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); (Fig. 5); the response was much more rapid, equilibrium oc- at higher concentrations the two phases merged. curring within 2 min. These relaxations were also unaffected Neurotensin (2-100 ,uM) and TRH (2-100 ,uM) caused con- by TTX (1 ,uM). tractions that peaked within 2 min but were poorly sustained (Fig. 3). If either peptide was left in contact with the tissue, DISCUSSION tone returned to base line within 5 min and, at this time, the This study has shown that a variety of neuropeptides pro- responses of the tissue to further doses of the peptide were duce distinctive changes in tone of the mouse anococcygeus. reduced greatly, although carbachol (4 ,uM) gave its normal Peptide-induced contractions were obtained in muscles that 250- to 350-mg rise in tone (Fig. 3 a and b). Peptide responses were both pretreated with guanethidine and maintained in were restored 20 min after washout. Because of the similar- phentolamine-containing Krebs solution, indicating that nor-

a) -

0 I 0.2 0.4 0.9 4 nM Oxy

b)i-~

I 2 0.5 0.9 2 4 23 230 nM AVP

C) i-i

0. 0.4 1 2 4 10 100 nM AVT I FIG. 1. Contractions of mouse anococcygeus muscle preparations exposed to oxytocin (Oxy; a), [Arg8]vasopressin (AVP; b), and [Arg]vasotocin (AVT; c). After each response the organ bath was washed out and muscle tone was allowed to return to base line. The time interval between each break in the trace was 20 min. Time calibration, 1 min; tension calibration, 0-400 mg. Downloaded by guest on September 29, 2021 Physiological Sciences: Gibson et aL Proc. NatL. Acad. Sci USA 81 (1984) 627

I 4 1 2 4 8 SP I 0.5 pM FIG. 2. Contractions of a mouse anococcygeus muscle preparation exposed to substance P (SP). After each response the organ bath was washed out and muscle tone was allowed to return to base line. The time interval between each break in the trace was 20 min. Time calibration, 1 min; tension calibration, 0-250 mg. epinephrine, released from sympathetic nerve endings, did workers found that vasopressin had no effect on this species not contribute to the effect. Relaxations were not blocked by but did cause the dog anococcygeus to contract (5). Further, TTX and, therefore, did not result from generation of action vasopressin is relaxant in the cat anococcygeus (3). It may potentials in NANC nerves, with subsequent release of in- be that the effects of these peptides are modified by certain hibitory transmitter. Thus, it is likely that the contractions conditions, such as hormonal status, as has been shown for and relaxations observed were due to a direct effect of the oxytocin acting on the uterus (11) and, possibly, on the semi- peptides on smooth muscle. nal vesicle (14). Certainly, the effects of neurohypophysial The mouse anococcygeus appears to be one of the few hormones on the anococcygeus merit further attention, since examples of nonvascular smooth muscle from male mam- they may represent an as-yet-unknown physiological func- mals to respond to low concentrations of neurohypophysial tion for this family of peptides and for the muscle itself. hormones and to have greater sensitivity to oxytocin than to TRH and neurotensin have been localized, by immunocy- vasopressin. The anococcygeus is associated with both the tochemistry and radioimmunoassay, in the gastrointestinal lower alimentary tract and male urogenital tract, since some tract and have been shown to modify gastrointestinal motil- of its smooth muscle cells merge with the longitudinal mus- ity (15-18). In the mouse anococcygeus they produce a dis- cle of the rectum while others, in some species, merge with tinctive pattern of contraction and display a selective cross- the retractor penis (1). This may be of significance because densensitization suggesting that, in this tissue, they may act vasopressin is known to cause certain gastrointestinal through a common mechanism, possibly on the same recep- smooth muscles to contract (11), albeit in some cases by an tor. They share a common NH2-terminal amino acid (pyro- indirect action (12), while there are reports that oxytocin glutamic acid), and there are recent reports of interaction may activate smooth muscle of the male reproductive sys- between these peptides in the brain (19, 20). tem (13). The literature on the effects of neurohypophysial Somatostatin and urotensin II also produced similar re- peptides on the anococcygeus muscles of species other than sponses in the mouse anococcygeus, in this case slow relax- the mouse is confusing. Vasopressin has been reported to ations, and this is consistent with their belonging to the same cause the rat anococcygeus to contract (3), although other family of peptides (10). Somatostatin has been found within

