Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with non-adrenergic, non-cholinergic nerve stimulation and ATP.

Substance P, somatostatin, enkephalin and vasoactive intestinal polypeptide (VIP) did not mimic the inhibitory responses to non-adrenergic, non-cholinergic nerve stimulation. Substance P (0.1-10 microgram/ml) always caused contraction, enkephalin (0.1-10 microgram/ml) and somatostatin (0.1 microgram/ml) were inactive, while VIP (0.1-1 microgram/ml) produced very slow relaxation, taking about 4 min to reach a maximum after a latency of about 60 sec. Low concentrations of neurotensin (1-10 ng/mg) caused contraction, but at higher concentrations (50-1000 ng/ml) it produced a biphasic response which consisted of an initial contraction followed by a slow relaxation. In high tone preparations, the slow relaxation did not mimic the nerve-mediated response, taking approximately 43 sec. to reach maximum, after a long latency of about 15 sec. In contrast, ATP (0.1-50 microgram/ml) mimicked closely the rapid responses to non-adrenergic, non-cholinergic nerve stimulation in all preparations, whether the tone was low, medium or high. The time for the inhibitory response to reach maximum was about 15 sec after a latency of approximately 1 sec. Indomethacin (3.4-34 microgram/ml) did not unmask any inhibitory responses to any of the peptides. It is concluded that ATP remains the most likely substance to be the inhibitory transmitter released from non-adrenergic, non-cholinergic nerves supplying the smooth muscle of the taenia coli.     

Potentiation of responses to sympathetic nerve stimulation and vasoconstrictor agents by SK&F 103829 in the feline mesenteric circulation.

1. The amplification of vasoconstrictor effects of several agonists and sympathetic nerve stimulation, caused by 5-HT2 receptor activation, was studied in the autoperfused mesenteric circulation of anaesthetized cats. To produce long lasting and selective 5-HT2 receptor stimulation we used SK&F 103829 (2,3,4,5 tetrahydro-8[methyl-sulphonyl]-1H3-benzazepin-7-ol methensulphonate). We assessed that SK&F 103829 was a strong contractile partial agonist in isolated preparations of rat tail artery and calf pulmonary artery. 2. The intrinsic activity of SK&F 103829 with respect to 5-hydroxytryptamine (5-HT) was 0.8 in rat tail artery and 0.6 in calf pulmonary artery. SK&F 103829-induced contractile responses were surmountably antagonized by ketanserin with a potency expected from its affinity for 5-HT2 receptors. SK&F 103829 surmountably antagonized the effects of 5-HT in rat tail artery with a pKp of 5.8. 3. Concentrations of SK&F 103829 causing greater than threshold constrictions enhanced vasoconstrictor responses of sympathetic nerve stimulation, noradrenaline, angiotensin II, methoxamine and alpha, beta-methylene ATP in the mesenteric arterial bed. Increases in mesenteric arterial pressure by noradrenaline, observed in the presence of prazosin, were also potentiated by SK&F 103829. 4. Ketanserin prevented both the constrictor effect of SK&F 103829 and the SK&F 103829-evoked potentiation of the responses to noradrenaline and angiotensin II in the mesenteric arterial bed. Ketanserin, however, failed to abolish (once established) the SK&F 103829-evoked potentiation of the constrictor effects caused by both noradrenaline and angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of alpha, beta-methylene ATP on electrical responses produced by ATP and nerve stimulation in smooth muscle cells of the guinea-pig mesenteric artery

1. In smooth muscle cells of the guinea-pig mesenteric artery, alpha, beta-methylene ATP(mATP) depolarized the membrane and decreased the membrane resistance. 2. Desensitization with mATP inhibited the excitatory junction potential (e.j.p.) and ATP-induced depolarization, while desensitization with ATP inhibited the e.j.p. but not the mATP-induced depolarization. 3. After desensitization with mATP, the response to mATP recovered rapidly in comparison with the e.j.p. and the ATP-induced depolarization. 4. The results suggest that mATP inhibits the ATP-induced depolarization by acting sites other than the ATP-receptor.     

