A non-selective cationic channel activated by diacylglycerol in mouse intestinal myocytes.

Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analogue of diacylglycerol (DAG) formed via M(3) muscarinic receptors, induced inward cationic currents via a protein kinase C-independent mechanism and produced membrane depolarization with increased action potential discharges in mouse intestinal myocytes. Outside-out patches from the myocytes responded to OAG with openings of 115-pS channels characterized by a mean open time (O(tau)) of 0.15 ms. M(3) receptor stimulation is reportedly capable of causing brief openings (O(tau)=0.23 ms) of 120-pS cationic channels in intestinal myocytes, thus the present results strongly support the idea that the M(3)-mediated 120-pS channel opening is brought about via DAG-dependent mechanisms.

Excitatory and inhibitory 5-hydroxytryptamine (5-HT) receptors expressed in the isolated porcine uterine muscles.

5-Hydroxytryptamine (5-HT) receptors mediating excitatory and inhibitory actions of 5-HT on contractility of uterine strips from non-pregnant pigs were characterized. Expression of 5-HT(2A) and 5-HT(7) receptors was examined by molecular biological study. 5-HT-containing cells were observed immunohistochemically. In the spontaneously contracting uterine circular muscle layers, 5-HT caused inhibition of contractile activity. SB269970 (5-HT(7) receptor antagonist, 10 nM) shifted the concentration-inhibition curve of 5-HT to the right, but higher concentrations of SB269970 (100 nM-1 microM) changed the monophasic curve to a biphasic curve (mixture of excitatory and inhibitory responses to 5-HT). Addition of ketanserin (5-HT(2) receptor antagonist, 10 nM-1 microM) decreased the excitatory effects of 5-HT. 5-HT was less effective in inhibiting the spontaneous contraction in the longitudinal muscles. Ketanserin enhanced the inhibitory responses and SB269970 reversed the inhibitory responses to excitatory responses. In the presence of SB269970, alpha-methyl-5-HT was equipotent to 5-HT in increasing contractility of longitudinal muscle and ketanserin competitively inhibited the responses to alpha-methyl-5-HT (pK(d)=8.78). Muscle layer-dependent expression of both 5-HT(2A) receptor and 5-HT(7) receptor mRNAs in the porcine uterine muscle layers was demonstrated by RT-PCR and real-time PCR. 5-HT immunoreactivity was detected only in uterine gland cells, which were localized near the uterine circular muscle layers. In the longitudinal and circular muscle layers with endometrium, compounds 48/80 and ketanserin did not change the spontaneous contractility, but SB269970 significantly increased the contractile activity of the circular muscle. In conclusion, excitatory 5-HT(2A) and inhibitory 5-HT(7) are present in the uterus of non-pregnant pigs. Endogenous 5-HT containing cells are mainly present in uterine glands of the pig. The possible roles of 5-HT and its receptors in regulation of porcine uterine spontaneous contractility are discussed.

Characterization of prostanoid receptors present on adrenergic neurons innervating the porcine uterine longitudinal muscle.

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.

Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle.

Functional muscarinic acetylcholine receptors present in the mouse uterus were characterized by pharmacological and molecular biological studies using control (DDY and wild-type) mice, muscarinic M2 or M3 single receptor knockout (M2KO, M3KO), and M2 and M3 receptor double knockout mice (M2/M3KO). Carbachol (10 nM-100 microM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (Emax) differed between cervical and ovarian regions of the uterus. The stage of the estrous cycle had no significant effect on carbachol concentration-response relationships. Tetrodotoxin did not decrease carbachol-induced contractions, but the muscarinic receptor antagonists (11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1,4]benzodiazepin6-one (AF-DX116), N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4] benzodiazepine-11-carboxamide (AF-DX384), 4-diphenylacetoxy-N-methyl-piperidine(4-DAMP), para-fluoro-hexa hydro-sila-diphenidol (p-F-HHSiD), himbacine, methoctramine, pirenzepine, and tropicamide) inhibited carbachol-induced contractions in a competitive fashion. The pKb values for these muscarinic receptor antagonists correlated well with the known pKi values of these antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 microg/kg, i.p. for 96 h), Emax values for carbachol were significantly decreased, but effective concentration that caused 50% of Emax values (EC50) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 microM), followed by subsequent washout of AF-DX116, neither carbachol nor N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M) caused any contractile responses. Both M2 and M3 muscarinic receptor messenger RNAs were detected in the mouse uterus via reverse transcription polymerase chain reaction. Carbachol also caused contraction of uterine strips isolated from M2KO mice, but the concentration-response curve was shifted to the right and downward compared with that for the corresponding wild-type mice. On the other hand, uterine strips isolated from M3KO and M2/M3 double KO mice were virtually insensitive to carbachol. In conclusion, although both M2 and M3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M3 receptor. Activation of M2 receptors alone did not cause uterine contractions; however, M2 receptor activation enhanced M3 receptor-mediated contractions in the mouse uterus.

Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells.

Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype-knockout (M2-KO) or M3-KO mice, carbachol (100 microM) evoked a muscarinic cationic current (mI(Cat)) as small as approximately 10% of mI(Cat) in wild-type (WT) cells. No appreciable current was evoked in M2/M3 double-KO cells. All mutant type cells preserved normal G-protein-cationic channel coupling. The M3-KO and WT mI(Cat) each showed a U-shaped current-voltage (I-V) relationship, whereas the M2-KO mI(Cat) displayed a linear I-V relationship. Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times O(tau) = 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (O(tau) = 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (O(tau) = 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U-shaped I-V curve similar to the WT mI(Cat). The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mI(Cat) generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2-Go protein and the M3-PLC systems predicted to control cationic channels.

Contractile effects of ghrelin-related peptides on the chicken gastrointestinal tract in vitro.

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R), and it stimulates growth hormone (GH) release, food intake and gastrointestinal motility in mammals. Ghrelin has also been identified in the chicken, but this peptide inhibits food intake in the chicken. We examined the effects of ghrelin and related peptides on contractility of the isolated chicken gastrointestinal tract in vitro. Among ghrelin-related peptides examined (1 microM of rat ghrelin, human ghrelin, chicken ghrelin and growth hormone releasing peptide-6 (GHRP-6)), only chicken ghrelin was effective on contraction of the chicken gastrointestinal tract. Des-acyl chicken ghrelin was ineffective, suggesting that octanoylation at Ser3 residue of chicken ghrelin was essential for inducing the contraction. Amplitude of chicken ghrelin-induced contraction was region-specific: highest in the crop and colon, moderate in the esophagus and proventriculus, and weak in the small intestine. The contractile response to chicken ghrelin in the crop was not affected by tetrodotoxin (TTX), but that in the proventriculus was decreased by TTX and atropine to the same extents. D-Lys3-GHRP-6 (a GHS-R antagonist) caused a transient contraction and inhibited the effect of chicken ghrelin without affecting the high-K+-induced contraction. Chicken ghrelin potentiated electrical field stimulation-induced cholinergic contraction without affecting the responsiveness to bath-applied carbachol in the proventriculus. The location of GHS-R differs in the crop (smooth muscle) and proventriculus (smooth muscle and enteric neurons). These results indicate that ghrelin has contractile activity on gastrointestinal tract in the chicken in vitro, and the effect was region-specific. The action would be mediated through the GHS-R, which is highly sensitive to chicken ghrelin.

Functional roles of muscarinic M2 and M3 receptors in mouse stomach motility: studies with muscarinic receptor knockout mice.

