Adenosine-5''-O-(2-thiodiphosphate) is a potent agonist at P2 purinoceptors mediating insulin secretion from perfused rat pancreas.

1. The effects of a P2 purinoceptor agonist, adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) have been studied on insulin secretion and flow rate of the isolated perfused pancreas of the rat. 2. In the presence of a moderately stimulating glucose concentration (8.3 mM), ADP-beta-S (4.95-495 nM) evoked a biphasic insulin response in a concentration-dependent manner. A comparison of relative potency between ADP-beta-S and adenosine 5'-triphosphate (ATP) showed that ADP-beta-S was 100 times more potent than ATP. On the other hand, in the presence of a non stimulatory glucose concentration (4.2 mM), ADP-beta-S (165 nM) did not modify the basal insulin secretion. 3. ADP-beta-S, at concentrations effective on insulin secretion and also at higher concentrations (1.65 and 16.5 microM), provoked an increase of the pancreatic flow rate in a concentration-dependent manner. 4. Our results show that ADP-beta-S is a potent insulin secretory P2 purinoceptor agonist. As it is resistant to hydrolysis it might be useful in studying the effect of activation of the P2 purinoceptor of beta cells on insulin secretion in vivo.     

5-Carboxamidotryptamine is a selective agonist at 5-hydroxytryptamine receptors mediating vasodilatation and tachycardia in anaesthetized cats.

We have attempted to characterize the 5-hydroxytryptamine (5-HT) receptors mediating bronchoconstriction, vasodilatation, vasodepression and tachycardia in anaesthetized cats following bilateral vagosympathectomy and beta-adrenoceptor blockade with propranolol. 5-HT (1-100 micrograms/kg-1 i.v.) caused dose-related bronchoconstriction and tachycardia but variable and complex effects on diastolic blood pressure and carotid arterial vascular resistance. In contrast, 5-carboxamidotryptamine (5-CT; 0.01-1 micrograms kg-1 i.v.) caused consistent, dose-related decreases in diastolic blood pressure and carotid arterial vascular resistance and increases in heart rate. 5-CT did not cause bronchoconstriction. The 5-HT-induced bronchoconstriction was dose-dependently antagonized by methiothepin, methysergide and ketanserin (10-100 micrograms kg-1 i.v.). The highest doses used of these antagonists did not antagonize bronchoconstriction induced by prostaglandin F2 alpha. The high potency of all three antagonists indicate a 5-HT2-receptor mediated effect. The 5-HT- and 5-CT-induced tachycardia as well as the 5-CT-induced vasodepressor and carotid arterial vasodilator responses were dose-dependently antagonized by low doses of methiothepin (10-100 micrograms kg-1 i.v.) and by high doses of methysergide (100-1000 micrograms kg-1 i.v.) but were little affected by ketanserin in doses up to 1000 micrograms kg-1 i.v. These selective effects of 5-CT appear to be mediated by '5-HT1-like' receptors.     

Contribution of P2-purinoceptors to neurogenic contraction of rat urinary bladder smooth muscle.

1. The contribution of P2-purinoceptors to neurogenic contraction was investigated in rat urinary bladder smooth muscle by measurement of isotonic tension. 2. Contraction of rat urinary bladder smooth muscle induced by electrical stimulation was decreased to 84.19 +/- 3.90% of the control (n = 16) in the presence of atropine (1 microM), which was further decreased to 38.80 +/- 2.75% of the control (n = 49) in the presence of both atropine and 10 microM alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-Me ATP). 3. The contractile response induced by electrical stimulation in the presence of atropine and alpha, beta-Me ATP was decreased to 27.81 +/- 4.07% (n = 23) and 26.63 +/- 5.01% (n = 15) of the control, by the addition of 100 microM cibacron blue 3GA and 100 microM suramin, respectively. The application of 100 microM adenosine 5'-o-2-thiodiphosphate (ADP beta S) in the presence of atropine and alpha, beta-Me ATP decreased the contractile response induced by electrical stimulations to 17.15 +/- 3.71% (n = 15) of the control. 4. Pretreatment of muscle strips with 100 microM ADP beta S significantly reduced the response to either 200 microM alpha, beta-methylene adenosine 5'-diphosphate or 200 microM ADP beta S. 5. Uridine 5'-triphosphate (100 microM to 1 mM) concentration-dependently contracted muscle strips, and this contraction was significantly antagonized by desensitization of P2-receptors with alpha, beta-Me ATP (10 microM), and completely antagonized by pretreatment of muscle strips with both alpha, beta-Me ATP and ADP beta S (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for 5-HT7 receptors mediating relaxation of human colonic circular smooth muscle