Adrenergic and purinergic neurotransmission in arterial resistance vessels of the cat intestinal circulation

The contribution of adrenoceptors and purine receptors in mediating neurogenic vasoconstriction was investigated in the autoperfused intestinal circulation of anaesthetised cats treated with atropine and propranolol. Prazosin (0.5 mg/kg) and yohimbine (1.5 mg/kg) reduced but did not abolish the vasoconstrictor responses to stimulation of the efferent sympathetic nerves. The inhibitory actions of the two antagonists were additive but even after alpha 1- and alpha 2-adrenoceptor blockade nerve stimulation still elicited a residual, frequency-related vasoconstriction. The initial, rapid, phase of this response was completely abolished after desensitisation of P2x-purinoceptors with a high dose (1.5 mg i.a.) of alpha,beta-methylene ATP. In the absence of alpha-adrenoceptor antagonists, alpha,beta-methylene ATP reduced neurogenic vasoconstriction particularly at low frequency (1 Hz) nerve stimulation, but also caused a short-lasting decrease in noradrenaline and methoxamine responses which indicates that the drug may have some non-specific effects on arterial smooth muscle. The results suggest that neurotransmission in arterial resistance vessels of the cat intestinal circulation is predominantly under adrenergic control mediated by postsynaptic alpha 1- and alpha 2-adrenoceptors, with a possible purine involvement in the initial rapid response of the blood vessels, particularly to low frequency nerve stimulation.     

Effects of alpha,beta-methylene ATP on potentiation of contractions to field stimulation of rat vas deferens by carbachol

Carbachol (0.1-300 mumol/L) potentiated contractions to field stimulation (0.1 Hz, 1 ms, supramaximal V) in the rat epididymal and prostatic vas deferens. Desensitization of P2-purinoceptors by exposure to alpha,beta-methylene ATP (30 mumol/L) markedly reduced (greater than 80%) the potentiating effect of carbachol in the prostatic vas deferens but only moderately reduced (about 20%) the maximal stimulated response to carbachol in the epididymal segment. The presence of prazosin (10 mumol/L) and yohimbine (10 mumol/L), being selective alpha 1- and alpha 2-adrenoceptor antagonists, did not modify the attenuation of carbachol potentiation caused by alpha,beta-methylene ATP treatment. At 0.1 mmol/L, alpha,beta-methylene ATP had no significant effect on the binding of [3H]QNB to muscarinic cholinergic receptors. It is concluded that carbachol may potentiate the contractions to field stimulation in the prostatic vas deferens via an enhancement of purinergic neurotransmission. The molecular mechanism of carbachol potentiation in the epididymal vas deferens remains to be established.     

Dual contractile effects of ATP released by field stimulation revealed by effects of alpha,beta-methylene ATP and suramin in rat tail artery.

1. The field stimulation-induced release of endogenous ATP and noradrenaline (NA) and contractile response in rat isolated tail artery were examined. The release of ATP was studied by extracellular electrophysiological recording and that of NA by a novel voltammetrical technique. The effects of the P2-purinceptor antagonist, suramin, on these parameters were compared with those of alpha,beta-methylene ATP, a P2X-purinoceptor desensitizing agent. 2. Neither alpha,beta-methylene ATP (10 microM) nor suramin (100-500 microM) had significant effects on the extracellularly recorded nerve terminal action potential but both abolished the ATP-induced excitatory junction current caused by stimulation at 0.1 Hz. Neither agent affected significantly the voltammetrically measured release of NA induced by 10 or 100 pulses at 20 Hz. 3. Combined blockade of both postjunctional alpha 1- and alpha 2-adrenoceptors by prazosin and yohimbine (both 0.1 microM) profoundly depressed the contractile response to 10 pulses at 20 Hz. The small and fast residual contraction in the presence of these agents was abolished by alpha,beta-methylene ATP (10 microM) and inhibited by suramin in a concentration-dependent manner (10-500 microM; IC50 75 microM) and was hence probably caused by ATP or a related nucleotide. 4. When added first, alpha,beta-methylene ATP (10 microM) or suramin (100-500 microM) delayed the onset and enhanced the amplitude of the neurogenic contraction. This enhanced response was abolished by further addition of prazosin and yohimbine (both 0.1 microM). 5. The K+ channel blocker, tetraethylammonium (10 mM), dramatically enhanced the contractile response to 100 pulses at 1 Hz and caused it to become diphasic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of human urotensin II in isolated vessels of various species; comparison with other vasoactive agents