Functional roles of muscarinic acetylcholine receptors in the regulation of mouse stomach motility were examined using mice genetically lacking muscarinic M(2) receptor and/or M(3) receptor and their corresponding wild-type (WT) mice. Single application of carbachol (1 nM-30 microM) produced concentration-dependent contraction in antral and fundus strips from muscarinic M(2) receptor knockout (M(2)R-KO) and M(3) receptor knockout (M(3)R-KO) mice but not in those from M(2) and M(3) receptors double knockout (M(2)/M(3)R-KO) mice. A comparison of the concentration-response curves with those for WT mice showed a significant decrease in the negative logarithm of EC(50) (pEC(50)) value (M(2)R-KO) or amplitude of maximum contraction (M(3)R-KO) in the muscarinic receptor-deficient mice. The tonic phase of carbachol-induced contraction was decreased in gastric strips from M(3)R-KO mice. Antagonistic affinity for 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116) indicated that the contractile responses in M(2)R-KO and M(3)R-KO mice were mediated by muscarinic M(3) and M(2) receptors, respectively. Electrical field stimulation (EFS, 0.5-32 Hz) elicited frequency-dependent contraction in physostigmine- and N(omega)-nitro-L-arginine methylester (l-NAME)-treated fundic and antral strips from M(2)R-KO and M(3)R-KO mice, but the cholinergic contractile components decreased significantly compared with those in WT mice. In gastric strips from M(2)/M(3)R-KO mice, cholinergic contractions elicited by EFS were not observed but atropine-resistant contractions were more conspicuous than those in gastric strips from WT mice. Gastric emptying in WT mice and that in M(2)/M(3)R-KO mice were comparable, suggesting that motor function of the stomach in the KO mice did not differ from that in the WT mice. The results indicate that both muscarinic M(2) and M(3) receptors but not other subtypes mediate carbachol- or EFS-induced contraction in the mouse stomach but that the contribution of each receptor to concentration-response relationships is distinguishable. Although there was impairment of nerve-mediated cholinergic responses in the stomach of KO mice, gastric emptying in KO mice was the same as that in WT mice probably due to the compensatory enhancement of the non-cholinergic contraction pathway.

Endogenous prostaglandins regulate spontaneous contractile activity of uterine strips isolated from non-pregnant pigs.

Myometrial strips isolated from non-pregnant pigs show spontaneous contractile activity. In the present study, the involvement of endogenous prostaglandins in regulation of uterine spontaneous contraction was investigated using mechanical, immunohistochemical and biochemical approaches. Immunohistochemical study and Western blot analysis for immunoreactive cyclooxygenase (COX) indicated that COX-1 but not COX-2 was expressed predominantly in the myometrium of non-pregnant pigs in a muscle layer-dependent manner (longitudinal muscle>circular muscle). Pretreatment of uterine strips with indomethacin and selective COX-1 inhibitors (SC-560 and FR122047) significantly reduced both the amplitude and frequency of spontaneous contraction in the longitudinal muscle, but inhibition by COX inhibitors was negligible in the circular muscle. On the other hand, CAY10404, a COX-2 inhibitor, did not change the spontaneous contraction in either of the muscle layers. Pretreatment with SC-560 reduced myometrial PGF(2alpha) and PGE(2) levels. Contractile FP and EP(3) receptors were expressed in a muscle layer-dependent manner (longitudinal muscle>circular muscle), similar to the expression pattern of COX-1. In conclusion, endogenous prostaglandins produced by COX-1 regulate spontaneous contractile activity of non-pregnant porcine uterine longitudinal muscle selectively due to the heterogeneous expression of contractile prostanoid receptors and COX-1.

Uterine region-dependent differences in responsiveness to prostaglandins in the non-pregnant porcine myometrium.

To clarify the uterine region-dependent distribution of prostanoid receptors, we compared the mechanical responses to selective prostanoid receptor agonists (FP, EP3, DP, EP2) and naturally occurring prostaglandins (PGF2alpha PGE2, PGD2) in longitudinal and circular muscles isolated from three different regions (cornu, corpus and cervix) of the non-pregnant porcine uterus. Expression levels of FP receptor and cyclooxygenase (COX-1 and COX-2) in the respective regions were also examined using RT-PCR and Western blotting. The contractile responses to fluprostenol (an FP agonist) and PGF2alpha in both longitudinal and circular muscles were strongest in the cornu but weak in the corpus and cervix. Expression levels of mRNA and protein of FP receptor were highest in the cornu, consistent with the contractile responses. ONO-AE-248 (an EP3 agonist) caused contraction of both muscle layers, but region-related difference in responsiveness was observed only in the longitudinal muscle. ONO-AE1-259 (an EP2 agonist) inhibited spontaneous contraction of the myometrium, and inhibition was conspicuously stronger in the cervix. PGE2 caused contraction (<100 nM, cornu > corpus = cervix) and inhibition (>300 nM, cornu = corpus < or = cervix) of contractility depending on the concentration in both muscle layers. BW245C (a DP agonist) inhibited the spontaneous contraction, and region-dependent different responsiveness was marked in the longitudinal muscle (cervix = corpus > cornu). COX-1 but not COX-2 was detected in the non-pregnant porcine uterus. Expression level of COX-1 was different in the longitudinal muscle (cornu > corpus = cervix) but the same in the circular muscle. SC-560 inhibited the spontaneous contraction of longitudinal muscles in all regions. The results of the present study indicate that there are region-related heterogeneous distributions of contractile (FP and EP3, cornu > cervix) and relaxant (EP2 and DP, cervix > cornu) prostanoid receptors and COX-1 in the porcine uterus. The results also suggest involvement of endogenous PGs in the regulation of spontaneous uterine contractility. Region-related differences in COX-1 and prostanoid receptors might be necessary to produce a gradient of uterine motility decreasing from the cornu to the cervix that manages movement of luminal contents.

