angiotensine II AT-1 receptor antagonist losartan on the senktide-induced antidiuretic effects. Injection of losartan by itself into the PVN Ž0.1 nmol to 10 nmol. did
Fig. 5. Autoradiogram of w 3 Hxsenktide binding in rat brain. Specific w 3 Hxsenktide was detected in the paraventricular nucleus ŽPVN., supraoptic nucleus ŽSON. and cortex.
T. Eguchi et al.r Brain Research 743 (1996) 49–55
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Fig. 6. Film ŽA. and emulsion ŽB. autoradiography of in situ hybridization using 32 P-labelled antisense probe for NK-3 receptor mRNA. ŽA.; strong signals are found in the hypothalamic paraventricular nucleus Žpvn. and supraoptic nucleus Žson.. Outside the hypothalamus, weak signals are detected in the cerebral cortex Žccx.. ŽB.; in the paraventricular nucleus, positive signals are found in the magnocellular part Žpvnm., but not in the parvocellular part Žpvnp.. v; third ventricle. ŽA. =10; ŽB. =100.
not cause any changes in urine output. Pretreatment with losartan completely inhibited the angiotensin II-induced antidiuretic effect, but it did not affect the senktide-induced antidiuretic action. ŽFig. 4.. Fig. 5 shows autoradiograms of w 3 Hxsenktide binding in rats. w 3 HxSenktide binding sites were detected in the PVN, and in the SON. In addition w 3 Hxsenktide binding sites
were also detected in the nucleus tractus solitarius Ždata not shown.. By investigating the coronal sections of the rat forebrain using an anti-sense probe, positive hybridization signals were detected in the cerebral cortex and hypothalamus. In the hypothalamus, strong hybridization signals were detectable in in the magnocellular part of the PVN and in the
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SON ŽFig. 6.. Weak signals were also detected in the zona incerta and cerebral cortex. In RNase-treated sections, no specific hybridization signals were detected with the sense NK-3 receptor cRNA probe. No specific hybridization was observed using the anti message sense cRNA probe.
4. Discussion The present study showed that the tachykinin NK-3 receptor in the PVN is involved in central water regulation by stimulating vasopressin secretion. Previously, we reported that i.c.v. injection of the tachykinin NK-3 receptor agonist senktide had an antidiuretic action in water-loaded rats with an increased plasma vasopressin level w17x. In addition, i.c.v. injection of the eledoisin was found to increase the plasma vasopressin level w2,8,16x, and Ciccocioppo et al. reported that NK-3 agonists inhibit alcohol intake in alcohol-preferring rats w3,14x. However, the site of action of NK-3 agonists in the brain has not been completely demonstrated. Microinjection of the NK-3 receptor agonist senktide had a dose-dependent antidiuretic action in water-loaded rats. This finding supports the idea that the site of the antidiuretic action of the NKB analogue senktide is in the PVN of the hypothalamus. Moreover, this results also support the PVN as a site of action for central effect of tachykinin peptides on plasma vasopressin w7x. In fact, w 3 Hxsenktide binding sites were detected in the PVN and the SON ŽFig. 5.. Moreover, we also detected NK-3 receptor mRNAs in the PVN and the SON by in situ hybridization ŽFig. 6.. These findings indicate that endogenous NKB stimulates vasopressin release via the NK-3 receptor in the PVN, because the PVN of the hypothalamus contains cell bodies of neurons that secrete vasopressin from the posterior pituitary gland. There are reports that senktide caused a marked increase of plasma vasopressin levels, but not of substance P in conscious rats w8,17x. Interestingly, NK-3 binding sites and NK-3 receptor mRNAs were detected in the SON and NTS as well as the PVN. In addition, positive hybridization signals were detected in the cerebral cortex. However, the functional roles of the SON and cortical sites are not yet known. Next, we examined the effects of peripheral vasopressin receptors on the antidiuretic action of the NK-3 agonist senktide using the selective, non-peptide vasopressin V1 and V2 receptor antagonists OPC-21268 and OPC-31260 w22,23x. Pretreatment with the V2 receptor antagonist OPC-31260 abolished the antidiuretic action of senktide, but pretreatment with the V1 antagonist did not ŽFig. 3.. These findings suggest that the antidiuretic action of the NKB analogue senktide may be due to stimulation of vasopressin secretion from the posterior pituitary gland and that plasma vasopressin then induces water reabsorbtion via the V2 receptor in the kidney. Our binding study showed that the V2 receptor antagonist OPC-31260 had a selective action on the V2 receptor in rat kidney w18x.