Urotensin II is a cyclic undecapeptide which activates the GPR14 receptor and exerts potent vasoconstrictor effects in some species of fish and mammals.The present study intended to investigate isolated vessels from various species in an attempt to find sensitive preparations to be used in studies of the human urotensin (hU-II)/GPR14 system.Contractile responses evoked by noradrenaline (NA), angiotensin II (Ang II), endothelin 1 (ET-1) and hU-II were measured in large vessels (aorta and some large arteries and veins) of rats, guinea pigs, rabbits, pigs and humans. Relaxing effects of hU-II, bradykinin (BK) and substance P (SP) were measured in pig coronary arteries contracted with KCl 30 mM. The rat mesenteric vasculature was investigated from the arterial and venous site to establish the function of ET-1 and hU-II receptors. Results indicate that the only preparation showing high sensitivity to hU-II (pEC(50)=8.27) is the rat aorta, whose contractions in response to hU-II develop slowly and persist for hours, similar to those of ET-1 (pEC(50)=8.35). Effects of NA (pEC(50)=8.12) and Ang II (pEC(50)=7.95) develop and reverse more rapidly. Tissues treated with ET-1 and hU-II show marked desensitization, in contrast to those treated with NA. Specific antagonists for alpha(1) (prazosin, p A(2)=10.46), AT(1) (EXP 3174, p A(2)=10.20), 5HT(2) (ketanserine, p A(2)=8.61) and ET(A)-ET(B) (bosentan, p A(2)=6.88) receptors were shown to block the effects of the respective agonists, while being inactive against hU-II. In some vessels, hU-II behaved as an highly potent but scarcely effective contractile agent. It is concluded that: the hU-II/GPR14 is not a functional contractile system in vessels of several species, in contrast with NA/alpha(1), Ang II/AT(1), 5HT/5HT(2) and ET-1/ET(A)-ET(B). The rat aorta appears however to be a sensitive and reliable preparation for evaluating biological activities of hU-II and related peptides.     

兔离体隐动脉交感神经共同传递：电刺激的影响及α，β—亚甲基ATP的增强作用

目的：分析亚最大电压、超短时程电场刺激诱发兔离体隐动脉收缩反应的神经共同传递特征。方法：兔离体隐动脉环等长收缩测定法及电场刺激诱发交感神经源性血管收缩法。结果：电场刺激诱发隐动脉交感神经源性血管收缩反应,具有频率依赖性。α1－受体阻断剂哌唑嗪（1μmol/L）不影响2Hz电刺激诱发的血管收缩反应,对8－16Hz电刺激诱发的血管收缩反应抑制约39．9－53．8％。另一方面,以α,β-亚甲基ATP（3μmol/L）脱敏P2X1受体后,完全抑制2Hz电刺激诱发的血管收缩反应,显著增强16Hz电刺激诱发 血管收缩反应,却不影响外源性NE的累积量效曲线。联合应用哌唑嗪（1μmol/L）和α,β－亚甲基ATP（3μmol/L）完全抑制了各频率电刺激诱发的血管收缩反应。结论：2Hz电刺激亦可诱发兔离体隐动脉神经源性收缩反应,2Hz电刺激诱发的收缩反应仅与嘌呤能神经递质ATP或相关核苷酸有关。较高频率电刺激诱发的收缩反应则与ATP和NE两种递质有关。此外,α,β-亚甲基ATP使P2X1受体脱敏,并通过突触前机制增强电刺激诱发的血管收缩反应。     

Effects of acute administration of and tachyphylaxis to alpha,beta-methylene ATP in the guinea-pig small intestine

The aim of the present study was to assess the acute motility effects and desensitizing activity of the stable ATP analogue and P(2X) purinoceptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) and the effect of alpha,beta-meATP desensitization on nerve-mediated cholinergic responses in the guinea-pig ileum in vitro. It was confirmed that alpha,beta-meATP (1-30 microM) causes neurally-mediated, cholinergic (tetrodotoxin- and atropine-sensitive) longitudinal contractions. These responses were not influenced by the ganglionic blocking drug hexamethonium (50 microM), or a combination of the adrenergic neurone blocking drug guanethidine (3 microM), the opioid receptor antagonist naloxone (0.5 microM) and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM), but were strongly reduced or abolished by the P2 purinoceptor antagonist PPADS (30 microM) or by tachyphylaxis evoked by 10 microM alpha,beta-meATP. The contractile effect of alpha,beta-meATP (3 microM) was moderately inhibited by 10 microM and strongly suppressed by 30 microM of NF 279, an antagonist predominantly affecting P2X1 purinoceptors, but left uninfluenced by the P2X(5,7) receptor antagonist Brilliant blue G. No relaxant effect of alpha,beta-meATP was detected in the concentration range of 1-30 microM. Tachyphylaxis to alpha,beta-meATP (1-10 microM) caused a moderate inhibition of the cholinergic (atropine-sensitive) contractile response of the ileum to electrical field stimulation (5 Hz for 5 sec.). This reduction was unaltered in the presence of guanethidine, naloxone and L-NOARG. Responses to nicotine (1 or 2 microM) were not reduced by alpha,beta-meATP tachyphylaxis. It is suggested that alpha,beta-meATP-sensitive P(2X) purinoceptors are involved in the prejunctional modulation of cholinergic neurotransmission between the myenteric plexus and longitudinal smooth muscle in the guinea-pig small intestine.     