a) ,-- N

4 10 20 100 4 100 pM I NT NT NT NT CARB NT b) I 4 8 20 40 40 40 4 40 TRH TRH TRH TRH TRH TRH CARB TRH FM c) I 20 40 2 20 40 2 20 40 2 FM TRH NT CARB TRH NT CARB TRH NT CARB FIG. 3. Contractions of mouse anococcygeus muscle preparations exposed to neurotensin (NT; a, c) and TRH (b, c). After each response the organ bath was washed out and muscle tone was allowed to return to base line. The time interval between each break in the trace was 20 min. In a and b, the second panel from the right shows that if either peptide was left in contact with the tissue, tone returned to base line and responses to further doses of the peptide were reduced greatly, although carbachol (CARB; 4 ,uM) produced a normal 250- to 350-mg rise in tension. Peptide responsiveness was restored 20 min after washout. In c, the middle trace shows that if TRH was left in contact with the tissue, the responses to NT, but not to CARB, were reduced greatly. Thus, TRH and NT showed selective cross-desensitization. Time calibration, 1 min; tension calibration, 0-300 mg. Downloaded by guest on September 29, 2021 628 Physiological Sciences: Gibson et aLPProc. NatL Acad ScL USA 81 (1984) 10 20 40 80 FM SS a) -A I 10 20 40 80 pM SS I b) _ I_ I 0.1 0.3 1 5 pM U 3 C)_ 1- II I 0.6 1 2 3 U H IfpM d)_I1 I FIG. 4. Relaxations of mouse anococcygeus muscle preparation to somatostatin (SS; a, b) and urotensin II (U II; c, d). Muscle tone was raised by carbachol (2-4 ,uM). After each response the organ bath was washed out and muscle tone was allowed to return to base line for 15 min before it was increased again with carbachol. The time interval between each break in the trace was 20 min. Time calibration, 1 min; tension calibration, 0-300 mg. nerves in the gut and may modify gastrointestinal motility Field stimulation of the mouse anococcygeus, using train (15). Urotensin II has hypertensive, smooth muscle-con- lengths of 60 s, produces a biphasic relaxation due to activa- tracting, and osmoregulatory effects (21) in fishes and birds tion of NANC nerves (9); there is an initial rapid reduction in but was thought to be devoid of significant activity in mam- tone during the first 10 s, followed by a slower relaxation malian systems (22). There is one report of urotensin II caus- over the succeeding 50 s. On the basis of immunocytochem- ing relaxation of rabbit aorta in vitro (23); however, this ef- istry, time course of relaxation, and sensitivity to block by fect was believed to be due to oxidation of the catechol- various procedures, it has been suggested that VIP might amines used to induce tone in the preparation, since the mediate the second-phase relaxation (8, 9), similar to its pro- effect was absent when tone was raised by noncatechola- posed role as mediator of delayed vasodilatation in cat sali- mines. In the present study, tone was raised by carbachol, a vary glands and tongue (26, 27). The results of the present noncatecholamine, and so the mouse anococcygeus appears study are consistent with this hypothesis since, of the pep- to be a mammalian smooth muscle tissue directly sensitive to tides studied, only VIP produced relaxations with a time urotensin II. The mammalian muscle proved to be 20-50 course and intensity likely to reflect a neurotransmitter role. times more sensitive to the piscine hormone than to somato- A high concentration of VIP has recently been reported in statin. The muscle also contracted in response to [Argivaso- the stimulated penis of several mammalian species (28), into tocin, which is found in some teleost fish urophyses in small which anococcygeal muscle fibers are known to pass (1). amounts (24). The second major neuropeptide of the fish Substance P has been reported to relax the rat anococcygeus caudal neurosecretory system, urotensin I, was not studied; (29), but was motor in the mouse. If VIP is responsible for however, its analogues, sauvagine and corticotropin-releas- the second-phase relaxation of the mouse anococcygeus, ing factor, had minimal or no effects (25). then the mediator of the first phase remains a mystery. It could be an as-yet-untested peptide, ATP (8, 30), or a sub- 0.1 0.2 0.4 IpM VIP . stance, extracted from rat anococcygeus and bovine retrac- l tor penis, which has still to be characterized chemically (31).

I This work was supported by National Science Foundation Grant PCM-81-10111 and by a grant from the Central Research Fund of London University. 1. Gillespie, J. S. (1980) Trends Pharmacol. Sci. 1, 453-457. 2. Gillespie, J. S. (1972) Br. J. Pharmacol. 45, 404-416. 3. Gillespie, J. S. & McGrath, J. C. (1974) Br. J. Pharmacol. 50, FIG. 5. Relaxations of a mouse anococcygeus muscle prepara- 109-118. tion exposed to VIP. Muscle tone was raised by carbachol (4 AM). 4. Creed, K. E., Gillespie, J. S. & McCaffery, H. (1977) J. Physi- After each response the organ bath was washed out and muscle tone ol. (London) 273, 121-135. was allowed to return to base line for 15 min before it was increased 5. Dehpour, A. R., Khoyi, M. A., Koutcheki, H. & Zarrindast, again with carbachol. The time interval between each break in the M. R. (1980) Br. J. Pharmacol. 71, 35-40. trace was 20 min. Time calibration, 1 min; tension calibration, 0-400 6. Gibson, A. & Wedmore, C. V. (1981) J. Autonom. Pharmacol. mg. 1, 225-233. Downloaded by guest on September 29, 2021 Physiological Sciences: Gibson et aL Proc. NatL. Acad. ScL USA 81 (1984) 629

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