Central angiotensin II is known to have an antidiuretuc action by stimulating the release of vasopressin. However, this central angiotensin II system is not involved in senktide-induced antidiuretic effects, because the AT-1 receptor antagonist losartan did not influence the senktide-induced antidiuretic effects of senktide. However, Perfumi et al. reported that the peptide angiotensin II receptor blocker completely inhibits the effect of eledoisin-induced vasopressin release w13x. This discrepancy may be due to the pharmacological properties of the antagonists used, for instance, selectivity for the AT-1 receptor. Recently, it has been reported that losartan shows affinity for tachykinin receptor w15x, so further studied needed. In conclusion, the present study suggests that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and vasopressin caused water reabsorbtion via the kidney V2 receptor.
Acknowledgements This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and by NIH Grant NS21937. We are grateful to Dr. K. Honda ŽFukuoka Univ.. for helpful discussions. We are grateful to Otsuka Pharmaceutical Co. for the generous gifts of OPC-21268 and OPC-31260, and to DuPont Merk Pharmaceutical Co. for losartan ŽDup 753..
References w1x Cantalamessa, F. de Caro, G., Massi, M. and Perfumi, M. Possible influence of tachykinins on body fluid homeostasis in the rat, J. Physiol. (Paris), 79 Ž1984. 524–530. w2x Chowdrey, H.S., Jesop, D.S. and Lightman, S.C., Substance P stimulates arginine vasopressin and inhibits adrenocorticotropin release in vivo in the rat, Neuroendocrinology, 52 Ž1990. 90–93. w3x Ciccocioppo, R., Panocka, I., Pompei, P., de Caro, G. and Massi, M., Selective agonists at NK-3 tachykinin receptors inhibit alcohol intake in sardinian alcohol-preferring rats, Brain Res. Bull., 33 Ž1994. 71–77. w4x Dam, T.V., Escher, E. and Quirion, R., Visualization of neurokinin-3 receptor sites in the rat brain using the highly selective ligand w 3 Hxsenktide, Brain Res., 506 Ž1990. 175–179. w5x Erspamer, V., The tachykinin peptide family, Trends Neurosci., 4 Ž1981. 267–269. w6x MaCarson, K.E. and Krause, J.E., NK-1 and NK-3 type tachykinin receptor mRNA expression in the rat spinal cord dorsal horn is increased during adjuvant or formalin-induced nociception, J. Neurosci., 14 Ž1994. 712–720. w7x Maggio, J.M., Tachykinins, Annu. ReÕ. Neurosci., 11 Ž1989. 13–28. w8x Massi, M., Saija, A., Polidori, C., Perfumi, M., Gentili, L., Costa, G. and de Caro, G., The hypothalamic paraventricular nucleus is a site of action for the central effect of tachykinins on plasma vasopressin, Brain Res. Bull., 26 Ž1991. 149–154. w9x Mizuta, A., Takano, Y., Honda, K., Saito, R., Matsumoto, T. and Kamiya, H., Nitric oxide is a mediator of tachykinin NK-3 receptorinduced relaxation in rat mesenteric artery, Br. J. Pharmacol., 116 Ž1995. 2919–2922.