5-HT4 receptors mediate relaxation of human colon circular muscle. However, after 5-HT4 receptor blockade (SB 204070 10 nM), 5-HT still induced a relaxation (pEC50 6.3). 5-HT4 receptors were sufficiently blocked, as the curves to 5-HT obtained in the presence of 10 and 100 nM SB 204070 were indistinguishable. This 5-HT-induced relaxation was tetrodotoxin-insensitive, indicative of a smooth muscle relaxant 5-HT receptor. This, and the rank order of potency (5-CT=5-MeOT=5-HT) suggested involvement of 5-HT1 or 5-HT7 receptors. Mesulergine, a 5-HT7 receptor antagonist at nanomolar concentrations, and a 5-HT1 receptor antagonist at micromolar concentrations, competitively antagonized the 5-HT-induced relaxation (pKB 8.3) and antagonized the relaxation to 5-CT. Methysergide antagonized the 5-HT-induced relaxation (pA2 7.6). It is concluded that the profile of the smooth muscle inhibitory 5-HT receptor resembles that of the 5-HT7 receptor. These data provide the first evidence for functional human 5-HT7 receptors.     

Evidence for two distinct P2-purinoceptors subserving contraction of the rat anococcygeus smooth muscle.

1. The effects of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP (alpha, beta-MeATP), beta, gamma-methylene ATP (beta, gamma-MeATP), L-beta, gamma-methylene ATP (L-beta, gamma-MeATP), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), and 2-methylthio ATP (2-MeSATP) were investigated on the isometric tension of the rat anococcygeus muscle. 2. Non-cumulative additions of ATP (100-1500 microM), alpha, beta-MeATP (1-300 microM), beta, gamma-MeATP (10-300 microM), L-beta, gamma-MeATP (3-100 microM) and ADP beta S (1-100 microM) produced concentration-dependent contractions, whereas 2-MeSATP (1-100 microM) had no effect. The rank order of potency was alpha, beta-MeATP > L-beta, gamma-MeATP > or = ADP beta S > beta, gamma-MeATP > > ATP > 2-MeSATP. 3. Contractions to cumulative additions of ATP, alpha, beta-MeATP, beta, gamma-MeATP and L-beta, gamma-MeATP were subject to desensitization whilst those to ADP beta S were unaffected. 4. Contractions to ATP, alpha, beta-MeATP, beta, gamma-MeATP and ADP beta S were abolished by the non-selective P2X/. P2Y-purinoceptor antagonist, suramin (100 microM). In contrast, contractions to ATP, alpha, beta-MeATP and beta, gamma-MeATP were not affected by the non-selective P1-purinoceptor antagonist 8-(p-sulphophenyl)-theophylline (8SPT, 30 microM). Blockade of P2X-purinoceptors with the selective P2X-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) or desensitization with L-beta, gamma-MeATP (10 microM) abolished contractions to alpha, beta-MeATP, but enhanced those to ADP beta S. The P2Y-purinoceptor antagonist, reactive blue 2 (RB2, 100 microM) enhanced contractions to ATP and alpha, beta-MeATP but abolished those to ADP beta S. 5. Simultaneous addition of alpha, beta-MeATP and ADP beta S produced an additive contraction. 6. The findings suggest that in the rat anococcygeus, smooth muscle cells are endowed with two distinct P2-purinoceptors which subserve contractions: a P2X-purinoceptor activated by ATP and its analogues, and another type of P2-purinoceptor activated by ADP beta S.     

Investigation into the 5-hydroxytryptamine receptor mediating smooth muscle relaxation in the rat oesophagus.

