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Neuropeptide-Induced Contraction and Relaxation of the Mouse

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Neuropeptide-Induced Contraction and Relaxation of the Mouse Proc. Natl. Acad. Sci. USA Vol. 81, pp. 625-629, January 1984 Physiological Sciences Neuropeptide-induced contraction and relaxation of the mouse anococcygeus muscle (neurohypophysial peptides/neurotensin/thyrotropin-releasing hormone/urotensin i/vasoactive intestinal polypeptide) ALAN GIBSON*, HOWARD A. BERNtt, MICHAEL GINSBURG*, AND JACK H. BOTTING* *Department of Pharmacology, Chelsea College, University of London, Manresa Road, London SW3 6LX, United Kingdom; and tDepartment of Zoology and Cancer Research Laboratory, University of California, Berkeley, CA 94720 Contributed by Howard A. Bern, September 26, 1983 ABSTRACT Isometric tension responses to neuropeptides fects of a wide range of neuropeptides on tone of the mouse were recorded from anococcygeus muscles isolated from male anococcygeus in vitro. mice. This smooth muscle tissue is innervated by inhibitory nonadrenergic, noncholinergic nerves that resemble, ultra- MATERIALS AND METHODS structurally, the peptidergic neurons of the gastrointestinal Male mice (LACA strain from A. Tuck & Son, Battles- tract; the physiological function of the anococcygeus is not bridge, Essex, U.K.; 25-35 g) were stunned and bled. Both known. Slow sustained contractions were produced by oxyto- anococcygeus muscles were dissected from the animal and cin (0.2-20 nM), [Arg8]vasopressin (0.4-200 nM), and [Arg]- set up in series, joined at the point of unification on the ven- vasotocin (0.4-100 nM); the mouse anococcygeus is, therefore, tral rectum, in 1-ml glass organ baths that contained Krebs one of the few examples of nonvascular smooth muscle from bicarbonate solution (mM: NaCl, 118.1; KCI, 4.7; MgSO4, male mammals to respond to low concentrations of oxytocin 1.0; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; glucose, 11.1). and related peptides. Substance P (0.5-8 ,AM) caused distinc- Organ bath temperature was maintained at 370C and the tive, biphasic increases in muscle tone of some, but not all, Krebs solution was gassed continuously with 95% 02/5% preparations. Other neuropeptides producing contractions CO2. A resting tension of 200-400 mg (1 mg tension = 9.8 were neurotensin (2-100 ,uM) and thyrotropin-releasing hor- p.N) was placed on the tissue, and changes in tension were mone (2-100 ,AM); the responses were of similar time course recorded by a Grass FT03 force-displacement transducer at- and displayed selective cross-desensitization, suggesting that tached to a Lectromed pen-recorder. Each preparation was these two peptides act through a common distinct mechanism. allowed to equilibrate for 30 min before the effects of neuro- Tetradecapeptide somatostatin (10-80 pM) and its analog peptides were tested. urotensin 11 (0.1-5 ,uM), a dodecapeptide from the urophysis The anococcygeus muscle is unusually sensitive to sub- of the teleost fish Gilichthys mirabilis, produced similar slowly stances that act by releasing norepinephrine from sympa- developing relaxations of carbachol-induced tone. Piscine uro- thetic nerve endings (1). To prevent such effects, each prepa- tensin II, of which there are no reported effects on nonvascular ration was incubated with the adrenergic neuron-blocking mammalian systems, was 20-50 times more potent than soma- drug guanethidine (30 ,uM) for 15 min before the experiment tostatin, a well-established mammalian hormone. Of the pep- was begun (during the 30-min equilibration period) and, in tides studied, only vasoactive intestinal polypeptide (0.05-1 addition, the Krebs solution contained the a-adrenoceptor ,uM) caused rapid powerful relaxations in low concentrations; antagonist phentolamine (1 ,uM) throughout. this is consistent with its proposed involvement in nonadrener- Peptides were added to the organ bath in volumes not ex- gic, noncholinergic neurotransmission in the mouse anococcy- ceeding 50 ,ul. Since the mouse anococcygeus, in vitro, has geus. no spontaneous tone, it was necessary to raise tone pharma- cologically in order to determine, initially, whether each The anococcygeus muscles are two thin sheets of smooth peptide would contract or relax the tissue. Tone was raised muscle that arise, separately, from tendonous origins on the by the muscarinic receptor agonist carbachol, which was posterior sacral vertebrae and run caudad around both sides added at submaximal concentrations (2-4 ,uM) in order to of the rectum to unite on its ventral aspect; a physiological produce a background tone of 250-350 mg. The maximal re- function of the tissue has yet to be elucidated (1). The mus- sponse to carbachol in this preparation is about 500 mg (6). cles of all species studied to date (rat, cat, rabbit, dog, If, in the initial