T. Eguchi et al.r Brain Research 743 (1996) 49–55 w10x Nagashima, A., Takano, Y., Tateishi, K., Matsuoka, Y., Hamaoka, T. and Kamiya, H., Central pressor actions of neurokinin B: increase in neurokinin B contents in discrete nuclei in spontaneously hypertensive rats, Brain Res., 499 Ž1989. 198–203. w11x Otsuka, M. and Yoshioka, K., Neurotransmitter functions of mammalian tachykinins, Physiol. ReÕ., 73 Ž1993. 229–308. w12x Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1982. w13x Perfumi, M., Sajia, A., Costa, G., Massi, M. and Polidori, C., Vasopressin release induced by intracranial injection of eledoisin is mediated by central angiotensin II, Pharmacol. Res. Commun., 20 Ž1988. 811–826. w14x Perfumi, M., Polidori, C., Pompei, P.L., de Caro, G. and Massi, M., The tachykinin NH 2-senktide inhibits alcohol intake in alcohol-preferring rats, Brain Res. Bull., 38 Ž1991. 881–887. w15x Picard, P., Chretien, L. and Couture, R., Functional interaction between losartan and central tachykinin NK3 receptors in conscious rat, Br. J. Pharmacol., 114 Ž1995. 1563–1570. w16x Polidori, C., Saija, A., Perfumi, M., Costa, G., de Caro, G. and Massi, M., Vasopressin release induced by intracranial injection of tachykinin is due to activation of central neurokinin-3 receptors, Neurosci. Lett., 103 Ž1989. 320–325. w17x Saigo, A., Takano, Y., Matsumoto, T., Tran, M., Nakayama, Y., Saito, R., Yamada, K. and Kamiya, H. Central administration of senktide, a tachykinin NK-3 agonist, has an antidiuretic action by stimulating AVP release in water-loaded rats, Neurosci. Lett., 159 Ž1993. 187–190.
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w18x Saito, R., Ishiharada, N., Ban, Y., Honda, K., Takano, Y. and Kamiya, H. Vasopressin V1 receptor in rat hippocampus is regulated by adrenocortical functions, Brain Res., 646 Ž1994. 170–174. w19x Shigeyoshi, Y., Okamura, H., Inatomi, T., Matsui, T., Ito, M., Kaji, H., Abe, H., Nakata, H., Chiba, T. and Chihara, K. Distribution of mRNA for CCK-B receptor in the brain of Mastomys natalensis; abundant expression in telencephalic neurons, Brain Res., 640 Ž1994. 81–92. w20x Takano, Y. and Kamiya, H. Tachykinin receptor subtypes: cardiovascular roles of tachykinin peptides, Asia Pacific J. Pharmacol., 6 Ž1991. 341–348. w21x Takano, Y., Nagashima, A., Hagio, T., Tateishi, K. and Kamiya, H. Role of central tachykinin peptides in cardiovascular regulation in rats, Brain Res., 528 Ž1990. 231–237. w22x Yamamura, Y., Ogawa, H., Chihara, T., Kondo, K., Onogawa, T., Mori, T., Tominaga, M. and Yabuuchi, M. OPC-21268, an orally effective, nonpeptide vasopressin V1 receptor antagonist, Science, 252 Ž1991. 572–574. w23x Yamamura, Y., Ogawa, H., Yamashita, H., Chihara, T., Miyamoto, H., Nakamura, S., Onogawa, T., Yamashita, T., Hosokawa, T., Mori, T. Tominaga, M. and Yabuuchi, Y. Characterization of a novel aquaretic agent, OPC-31260, as an orally effective, nonpeptide vasopressin V2 receptor antagonist, Br. J. Pharmacol., 105 Ž1992. 787–791. w24x Wormser, U., Laufer, R., Hart, Y., Chorev, M., Gilon, C. and Selinger, Z. Highly selective agonists for substance P receptor subtypes, EMBO J., 5 Ž1986. 2805–2808.