1. An investigation has been made into the 5-hydroxytryptamine (5-HT) receptor mediating relaxation of rat oesophagus in preparations precontracted with carbachol. 2. In tissues treated with pargyline (100 microM) and in the presence of corticosterone (30 microM) and cocaine (30 microM) the potency of 5-HT and 5-methoxytyramine (5-MeOT) was not changed but the maximum response to these agonists was reduced. Thus there was no evidence of metabolism and/or uptake through an amine depleting mechanism. 3. The relaxant concentration-effect curves to 5-HT were shifted to the left in a concentration-related manner by isobutylmethylxanthine (1 and 10 microM), suggesting the involvement of adenosine 3':5'-cyclic monophosphate in these responses. 4. 5-HT produced concentration-related relaxations of rat oesophagus with an EC50 value of 0.24 microM. Several indole agonists were tested and the following rank order of potency of key agonists obtained: 5-HT greater than alpha-methyl-5-hydroxytryptamine = 5-carboxamidotryptamine (5-CT) greater than 5-MeOT. In contrast, 2-methyl-5-hydroxytryptamine, sumatriptan and 8-hydroxy-2-(di-n-propylamino) tetralin were weak or inactive. 5. The substituted benzamides, metoclopramide, cisapride, renzapride and R,S-zacopride acted as partial agonists, producing 60-70% of the 5-HT maximum. 6. The relaxation responses to 5-HT were neither inhibited by antagonists selective for 5-HT1 or 5-HT2 receptors nor by the 5-HT3 receptor antagonists, ondansetron, granisetron or MDL 72222. 7. The relaxation responses induced by 5-HT, 5-CT, 5-MeOT and renzapride were selectively inhibited by high concentrations of ICS 205-930 with pKB values of approximately 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

The signalling pathway which causes contraction via P2-purinoceptors in rat urinary bladder smooth muscle

1. The signalling pathway which causes contractions to adenosine 5′-O-2-thiodiphosphate (ADPβS) and α,β-methylene adenosine 5′-diphosphate (α,β-Me ADP) was investigated in rat urinary bladder smooth muscle by measuring isotonic tension.
2. The responses to 10 μM α,β-methylene adenosine 5′-triphosphate (α,β-Me ATP) in 0 and 3.6 mM Ca²⁺ were 5.9±1.3 (n=10) and 122.2±6.4 (n=8) % respectively of those obtained in 1.8 mM Ca²⁺, whereas those to 100 μM ADPβS were 34.6±3.3 (n=8) and 96.8±7.2 (n=8) %, in 0 and 3.6 mM Ca²⁺, respectively. In both experimental conditions, the responses to the two agonists expressed as % of the control responses were significantly different (P<0.01).
3. Indomethacin at high concentrations (>1 μM) decreased the responses to α,β-Me ATP (10 μM), ADPβS (100 μM) and α,β-Me ADP (100 μM). However, no significant difference was obtained between the responses to all the agonists at 30 μM indomethacin.
4. 2-Nitro-4-carboxphenyl n,n-diphenylcarbamate (NCDC) at concentrations between 1 μM and 100 μM concentration-dependently decreased the responses to ADPβS (100 μM) and α,β-Me ADP (100 μM) and almost completely inhibited them at 100 μM. Although the responses to α,β-Me ATP (10 μM) were also inhibited by the drug, at 50 and 100 μM NCDC the responses to α,β-Me ATP were significantly larger than those to ADPβS and α,β-Me ADP (P<0.01).
5. NCDC 100 μM significantly inhibited the KCl-induced contraction to 65.9±4.9% (n=6) of the control (P<0.01).
6. It is suggested that the contraction via ADPβS-sensitive receptors in the rat urinary bladder smooth muscle mainly depends on Ca²⁺ ions liberated from intracellular Ca²⁺ stores, though the contribution of Ca²⁺ ions from the extracellular space cannot be neglected. The release of Ca²⁺ ions from stores is mainly mediated by the production of inositol trisphosphate (IP₃) via the activation of phospholipase C.

     

Direct effects of adenylyl 5''-(beta,gamma-methylene)diphosphonate, a stable ATP analogue, on relaxant P1-purinoceptors in smooth muscle.