experiment, the peptide produced further mouse, and ox) are innervated by motor sympathetic fibers, contraction of the tissue, then the experiment was continued of which the transmitter is norepinephrine, and by inhibitory in the absence of carbachol. If, however, the peptide relaxed nonadrenergic, noncholinergic (NANC) fibers, of which the the tissue, then carbachol was used routinely to raise tone. transmitter is unidentified (1-6). Ultrastructurally, the The peptide was not added until carbachol had produced a NANC nerves of the anococcygeus resemble the p-type fi- steady, sustained rise in tone (usually within 2-3 min). When bers of the gastrointestinal tract, suggesting that the trans- the response to the peptide had been obtained, both it and mitter may be a peptide (7). It has been proposed recently the carbachol were washed out of the bath and muscle tone that vasoactive intestinal polypeptide (VIP) might be in- was allowed to return to base line for 15 min before it was volved in NANC transmission in the mouse anococcygeus raised again with carbachol. A peptide was considered inac- (8). However, NANC relaxations of the mouse anococcy- tive if it caused no significant change in carbachol-induced geus in response to field stimulation are complex, and it is tone within 5 min. Each peptide was studied on a minimum possible that there is more than one NANC transmitter in of four separate muscle preparations. this tissue (9). Consequently, we have investigated the ef- Abbreviations: NANC, nonadrenergic noncholinergic; TRH, thyro- The publication costs of this article were defrayed in part by page charge tropin-releasing hormone; TTX, tetrodotoxin; VIP, vasoactive in- payment. This article must therefore be hereby marked "advertisement" testinal polypeptide. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 625 Downloaded by guest on September 29, 2021 626 Physiological Sciences: Gibson et al. Proc. Natl Acad Sci. USA 81 (1984) The following drugs were used: carbachol (Koch-Light ity of the responses to neurotensin and TRH, it seemed pos- Laboratories, Bucks, England), glucagon (Sigma), luteiniz- sible that these peptides were acting via a common mecha- ing hormone-releasing hormone (LHRH; Sigma), [Met]en- nism. This possibility was confirmed by the type of experi- kephalin (Cambridge Research Biochemicals), neurotensin ment shown in Fig. 3c. When the muscle was exposed to (Sigma), oxytocin (preservative-free; Sandoz Pharmaceuti- TRH it became desensitized to neurotensin and vice versa, cal), somatostatin (tetradecapeptide; Sigma), substance P but in neither case was the response to carbachol reduced. In (Sigma), tetrodotoxin (TTX; Sigma), thyrotropin-releasing other experiments it was found that contractions in response hormone (TRH; Sigma), urotensin II (synthetic Gillichthys; to [Arg]vasotocin were normal in muscles desensitized to Peninsula Laboratories, San Carlos, CA), VIP (Sigma), TRH and neurotensin. These experiments show that the re- [Arg8]vasopressin (Sigma), [Arg]vasotocin (Sigma). sponses of the mouse anococcygeus to TRH and neurotensin display selective cross-desensitization. RESULTS Peptides Producing Relaxation. Tetradecapeptide somato- Peptides Having no Effect. The following peptides neither statin (10-80 ,uM) produced slowly developing relaxations of increased nor decreased carbachol-induced tone (all in con- carbachol-induced tone (Fig. 4 a and b), which required 5 centrations up to 10 MxM): luteinizing hormone-releasing hor- min to reach equilibrium. At higher concentrations these re- mone, [Met]enkephalin, and glucagon. laxations were preceded by small transient contractions. The Peptides Producing Contraction. Oxytocin (0.2-20 nM), somatostatin analog urotensin 11 (0.1-6 AM), a 12-amino- [Arg8]vasopressin (0.4-200 nM), and [Arg]vasotocin (0.4- acid neuropeptide from the urophysis of the teleost fish Gil- 100 nM) produced slow, sustained contractions (Fig. 1). The lichthys mirabilis (10), produced changes in tone similar to characteristics of the responses to all three peptides were those of somatostatin but was 20-50 times more potent (Fig. similar, with the exception that the contractions to oxytocin 4 c and d). The relaxations produced by both peptides were required a longer time (3-4 min) to reach equilibrium than well sustained and showed no sign of desensitization. The those to the other two peptides (2-3 min). effects of somatostatin and urotensin II were not blocked by Substance P (0.5-8 ,uM) produced a variable response, TTX (1 A.M), which abolishes nerve-mediated relaxation of causing three preparations to contract but having no effect the mouse anococcygeus (9). on two others. When present, the contractions produced by VIP (0.05-1 ,uM) produced a different pattern of relaxation lower concentrations of substance P were biphasic (Fig. 2);
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