Research report
Antidiuretic action of tachykinin NK-3 receptor in the rat paraventricular nucleus Takashi Eguchi a , Yukio Takano a, ) , Takashi Hatae a,1, Ryo Saito a , Yasuhisa Nakayama Yasufumi Shigeyoshi b , Hitoshi Okamura b , James E. Krause c , Hiro-o Kamiya a a
c
a,2
,
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka UniÕersity, Fukuoka 814-80, Japan b Department of Anatomy and Neurobiology, Kobe UniÕersity School of Medicine, Kobe 650, Japan Department of Anatomy and Neurobiology, Washington UniÕersity School of Medicine, St. Louis, MO 63110, USA Accepted 13 August 1996
Abstract Studies were performed on the central antidiuretic actions via the tachykinin NK-3 receptor in the rat hypothalamic paraventricular nucleus ŽPVN.. Microinjections of the selective tachykinin NK-3 receptor agonist senktide Ž2–200 pmol. into the PVN resulted in prolonged inhibition of urine output in water-loaded rats, its effect being dose-dependent. The antidiuretic action of senktide was blocked by pretreatment with the vasopressin V2 receptor antagonist OPC-31260 Ž1 mgrkg, i.v.., but not by microinjection of the angiotensin II AT-1 receptor antagonist losartan Ž1 nmol. into the PVN. NK-3 receptor mRNA was strongly detected in the magnocellular part of the PVN and the supraoptic nucleus ŽSON. of the hypothalamus as detected by in situ hybridization histochemistry. Moreover, w 3 Hxsenktide binding sites were also detected in the PVN and the SON by receptor autoradiography. These findings suggest that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and that vasopressin caused water reabsorbtion via the kidney V2 receptor. Keywords: Tachykinin; NK-3 receptor; Antidiuretic action; Paraventricular nucleus; Vasopressin V2 receptor; Senktide
1. Introduction Tachykinin peptides have a common C-terminal amino acid sequence ŽPhe-X-Gly-Leu-Met-NH 2 ., and have a variety of pharmacological effects. Several lines of evidence indicate the existence of three major tachykinin receptors, NK-1, NK-2 and NK-3, which have highest affinities for substance P ŽSP., neurokinin A ŽNKA. and neurokinin B ŽNKB., respectively w5,7,11,20x. The distributions of these receptor subtypes in the central nervous system ŽCNS. has been extensively studied by biochemical and autoradiographic analyses of binding sites. Binding studies demonstrated the presence of the NK-3 receptors in the paraventricular ŽPVN. and supraoptic nuclei ŽSON. of the hypo)
Corresponding author. Fax: q81 Ž92. 863-0389. Present address: Department of Anatomy, Saga Medical School, Saga 849, Japan. 2 Present address: Division of Pediatric Pharmacology, National Children’s Medical Resaerch Center, Tokyo 154, Japan. 1
thalamus w4x. In addition, we found previously that the PVN, the SON and nucleus tractus solitarius ŽNTS. have high contents of NKB-like immunoreactivity w10x. Several lines of evidence have indicated that the NKB agonist senktide has a potent effect in causing endothelium-dependent relaxation of mesenteric arteries w9x and that central administration of senktide increases the blood pressure w21x. However, little is known about the physiological role of NKB in the CNS. Previous studies have shown that intracerebroventricular Ži.c.v.. injection of the tachykinin NK-3 agonist senktide had an antidiuretic action by stimulating argininevasopressin ŽAVP. release in water-loaded rats w17x. In addition, eledoisin reduced urine output in Wistar rats, but not in Brattleboro rats w1x. Therefore, in the present study, we examined the site of action of the tachykinin NK-3 receptor in central water regulation after a water-load and spontaneous urine output. We used senktide rather than NKB as an NK 3 agonist because NKB is essentially insoluble in water w24x.