1. Previous results obtained with the rat colon muscularis mucosae, which contracts in response to adenosine and adenosine 5'-triphosphate (ATP), had suggested that adenylyl 5'-(beta,gamma-methylene)diphosphonate (AMPPCP), a stable ATP analogue, acted on P1-purinoceptors rather than, as expected, on P2-purinoceptors. This possibility has been examined in two tissues in which adenosine and ATP both cause relaxation, the guinea-pig taenia caeci and the rat duodenum. 2. ATP, 2-methylthio-ATP (2-MeSATP), AMPPCP, adenosine 5'-(alpha,beta-methylene)triphosphonate (AMPCPP) and adenosine each relaxed the taenia caeci and the duodenum, and the order of potency of the nucleotides in each tissue was 2-MeSATP greater than ATP greater than AMPCPP greater than AMPPCP, indicating that these effects were mediated by P2Y-purinoceptors. 3. The P1 antagonist 8-(p-sulphophenyl)theophylline (8-SPT) (100 microM) did not affect the responses to ATP, 2-MeSATP or AMPCPP in either tissue, but inhibited the responses of adenosine and of AMPPCP in both tissues. In the duodenum a lower concentration of 8-SPT caused a parallel shift to the right of the concentration-response curve to adenosine and to AMPPCP but to different extents, with AMPPCP being inhibited more powerfully than adenosine. A dose-ratio of around 5 was observed for adenosine and AMPPCP at concentrations of 8-SPT of 20 microM and 2 microM respectively, but Schild analysis resulted in plots with slopes greater than unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine 5'-tetraphosphate (Ap(4)), a new agonist on rat midbrain synaptic terminal P2 receptors

The aim of this study was to see whether the compound adenosine 5'-tetraphosphate (Ap(4)) is active in the central nervous system by examining its effect on isolated rat brain synaptic terminals. Ap(4) proved to be more resistant to ecto-enzymatic hydrolysis than adenosine triphosphate (ATP), showing only 2% hydrolysis after a 2-min incubation, compared to 75% for ATP.In addition, Ap(4) was able to produce concentration-dependent increases in intracellular Ca(2+) when applied extracellularly. This action was dependent upon the presence of extracellular calcium. Ap(4) acts through ionotropic ATP receptors (P2X receptors) and not through diadenosine polyphosphate receptors, since ATP abolished the response elicited by Ap(4) whereas Ap(5)A did not. Ap(4), ATP and ATP-gamma-S were of similar potency (EC(50) approximately 20 microM) while 2MeSATP, alpha,beta-meATP and ADP-beta-S possessed slightly lower potency (EC(50) approximately 50 microM).The P2-purinoceptor antagonists suramin and PPADS blocked the Ap(4) effect. The IC(50) values for these compounds were 35.5 and 7.8 microM respectively. Diinosine polyphosphates and inosine tetraphosphate inhibited the response elicited by Ap(4) with IC(50) values that varied between approximately 40 and 50 microM.These results show that Ap(4) is as good an agonist as ATP on synaptosomal P2X receptors, being more resistant to extracellular hydrolysis by ecto-nucleotidases.     

5-Hydroxytryptamine-induced relaxation of isolated mammalian smooth muscle

5-HT caused concentration-dependent relaxation of methoxamine contracted cat isolated saphenous vein and atropine pretreated guinea-pig isolated ileum contracted with histamine. The concentration required to cause 50% of its maximum effect was 7.4 X 10(-7) and 1.4 X 10(-6) mol/l respectively. alpha-Methyl 5-HT was at least 200 times weaker than 5-HT in this respect. The relaxant action of 5-HT in both preparations was not antagonised by atropine, indomethacin, cimetidine, propranolol or tetrodotoxin. In both preparations the relaxant effect was not specifically antagonised by cyproheptadine but was specifically and competitively antagonised by methysergide (pA2 values of 6.75 and 7.37 in cat saphenous vein and guinea-pig ileum, respectively). The results suggest that the 5-HT mediated relaxation in both preparations is mediated directly via a similar 5-HT receptor. The weak antagonistic action of methysergide and lack of relaxant effects with alpha-methyl 5-HT is consistent with the view that this receptor is not a 'D' or 'M' 5-HT-receptor.     

Pharmacological characterization of 5-HT4 receptors mediating relaxation of canine isolated rectum circular smooth muscle.