Intracerebroventricular administration of chicken motilin does not induce hyperphagia in meat-type chicks.

The effect of chicken motilin on food intake was investigated in meat-type chicks under ad libitum feeding, refeeding, and fasting conditions. We found that the intracerebroventricular injection of chicken motilin (0.1 and 0.2 microg) tended to increase food intake under ad libitum feeding and refeeding conditions at 60 min postinjection, but the differences were not significant (P>.05). On the other hand, central administration of chicken motilin (0.2 and 0.4 microg) showed a tendency to suppress feeding of fasted chicks as well as the result of high dose (5.0 microg) under ad libitum feeding conditions. Therefore, the results presented here suggest that central motilin alone does not induce hyperphagia in meat-type chicks.

Thromboxane A2 (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity.

Although there are species-related differences in uterine prostanoid receptor subtypes, functional prostanoid receptors in the porcine uterus are similar with those in the human uterus (FP, TP, EP(1), EP(2), EP(3), DP and IP) except for the TP receptor. These similarities promoted us to determine whether TP receptors are present in the non-pregnant porcine uterus. For this purpose, the effects of TP receptor agonists and antagonists were investigated by a contraction study and by a binding study. 9,11-Dideoxy-9 alpha, 11 alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619, 1 nM-10 microM), a stable thromboxane A(2) mimetic, caused tetrodotoxin-resistant contraction in both longitudinal and circular muscles of the uterine cornu. The pEC(50) value in the longitudinal muscle (6.69) was lower than that in the circular muscle (7.62), but the maximum response in the longitudinal muscle was two times larger than that in the circular muscle. The longitudinal and circular muscles of other regions (corpus and cervix) also responded to U46619, and region-related difference in contractile responses was observed only in the longitudinal muscles. 4(Z)-6-(2-o-Chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl) hexenoic acid (ICI192605) and 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-,[1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-]5-heptenoic acid (SQ29548) inhibited the contractile responses to U46619 competitively. The longitudinal and circular muscles in the cornu contained a single class of [3H]SQ29548 binding site with similar K(d) values (30 nM), but B(max) in the circular muscle (90.9+/-8.6 fmol/mg protein) was two times higher than that in the longitudinal muscle (58.2+/-8.6 fmol/mg protein). The ranking order of competition by TP receptor agonists and antagonists (with pK(i) values in parentheses) was [1S-[1,2(Z),3(1E,3S*),4]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP, 7.70)>SQ29548 (7.39)>7-[3-(3-Hydroxy-1-octenyl)bicycle[3.1.1]hept-2-yl]-,[2S-[2 alpha(Z),3 beta(1E,3R*)]]-5-heptenoic acid (CTA(2), 6.55)>7-[3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl-,[1S-[1 alpha,2 beta(Z),3 alpha(1E,3R*),5 alpha]]-5-heptenoic acid (PTA(2), 6.50)>U46619 (6.41)>7-[5-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1] hept-6yl]-,[1S-[1 alpha,4 alpha,5 alpha(1E,3R*),6 beta(Z)]]-5-heptenoic acid (U44069, 6.34), and this order is consistent with current TP receptors. Treatment with indomethacin (100 nM) and N-tert-butyl-N cent -[(2-cyclohexylamino-5-nitrobenzene) sulfonyl] urea (BM-531, 10 microM) inhibited the spontaneous contractile activities of both longitudinal and circular muscles. The present results indicate that contractile TP receptors are present in the non-pregnant porcine uterus. Therefore, the prostanoid receptor subtypes that exist in the porcine uterus (TP, IP, DP, FP, EP(1), EP(2) and EP(3)) are the same as those present in the human uterus. The distribution of TP receptors in the porcine uterus differed depending on the type of myometrium (longitudinal and circular muscles) and region of the uterus. The endogenous thromboxane A(2)-TP receptor pathway is thought to play a physiological role in regulation of spontaneous contractile activity in the porcine uterus.