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 2 0 - 7
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T. Eguchi et al.r Brain Research 743 (1996) 49–55
2. Materials and methods 2.1. Animals Male Wistar rats Ž200–350 g. were purchased from Kyudo ŽKumamoto, Japan.. The animals were kept in a room at 24 " 28C with a 12 h light–dark cycle Žlight on at 07.00 h. and were given free access to commercial food and tap water. 2.2. Antidiuretic responses For microinjections into the PVN, rats were anesthetized with 1–3% halothane and placed in a stereotaxic apparatus ŽNarishige, Japan.. A guide cannula Ž26-gauge stainless steel. was inserted into the right PVN ŽA: 7.2, L: 0.6, V: 2.1. from the interaural region and secured to the skull with dental acrylic cement. A silicone catheter ŽSilastic, MI, USA. was inserted into the right external jugular vein for intravenous injections. Rats were used at least 5 days after surgery. Experiments on antidiuretic responses were performed as described previously w17x. Rats were first given water orally Ž4.5% body weight. and placed in a metabolic cage. From l h later, urine was collected every 30 min for 2–2.5 h for measurement of its volume. All experiments were carried out between 09.00 and 14.00 h. In an experiment on the effects of injections of senktide and substance P into the PVN on spontaneous urine output, these peptides were injected into rats at 19:00 and the volume of urine and water intake was measured every 30 min for 24 h. The non-peptide angiotensin II AT-1 receptor antagonist losartan was injected 10 min before injection of senktide or angiotensin II. Senktide, substance P, angiotensin II and losartan were dissolved in artificial cerebrospinal fluid ŽACSF: NaCl 128.5 mM, KCl 3 mM, CaCl 2 1.2 mM, MgCl 2 0.8 mM, NaH 2 PO4 0.25 mM, NaHCO 3 21 mM, glucose 3.4 mM, pH 7.4. and 500 nl volumes of solution were injected into the PVN with a microinjection pump over a period of 150 s. The non-peptide vasopressin receptor antagonists, OPC21268 and OPC-31260, were dissolved in isotonic saline contained 10% dimethylsulphoxide ŽDMSO.. After each experiment, malachite-green was injected to confirm the site of microinjection. 2.3. [ 3 H]Senktide binding Receptor autoradiography was carried out according to the protocol developed by Dam et al. w4x. Male Wistar rats were sacrificed by decapitation and their brains were removed, rapidly frozen in powdered dry-ice, mounted on cryostat chuks and cut into 20-m m-thick sections at y228C ŽCryocut 1800, Reichert-Jung.. Sections were thawmounted on precleaned gelatin-coated slide glasses, dried
overnight in a desiccator at 48C and then incubated for 90 min at 258C in 50 mM Tris-HCl buffer ŽpH 7.4., containing 3 mM MnCl 2 , 0.02% Žwrv. bovine serum albumin, 40 m grml bacitracin, 2 m grml chymostatin and 4 m grml leupeptin in the presence of 3 nM w 3 Hxsenktide Ž2738.0 GBqrmmol; Dupont NEN.. Non-specific binding was determined by the addition of 1.0 m M unlabelled senktide to the incubation medium. At the end of the incubation period, slides were placed in racks and transferred sequentially through four rinses Ž1 min each. of ice-cold 50 mM Tris-HCl buffer ŽpH 7.4., followed by rapid dip in cold deionized water to remove ions. Incubated slides were then rapidly dried under a stream of cold air, juxaposed tightly against tritium-sensitive film ŽUltrofilm- 3 H, PharmaciaLKB. and stored at y808C for 6 months. Computerized densitometry ŽMCID, Imazing Research, Canada. was performed using 3 H-microscales as standards. Anatomical localization was determined using the atlas of Paxinos