This study aimed to characterize for the first time in vitro 5-HT4 receptors in the canine gastrointestinal tract. For this purpose, we used circular muscle strips of the canine isolated rectum. In the presence of methysergide (60 microM), 5-HT induced relaxation of methacholine (1 microM)-precontracted muscle strips, yielding a monophasic sigmoidal concentration-relaxation curve (pEC50 7.2+/-0.07). Tetrodotoxin (0.3 microM) did not affect the curve to 5-HT, suggesting the inhibitory 5-HT receptor is located on the smooth muscle. Granisetron (0.3 microM) did also not affect the curve to 5-HT, which excludes the 5-HT3 receptor mediating the relaxation to 5-HT. The presence of methysergide rules out the involvement of 5-HT1, 5-HT2 or 5-HT7 receptors. 5-HT, the selective 5-HT4 receptor agonists R076186, prucalopride (R093877) and SDZ HTF-919 and the 5-HT4 receptor agonists cisapride and 5-MeOT relaxed the muscle strips with a rank order of potency R076186 = 5-HT > cisapride > prucalopride > or = SDZ HTF-919 > 5-MeOT. The selective 5-HT4 receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5-HT, yielding pK(B) estimates of 9.7, 7.9 and 9.1, respectively. The selective 5-HT4 receptor antagonist SB 204070 shifted the curve to 5-HT rightward and depressed the maximal response (apparent pA2 10.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5-HT4 receptor agonists R076186 (pA2 8.8). It is concluded that 5-HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5-HT4 receptors. For the first time, a nonhuman species was shown to exhibit relaxant 5-HT4 receptors in the large intestine.     

Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle

1. The effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), α,β-methylene-ATP (α,β-MeATP) and 2-methylthio-ATP (2-MeSATP) on longitudinally orientated smooth muscle strips from marmoset urinary bladder were investigated by use of standard organ bath techniques.
2. After being mounted in superfusion organ baths, 66.7% (n=249) of marmoset detrusor smooth muscle strips developed spontaneous tone, 48.2% of all strips examined developed tone equivalent to greater than 0.1 g mg⁻¹ of tissue and were subsequently utilized in the present investigation.
3. On exposure to ATP, muscle strips exhibited a biphasic response, a rapid and transient contraction followed by a more prolonged relaxation. Both responses were found to be concentration-dependent. ADP and 2-MeSATP elicited a similar response (contraction followed by relaxation), whereas application of α,β-MeATP only produced a contraction. The potency order for each effect was α,β-MeATP>>2-MeSATP⩾ATP>ADP (contractile response) and ATP=2-MeSATP⩾ADP>>α,β-MeATP (relaxational response).
4. Desensitization with α,β-MeATP (10 μM) abolished the contractile phase of the response to ATP, but had no effect on the level of relaxation evoked by this agonist. On the other hand, the G-protein inactivator, GDPβS (100 μM) abolished only the relaxation response to ATP. Suramin (general P2 antagonist, 100 μM) shifted both the contractile and relaxation ATP concentration-response curves to the right, whereas cibacron blue (P2Y antagonist, 10 μM) only antagonized the relaxation response to ATP. In contrast, the adenosine receptor antagonist, 8-phenyltheophylline (10 μM), had no effect on the relaxation response curve to ATP.
5. Incubation with tetrodotoxin (TTX, 3 μM) or depolarization of the muscle strip with 40 mM K⁺ Krebs failed to abolish the relaxation to ATP. In addition, neither N_ω-nitro-L-arginine (L-NOARG, 10 μM) nor methylene blue (10 μM) had any effect on the relaxation response curve. However, tos-phe-chloromethylketone (TPCK, 3 μM), an inhibitor of cyclicAMP-dependent protein kinase A (PKA), significantly (P<0.01) shifted the curve for the ATP-induced relaxation to the right.
6. It is proposed that marmoset detrusor smooth muscle contains two receptors for ATP, a classical P2X-type receptor mediating smooth muscle contraction, and a P2Y (G-protein linked) receptor mediating smooth muscle relaxation. The results also indicate that the ATP-evoked relaxation may occur through the activation of cyclicAMP-dependent PKA.

     

P2-purinoceptors mediating spasm of the isolated uterus of the non-pregnant guinea-pig.