Pregnancy-associated changes in responsiveness of the porcine myometrium to bioactive substances.

To determine the pregnancy-associated changes in the porcine uterine contractility, the spontaneous contraction and the mechanical responses to bioactive substances of uteri in nonpregnant proestrus and pregnant pigs (25-60 days of gestation) were compared in vitro. Longitudinal muscle (LM) and circular muscle (CM) of the uterus exhibited spontaneous contraction, but the frequency in pregnant pigs was lower than that in the nonpregnant pigs. The duration and force of spontaneous contraction in the pregnant pigs were long and large compared with both in the nonpregnant pigs. L-Nitroarginine methylester (L-NAME) and 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydro-benzo[cd]indol-2(1H)-one (DR4004) did not change the spontaneous contraction in the uteri of nonpregnant pigs but increased its amplitude in the uteri of pregnant pigs. Isoprenaline inhibited the uterine spontaneous contraction of the nonpregnant and pregnant pigs, and the inhibition was stronger in the pregnant than in the nonpregnant pigs. 5-Hydroxytryptamine also caused inhibition of spontaneous contraction in the uteri of nonpregnant pigs (CM>LM). In the pregnant pigs, sensitivity to 5-hydroxytryptamine increased in a muscle layer-dependent manner (LM>CM) and difference in the responsiveness between LM and CM decreased. Acetylcholine contracted the uterine LM and CM strips of the pregnant and nonpregnant pigs. The responsiveness of CM increased slightly during pregnancy, but that of the LM did not change. 5-Bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine (UK14304) caused contraction of only LM in the uteri of nonpregnant pigs, but contracted both LM and CM strips in the pregnant pigs. Oxytocin and prostaglandin F(2 alpha) also contracted the uteri of nonpregnant pigs (LM>CM). Pregnancy increased the contraction of both agents in the LM and CM, but the increment was marked in the CM. The contractile forces induced by all stimulants were increased (by 1.7- to 2.5-fold) in the LM and CM of pregnant pigs. In conclusion, (1) low frequency, slow kinetics and large force of spontaneous contraction are characteristics of the pregnant porcine uteri, and nitric oxide and 5-hydroxytryptamine are supposed to be partially involved in the regulation of spontaneous contraction, and (2) responses to both contractile and inhibitory agents are increased in the pregnant pigs. Increment of the responsiveness is conspicuous in the muscle layer that is less sensitive to each agonist in the uteri of nonpregnant pigs. According to the pregnancy-associated changes, muscle layer-related differences of responsiveness to bioactive substances in the nonpregnant pigs tend to decrease in the pregnant pigs.

5-HT7 receptor-mediated relaxation of the oviduct in nonpregnant proestrus pigs.