and Watson w12x. 2.4. In situ hybridization A rat NK-3 receptor cDNA fragments Žcoding region bases 1014–1359. was previously isolated and cloned into the plasmid Bluescript SK9-vector w6x. The cDNA-containing vector was linearized with restriction enzymes and were used as template for sense or antisense cRNA probes. Radiolabeled probes for NK-3 receptor were made by using T7 or T3 polymerase in the presence of w a- 32 PxCTP ŽNew England Nuclear, USA.. Male albino rats were anesthetized with pentobarbital and perfused intracardially with 100 ml of ice-cold saline and 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB., pH 7.4. Brains were removed, postfixed for 2 h at 48C, and placed in 0.1 M PB with 20% sucrose for 4 h. Then they were frozen in dry ice and stored at y808C until use. Tissues were cut at 25 m m thickness on a cryostat and slices were collected in 0.1 M PBS. In situ hybridization histochemistry was performed as described previously w19x. Briefly, tissue sections were placed in 4 = SSC, proteinase K Ž1 m grml, 0.1 M Tris buffer pH 8.0; 50 mM EDTA. for 15 min at 378C, 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, and 4 = SSC for 10 min. The sections were then incubated in a hybridization buffer w55% formamide, 10% dextran sulfate, 10 mM Tris-HCl ŽpH 8.0., 1 mM EDTA ŽpH 8.0., 0.6 M NaCl, 0.2% N-laurylsarcosine, 500 mgrml, 200 m grml tRNA, 1 = Denhardt’s, 0.25% SDS, 10 mM dithiothreitol ŽDTT.x containing radio-labeled cRNA for 16 h at 608C. After 2 rinses in 2 = SSC 50% formamide at 608C for 15 min, the sections were treated with RNase solution Ž10 m grml RNase A., 10 mM Tris-HCl ŽpH 8.0., 1 mM EDTA, 0.5 M NaCl for 30 min at 378C. The sections were further washed two times each in 2 = SSC 50% formamide at 608C for 15 min, and 0.4 = SSC Ž608C, 15 min.. Air-dried
T. Eguchi et al.r Brain Research 743 (1996) 49–55
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sections were exposed to b-max film ŽAmersham, UK. for 7 days. They were then dipped in nuclear track emulsion ŽIllford K5. and exposed for 4 weeks at 48C. Following development of the tissue autoradiograms, the sections were stained with cresyl violet and analyzed under brightfield and darkfield microscopy. For confirmation of the specificity of the NK-3 receptor cRNA probes for in situ hybridization method, in situ hybridization was performed using message sense probe and using antisense probe on RNase pretreated sections. 2.5. Drugs Senktide was obtained from Peninsula ŽCA, USA.. Substance P and angiotensin II were obtained from the Peptide Institute ŽOsaka, Japan.. OPC-21268 and OPC31260 were gifts from Otsuka Pharmaceutical Co. ŽTokushima, Japan.. Losartan was a gift from Du Pont Merk Pharnaceutical Co. w 3 Hxsenktide was obtained from
Fig. 2. Effects of senkide ŽA. and substance P ŽB. on diuresis in water-loaded rats. Senktide Ž2–200 pmol. and Substance P Ž0.2–2 nmol. were injected into the PVN 1 h after water loading. Values are means" S.E.M. for 6 rats. ) P - 0.01 and † P - 0.05 vs. ACSF control by Dunnett’s test.
Fig. 1. Effects of senkide ŽA. and substance P ŽB. on spontaneous urine output in rats. Senktide and substance P were injected into the PVN at 19:00h. Values are means"S.E.M. for 6 rats. ) P - 0.01 vs ACSF control by Student’s t-test.
Fig. 3. Effects of the vasopressin receptor antagonists OPC-21268 and OPC-31260 on the antidiuretic action of senktide in water-loaded rats. OPC-21268 and OPC-31260 were injected intravenously 10 min before senktide injection. Values are means"S.E.Ms. for 6 rats. ) P - 0.01 and † P - 0.05 vs. ACSF control by Dunnett’s test.