1. The isolated uterus of the non-pregnant guinea-pig has been suggested to contain P1-, and possibly P2-purinoceptors mediating spasm. The presence of P1-purinoceptors has been confirmed and these receptors have been further characterized. 2. In the presence of the adenosine uptake inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 300 nM) and a pA100 concentration of the P1-purinoceptor antagonist 8-sulphophenyltheophylline (140 microM), the potency order of agonists as spasmogens was: 2 methylthio ATP >> alpha,beta methylene ATP = UTP = ATP >> beta,gamma methylene ATP. This order is not consistent with any single recognised P2-purinoceptor subtype. 3. Indomethacin (1 microM) treatment abolished responses to 2 methylthio ATP, alpha,beta methylene ATP and UTP, while spasm to ATP was significantly inhibited. When the endometrial and circular smooth muscle cell layers were removed, spasmogenic responses to ATP, 2 methylthio ATP, alpha,beta methylene ATP and UTP were significantly reduced. 4. 2-methylthio ATP was able to cause desensitization to itself, but not to UTP, indicating that these agonists act at different receptor sites. 5. The P2-purinoceptor antagonist, suramin antagonized 2 methylthio ATP with a PA2 of 5.9 +/- 0.3. Suramin was also an antagonist of ATP and UTP. In the case of ATP, the antagonism was not dependent on suramin concentration, while for UTP the interaction appeared to be non-equilibrium. Pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS, 10 microM) had no effect on spasm to ATP, UTP or 2 methythio ATP. 6. In the presence of indomethacin, responses to ATP were unaffected by 8-sulphophenyltheophylline (140 microM) or by suramin (100 microM), but PPADS (10 microM) antagonized ATP. 7. These results suggest that the isolated uterus of the non-pregnant guinea-pig contains a mixture of P2-purinoceptors. P2U- (or UTP-selective pyrimidinoceptors) and P2Y-purinoceptors appear to be present, probably mainly located on the endometrial or circular smooth muscle layer. Activation of these receptors leads to spasm via increases in prostanoid generation. There appears also to be a third class of non-P2X-, non p2Y-purinoceptor present, at which ATP is an agonist and PPADS is an antagonist, located on the longitudinal smooth muscle, activation of which causes spasm independent of changes in prostanoids.     

Pharmacological evidence for the 5-HT7 receptor mediating smooth muscle relaxation in canine cerebral arteries.

1. We investigated in the present study whether 5-HT is able to exert direct relaxant responses in canine basilar and middle cerebral arteries via the 5-HT7 receptor. 2. In arterial rings deprived of endothelium and pre-contracted with prostaglandin F2 alpha (2 microM), 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine, sumatriptan or alpha-methyl-5-HT produced further increase in tone and/or slight relaxation. Blockade of 5-HT1B 1D and 5-HT2A receptors with GR127935 (1 microM) and ketanserin (0.1 microM), respectively, antagonized the vasoconstrictor component of the response and unmasked a concentration-dependent relaxation to 5-HT, 5-CT and 5-methoxytryptamine; sumatriptan and alpha-methyl-5-HT remained inactive as relaxant agonists. The rank order of agonist potency in both arteries was 5-CT > 5-HT > 5-methoxytryptamine > sumatriptan > or = alpha-methyl-5-HT. 3. In dog basilar artery, pre-incubated with GR127935 (1 microM) and ketanserin (0.1 microM) and precontracted with prostaglandin F2 alpha (2 microM), the 5-HT7 ligands, clozapine (1 microM), mesulergine (0.3 microM), methiothepin (3 nM), risperidone (3 nM), spiperone (1 microM) and LY215840 (10-100 nM), produced significant rightward shifts of the concentration-response curves for 5-HT and 5-CT. Only methiothepin and risperidone reduced significantly the maximum relaxant response (Emax), whilst the other drugs behaved as competitive antagonists with affinity values (pKB) that significantly correlated with their binding affinity (pKi) at recombinant 5-HT7 receptors. 4. These data disclosing the involvement of the 5-HT7 receptor in cerebrovascular relaxation may be strongly relevant in the light of: (1) the involvement of 5-HT in migraine; (2) the putative linkage between cephalovascular vasodilatation and migraine headache; and (3) the relatively high 5-HT7 receptor affinity of migraine prophylactic 5-HT antagonists.     

Adenosine 5'-triphosphate (ATP) is an excitatory cotransmitter with noradrenaline to the smooth muscle of the rat prostate gland