The effects of 5-hydroxytryptamine (5-HT) on the muscle tonus of the ampulla and isthmus of the oviduct isolated from nonpregnant proestrus pigs were investigated, and the 5-HT receptor subtype and mechanisms of the responses were analyzed. 5-HT (1 nM-10 microM) caused a relaxation of longitudinal and circular muscles of the isthmus in a concentration-dependent manner. Tetrodotoxin did not change the relaxation, indicating a direct action of 5-HT on smooth muscle cells. The EC(50) value in the longitudinal muscle was significantly lower than that in the circular muscle but the maximum relaxations were similar. 5-HT also caused a relaxation of both muscle layers in the ampulla but the maximum relaxation of both muscles was smaller than that of the isthmus. 5-Carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) mimicked the relaxation of the isthmic longitudinal muscle by 5-HT, and the ranking order was 5-CT>5-HT>5-MeOT>8-OH-DPAT. On the other hand, oxymethazoline, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT), [endo-N-8-methyl-8-azabicyclo-(3,2,1) oct-3-yl]-2,3-dihydro-3-ethyl-2-oxo-1H-benzimidazol-1-carboxamide (BIMU-1), ergotamine and dihydroergotamine were less effective. The relaxation by 5-HT was not decreased by ketanserin, 2-methoxy-4-amino-5-chlorobenzoic acid 2-(diethylamino)ethyl ester (tropisetron) or [1[2-(methylsulphonyl) amino ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate (GR113808) but was antagonized by the following compounds in a competitive manner (with pK(b) values in parentheses): 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydro-benzo[cd]indol-2(1H)-one (DR4004, 9.31), methiothepin (8.91), methysergide (7.95), metergoline (7.98), mianserin (7.69), mesulergine (8.4), spiperone (6.86) and clozapine (7.4). The correlation of these pK(b) values with pK(i) values of cloned 5-HT(7) receptor or pA(2) values of porcine uterus was high and significant. 4-(3-Butoxy-4-methoxybenzyl)-imidazolidin-2-one (Ro20-1724) significantly enhanced the relaxation by 5-HT but zaprinast, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and L-nitroarginine methylester (L-NAME) did not change the responses to 5-HT. 5-HT increased cyclic AMP in the isthmic oviduct. Ampulla and isthmus contained a single class of [3H]5-CT binding sites with a similar K(d) value (0.4 nM), but the density of the receptors in the isthmus was 2.4 times higher than that in the ampulla. A significant correlation was found between the pK(i) values in the oviduct and those of the cloned 5-HT(7) receptors. Isoprenaline, sodium nitroprusside, vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide were less effective in causing the relaxation of the oviduct. In conclusion, the 5-HT receptor, functionally correlated to the 5-HT(7) type, mediates the relaxation of the porcine oviduct by 5-HT through an increase in intracellular cyclic AMP. The degrees of 5-HT-induced relaxation in the isthmus and ampulla of the oviduct were different due to the heterogeneous distribution of 5-HT(7) receptors. The strongest relaxation through 5-HT(7) receptor activation suggests that 5-HT plays an important physiological role in the regulation of porcine oviduct contractility.

In vitro pharmacological characterization of the prostanoid receptor population in the non-pregnant porcine myometrium.

In order to characterize prostanoid receptors present in the non-pregnant porcine uterus, the effects of naturally occurring prostaglandins (D2, E2, F2alpha, I2) and synthetic prostanoid receptor agonists on contractility of the longitudinal and circular muscles were examined in vitro. The potent contractile actions of prostaglandin F2alpha and cloprostenol indicate the presence of excitatory FP receptors in the porcine uterus. The longitudinal muscle was more sensitive to FP receptor agonists than was the circular muscle. Prostaglandin D2 produced an excitatory response in the longitudinal muscle but completely inhibited the spontaneous contraction of the circular muscle. BW-245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin, 1 nM-10 microM, a DP receptor agonist) inhibited the spontaneous contractions of both muscles, but the inhibition was conspicuously stronger in the circular muscle. Prostaglandin I2 caused excitatory and inhibitory responses in the longitudinal and circular muscles, respectively, at relatively high concentrations (10-100 microM). Cicaprost, an IP receptor agonist caused inhibition of the contraction in the circular muscle but contracted the longitudinal muscle. Iloprost, an EP(1)/IP receptor agonist, caused excitatory responses in both muscles at relative high concentrations. Prostaglandin E2 caused excitatory responses at 1-100 nM and inhibitory responses at 100 nM-10 microM in both muscle layers. ONO-DI-004 ((17S)-2,5-ethano-6-oxo-17,20-dimethyl prostaglandin E1, an EP1 receptor agonist) and ONO-AE-248 ((16S)-9-deoxy-9beta-chloro-15-deoxy-16-hyfroxy-17,17-trimethylene-19,20-didehydro prostaglandin F2, an EP3 receptor agonist) contracted the longitudinal muscle but had little effect on the circular muscle. ONO-AE1-259 (11,15-O-dimethyl prostaglandin E2, an EP2 receptor agonist) inhibited the spontaneous contractions of both muscle layers to almost the same degree, but ONO-AE1-329 (16-(3-methoxymethyl)phenyl-omega-tetranor-3,7-dithia prostaglandin E1, an EP4 receptor agonist) did not inhibit the myometrial contraction. The present results indicate that contractile (FP, EP1, EP3) and relaxatory (DP, IP, EP2) prostanoid receptors are present in the non-pregnant porcine uterus. There are marked muscle layer-related differences in the degree of responsiveness of prostanoid receptor agonists, and these differences suggest that there is a heterogeneous distribution of prostanoid receptors in the longitudinal and circular muscles (FP, EP1 and EP3, longitudinal muscle>circular muscle; DP, circular muscle>longitudinal muscle).