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3. Results
Fig. 4. Effects of the angiotensin II AT-1 receptor antagonist losartan on the antidiuretic actions of senktide and angiotensin II in water-loaded rats. Values are means"S.E.M. for 6 rats. ) P - 0.01 vs. ACSF control by Dunnett’s test.
Du pont NEN ŽBoston, MA.. Data are expressed as means " S.E.M. Statistical anaysis of data were performed by Dunnett’s test or Student’s t-test.
Fig. 1 shows the effect of the NK-3 agonist senktide on spontaneous urine output in rats. The volume of urine and water intake was obviously increased in the dark period Ž19.00–07.00 h., because rats are nocturnal animals. Microinjection of the NK-3 agonist senktide Ž200 pmol. into the PVN at 19.00 h completely inhibited spontaneous urine output and water intake for 5 h ŽFig. 1A.. The endogenous NK-1 agonist substance P Ž200 pmol., however, did not inhibit spontaneous urine output or water intake ŽFig. 1B.. In water-loaded rats, injection of senktide Ž2–200 pmol. caused a dose-dependent prolonged inhibition of urine output ŽFig. 2A.. But substance P Ž200 pmol. did not affect urine output, although at a high dose Ž2 nmol., it had a slight antidiuretic effect ŽFig. 2B.. To determine whether vasopressin receptors in the kidney are involved in the antidiuretic response to senktide, we injected vasopressin receptor antagonists Ži.v.. 10 min before the injection of senktide into water-loaded rats. Pretreatment with the selective vasopressin V2 receptor antagonist OPC-31260 Ž1 mgrkg, i.v.. significantly inhibited the antidiuretic effect of senktide Ž0.2 nmol, PVN. in water-loaded rats. However, pretreatment with a vasopressin V1 receptor antagonist OPC-21268 Ž1 mgrkg, i.v.. did not prevent senktide’s antidiuretic effect ŽFig. 3.. And the vasopressin antagonist, OPC-31260 or OPC-21268 itself did not cause diuretic response. In the brain angiotensin II stimulates the releases of vasopressin, oxytocin and adrenocorticotropic hormone, and has an antidiuretic action. To determine the contribution of the central angiotensin II system on the antidiuretic action of senktide, we examined the effect of pretreatment with the angiotensine II AT-1 receptor antagonist losartan on the senktide-induced antidiuretic effects. Injection of losartan by itself into the PVN Ž0.1 nmol to 10 nmol. did
Fig. 5. Autoradiogram of w 3 Hxsenktide binding in rat brain. Specific w 3 Hxsenktide was detected in the paraventricular nucleus ŽPVN., supraoptic nucleus ŽSON. and cortex.
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Fig. 6. Film ŽA. and emulsion ŽB. autoradiography of in situ hybridization using 32 P-labelled antisense probe for NK-3 receptor mRNA. ŽA.; strong signals are found in the hypothalamic paraventricular nucleus Žpvn. and supraoptic nucleus Žson.. Outside the hypothalamus, weak signals are detected in the cerebral cortex Žccx.. ŽB.; in the paraventricular nucleus, positive signals are found in the magnocellular part Žpvnm., but not in the parvocellular part Žpvnp.. v; third ventricle. ŽA. =10; ŽB. =100.
not cause any changes in urine output. Pretreatment with losartan completely inhibited the angiotensin II-induced antidiuretic effect, but it did not affect the senktide-induced antidiuretic action. ŽFig. 4.. Fig. 5 shows autoradiograms of w 3 Hxsenktide binding in rats. w 3 HxSenktide binding sites were detected in the PVN, and in the SON. In addition w 3 Hxsenktide binding sites
were also detected in the nucleus tractus solitarius Ždata not shown.. By investigating the coronal sections of the rat forebrain using an anti-sense probe, positive hybridization signals were detected in the cerebral cortex and hypothalamus. In the hypothalamus, strong hybridization signals were detectable in in the magnocellular part of the PVN and in the
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SON ŽFig. 6.. Weak signals were also detected in the zona incerta and cerebral cortex. In RNase-treated sections, no specific hybridization signals were detected with the sense NK-3 receptor cRNA probe. No specific hybridization was observed using the anti message sense cRNA probe.