1. This study investigated whether adenosine 5'-triphosphate (ATP) is involved in neurotransmission to the rat prostate gland. 2. Fluorescence immunohistochemistry carried out on formaldehyde-fixed and frozen sections of rat prostate showed immunoreactivity for the P2X(1)-receptor in the fibromuscular stroma surrounding the secretory acini but not in the glandular epithelium. P2X(2)-, P2X(3)-, P2X(4)- and P2X(7)-receptors were immunonegative in the rat prostate stroma. Double-staining procedures showed P2X(1)-receptor immunoreactivity to be colocalized with alpha-actin immunoreactivity. 3. Isolated organ bath studies investigated whether drugs, which modify purinergic mechanisms, are able to affect contractility of the rat prostate gland. Suramin (100 micro M) and alphabetamethylene ATP (10 micro M) inhibited contractile responses to trains of electrical-field stimulation (70 V, 0.5 ms, 0.1-2 Hz) in the absence and presence of prazosin (300 nM). Responses to 5-20 Hz were unaffected by suramin or alphabetamethylene ATP. 4. Exogenous application of ATP analogues to unstimulated isolated preparations of rat prostate produced concentration-dependent suramin (100 micro M) sensitive transient contractions with a relative order of potency: alphabetamethylene ATP>betagammamethylene ATP>ATP. 5. Adenosine and adenosine 5'-monophosphate (AMP) did not produce contractile responses. 6. These results suggest that P2X(1)-receptors for ATP, which mediate contractions are present in the fibromuscular stroma of the rat prostate. The relative order of potency of ATP analogues in producing contractions of the rat prostate is consistent with the activation of P2X(1)-receptors. Inhibition by suramin and alphabetamethylene ATP of electrically evoked nerve-mediated contractions of the rat prostate implies that ATP contributes to this contractile response and is therefore a cotransmitter with noradrenaline during low-frequency stimulation.     

Actions of non-peptide ergot alkaloids at 5-HT1-like and 5-HT2 receptors mediating vascular smooth muscle contraction

Summary This report describes the actions of the non-peptide ergot alkaloids methysergide, methylergometrine and ergometrine at two types of 5-HT receptor mediating vascular contraction; the well established 5-HT₂ receptor in rabbit aorta and a non-5-HT₂ receptor in rabbit saphenous vein which resembles the 5-HT₁-like receptor in dog saphenous vein.In the rabbit aorta ergometrine (1 mol/l) and methylergometrine (0.3 mol/l), but not methysergide, produced small contractions (14% and 7% respectively of the maximal response to 5-HT). This contraction was not related to activation of 5-HT₂ receptors since it was resistant to blockade by ketanserin (0.3 mol/l). When examined as antagonists of 5-HT-induced contractions of rabbit aorta, each ergot displayed nanomolar affinity at the 5-HT₂ receptor but only methysergide behaved as a simple competitive antagonist (pK_B = 8.25). Methylergometrine and ergometrine produced surmountable blockade which was accompanied by a non-parallel displacement of the 5-HT concentration-effect curves. The selective 5-HT₁-like receptor agonist GR43175 ( 30 mol/l) was devoid of affinity at the 5-HT₂ receptor in rabbit aorta.In the rabbit saphenous vein each of the ergots produced concentration-dependent contractions which resulted in overtly biphasic concentration-effect curves. Only the first phase of contraction mimicked the effects of 5-HT and GR43175 since contractions were not blocked by MDL 72222 (1 mol/l), but were surmountably antagonised by methiothepin (10 nmol/1), ketanserin (0.3 mol/l) and spiperone (0.3 mol/l). These results are expected for interactions at the 5-HT₁-like receptor in this preparation (Martin and MacLennan 1990). The mechanism(s) underlying the second phase of contraction with the ergots remains to be established. Receptor inactivation studies using the alkylating agent benextramine tetrahydrochloride enabled each agonists' affinity and efficacy at the 5-HT₁-like receptor to be estimated. Affinity estimates (pK_A) decreased in the order: methylergo metrine (7.79), ergometrine (7.75), 5-HT (7.19), methysergide (6.76), GR43175 (6.20), whereas efficacies () decreased in the order: 5-HT (3.28), methylergometrine (2.24), GR43175 (2.14), ergometrine (1.94), methysergide (0.99). Of particular interest, methysergide was significantly lower in affinity and efficacy than its primary demethylated metabolite methylergometrine. Evidently, at the 5-HT₁-like receptor mediating vascular contraction the ergots ergometrine and methylergometrine are both higher in affinity than, and comparable in efficacy to, the natural receptor agonist 5-HT. This contrasts with their actions at the 5-HT₂ receptor in rabbit aorta where they demonstrated a higher affinity but much lower intrinsic efficacy than 5-HT. These results favour the view that vascular contraction induced by these ergots is more likely to be mediated by 5-HT₁-like, rather than 5-HT₂ receptors. These results are discussed in relation to the therapeutic applications of these ergots, particularly in obstetrics and in migraine, and to their utility as diagnostic agents in patients with Prinzmetal's variant form of angina.Send offprint requests to S. J. MacLennan at the above address     