4. Discussion The present study showed that the tachykinin NK-3 receptor in the PVN is involved in central water regulation by stimulating vasopressin secretion. Previously, we reported that i.c.v. injection of the tachykinin NK-3 receptor agonist senktide had an antidiuretic action in water-loaded rats with an increased plasma vasopressin level w17x. In addition, i.c.v. injection of the eledoisin was found to increase the plasma vasopressin level w2,8,16x, and Ciccocioppo et al. reported that NK-3 agonists inhibit alcohol intake in alcohol-preferring rats w3,14x. However, the site of action of NK-3 agonists in the brain has not been completely demonstrated. Microinjection of the NK-3 receptor agonist senktide had a dose-dependent antidiuretic action in water-loaded rats. This finding supports the idea that the site of the antidiuretic action of the NKB analogue senktide is in the PVN of the hypothalamus. Moreover, this results also support the PVN as a site of action for central effect of tachykinin peptides on plasma vasopressin w7x. In fact, w 3 Hxsenktide binding sites were detected in the PVN and the SON ŽFig. 5.. Moreover, we also detected NK-3 receptor mRNAs in the PVN and the SON by in situ hybridization ŽFig. 6.. These findings indicate that endogenous NKB stimulates vasopressin release via the NK-3 receptor in the PVN, because the PVN of the hypothalamus contains cell bodies of neurons that secrete vasopressin from the posterior pituitary gland. There are reports that senktide caused a marked increase of plasma vasopressin levels, but not of substance P in conscious rats w8,17x. Interestingly, NK-3 binding sites and NK-3 receptor mRNAs were detected in the SON and NTS as well as the PVN. In addition, positive hybridization signals were detected in the cerebral cortex. However, the functional roles of the SON and cortical sites are not yet known. Next, we examined the effects of peripheral vasopressin receptors on the antidiuretic action of the NK-3 agonist senktide using the selective, non-peptide vasopressin V1 and V2 receptor antagonists OPC-21268 and OPC-31260 w22,23x. Pretreatment with the V2 receptor antagonist OPC-31260 abolished the antidiuretic action of senktide, but pretreatment with the V1 antagonist did not ŽFig. 3.. These findings suggest that the antidiuretic action of the NKB analogue senktide may be due to stimulation of vasopressin secretion from the posterior pituitary gland and that plasma vasopressin then induces water reabsorbtion via the V2 receptor in the kidney. Our binding study showed that the V2 receptor antagonist OPC-31260 had a selective action on the V2 receptor in rat kidney w18x.
Central angiotensin II is known to have an antidiuretuc action by stimulating the release of vasopressin. However, this central angiotensin II system is not involved in senktide-induced antidiuretic effects, because the AT-1 receptor antagonist losartan did not influence the senktide-induced antidiuretic effects of senktide. However, Perfumi et al. reported that the peptide angiotensin II receptor blocker completely inhibits the effect of eledoisin-induced vasopressin release w13x. This discrepancy may be due to the pharmacological properties of the antagonists used, for instance, selectivity for the AT-1 receptor. Recently, it has been reported that losartan shows affinity for tachykinin receptor w15x, so further studied needed. In conclusion, the present study suggests that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and vasopressin caused water reabsorbtion via the kidney V2 receptor.
Acknowledgements This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and by NIH Grant NS21937. We are grateful to Dr. K. Honda ŽFukuoka Univ.. for helpful discussions. We are grateful to Otsuka Pharmaceutical Co. for the generous gifts of OPC-21268 and OPC-31260, and to DuPont Merk Pharmaceutical Co. for losartan ŽDup 753..
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