Pharmacological characterization of the 5-HT receptors mediating contraction and relaxation of canine isolated proximal stomach smooth muscle

1. We aimed to characterize 5-HT receptors mediating contraction and relaxation to 5-HT in dog proximal stomach longitudinal muscle (LM) strips. 2. Of the tryptamine analogues tested, 5-HT was the most potent contractile agent at basal length, while 5-CT was the most potent relaxant of PGF(2alpha)-induced contraction. Neither the contractions to 5-HT, nor the relaxations to 5-CT were influenced by tetrodotoxin, illustrating that action potential propagation is not involved. 3. The 5-HT-induced contraction was antagonized by mesulergine (0.03 to 0.3 microM) and ketanserin (2 - 20 nM), but the antagonism was not of a simple competitive nature, indicating multiple receptor involvement. Ketanserin (3 to 30 nM) and mesulergine (30 nM) competitively antagonized the alpha-Me-5-HT-induced contraction (pK(B): 8.83+/-0.09 and pA(2): 8.25+/-0.06 respectively). These affinity values are in line with literature affinities of ketanserin and mesulergine at 5-HT(2A) receptors in various bioassays. 4. The 5-CT-induced inhibition of PGF(2alpha)-induced contraction was competitively antagonized by mesulergine (pK(B) estimate: 8.52+/-0.12) and by the selective 5-HT(7) receptor antagonist SB-269970 (pK(B) estimate: 9.36+/-0.14). Both pK(B) estimates are in line with literature affinities of these compounds for 5-HT(7) receptors. Mesulergine (30 nM) and SB-269970 (10 nM) shifted the relaxant curve to 5-HT parallel to the right in the presence of ketanserin (0.3 microM) (pA(2) estimates of 8.08+/-0.10 and 8.75+/-0.14 respectively), indicative of 5-HT(7) receptor involvement. 5. It is concluded that 5-HT induces dog proximal stomach (LM) contraction via smooth muscle 5-HT(2A) receptors and relaxation via smooth muscle 5-HT(7) receptors.     

Neocuproine,a selective Cu(I) chelator,and the relaxation of rat vascular smooth muscle by S-nitrosothiols

1. A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and S-nitroso-glutathione (GSNO).
2. Bolus injections (10 μl) of SNAP or GSNO (10⁻⁷–10⁻³ M) were delivered into the lumen of perfused vessels pre-contracted with sufficient phenylephrine (1–7 μM) to develop pressures of 100–120 mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre-mixed with neocuproine (10⁻⁴ M) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10⁻⁵ M) and then injected with either pure SNAP or GSNO.
3. In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)-dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.

     

Characterization of P2-purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses.

1. The electrophysiological actions of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), 2-methylthioATP (2-meSATP), alpha, beta-methyleneATP (alpha, beta-meATP) and uridine 5'-triphosphate (UTP) were studied under concentration and voltage-clamp conditions in acutely dissociated rat tail artery smooth muscle cells. For comparison, their actions as vasoconstrictors were studied in intact ring preparations. 2. Rapid application of ATP (100 nM-1 microM) via a U-tube superfusion system activated concentration-dependent inward currents with a latency to onset of less than 3 ms. The inward current decayed by more than 95% during a 2 s application of 300 nM and 1 microM ATP. 3. 2-meSATP (100 mM-1 microM) and alpha, beta-meATP (100 nM-1 microM) also evoked transient inward currents. The agonist order of potency was ATP = 2-meSATP > or = alpha, beta-meATP. UTP (300 nM-1 microM) did not produce a change in the holding current. 4. A second application of ATP (300 nM and 1 microM) 10 min after the first, evoked currents which were one third of the initial amplitude. This decline was dependent upon activation of the P2-purinoceptor. Similar results were seen with 2-meSATP and alpha, beta-meATP (both 300 nM and 1 microM). Cross-desensitization was seen between ATP and 2-meSATP or alpha, beta-meATP. 5. Inward currents evoked by ATP, 2-meSATP and alpha, beta-meATP (all 1 microM) were abolished by the P2-purinoceptor antagonist suramin (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)