Effects of calcium antagonists on myogenic and neurogenic control of resistance and capacitance vessels in cat skeletal muscle

The effects of five different calcium antagonists (diltiazem, felodipine, nifedipine, nimodipine, and verapamil) on cat skeletal muscle resistance and capacitance vessels were studied in a whole organ preparation. These calcium antagonists seemed to have the similar qualitative effects on these vascular functions. Calcium antagonists were found to be potent inhibitors of myogenic vascular reactivity (here defined as the maximal increase in flow resistance evoked by a sudden rise of transmural pressure). Basal vascular tone and vascular tone induced by low frequency stimulation of sympathetic nerves were both less sensitive to these drugs than vascular tone induced by myogenic vascular reactivity. Sympathetically mediated vascular tone at high stimulation frequencies seemed to be least sensitive. Further, resistance vessels were much more sensitive to these drugs than capacitance vessels. Finally, basal tone in the large bore arterioles were more sensitive than in the small bore arterioles, a surprising finding which was interpreted with the aid of computer simulations using a mathematical model of local vascular control in cat skeletal muscle. The model suggested that this difference could be due to a delicate interaction between myogenic vascular reactivity and metabolic vascular control. It is suggested that the inhibition of myogenic vascular reactivity is a factor contributing to the edema formation of calcium antagonists.     

Actions of alpha, beta-methylene ATP and 6-hydroxydopamine on sympathetic neurotransmission in the vas deferens of the guinea-pig, rat and mouse: support for cotransmission.

alpha-Adrenoceptor antagonists (prazosin or phentolamine) reduced the contractile response to field stimulation of the isolated vasa deferentia of guinea-pig, rat and mouse. alpha, beta-Methylene ATP (alpha, beta-MeATP) reduced that portion of the contraction which was resistant to alpha-adrenoceptor blockade. alpha, beta-MeATP (1-800 microM) did not affect action potential conduction in the guinea-pig vas deferens nerves, and (up to 10 microM) did not reduce the stimulation-evoked overflow of [3H]-noradrenaline from this tissue. Spontaneous excitatory junction potentials (s.e.j.ps) in the majority of cells of guinea-pig, rat, and mouse vasa were abolished by alpha, beta-MeATP (0.1-10 microM). In a small number of cells s.e.j.ps were resistant to the actions of alpha, beta-MeATP (10 microM). Excitatory junction potentials (e.j.ps) in the majority of cells in vasa of all species studied were abolished by alpha, beta-MeATP (1-10 microM). E.j.ps elicited in some 'resistant' cells demonstrated marked facilitation characteristics. It is concluded that alpha, beta-MeATP inhibits s.e.j.ps and e.j.ps by a postjunctional action. In all species pretreatment of animals with 6-hydroxydopamine produced a marked reduction in noradrenaline (NA) content (as determined by fluorescence histochemistry) and abolished e.j.ps, findings which suggest that e.j.ps originated from sympathetic nerves. The results support the hypothesis that NA and ATP are co-transmitters in the sympathetic nerves of rodent vasa.     

Effects of prolonged exposure to alpha,beta-methylene ATP on non-adrenergic, non-cholinergic excitatory transmission in the rectum of the chicken.

1. Effects of prolonged exposure to alpha,beta-methylene ATP (alpha,beta-Me ATP) on contractions and excitatory junction potentials (e.j.ps) evoked by non-adrenergic, non-cholinergic (NANC) excitatory nerve stimulation have been investigated in the chicken isolated rectum and longitudinal muscle strip from chicken rectum pretreated with atropine (0.5 microM), methysergide (2 microM) and pyrilamine (3 microM). 2. Alpha,beta-Me ATP (20 nM-4 microM) caused a rapid rise in tension of the longitudinal muscle of the isolated rectum preparation which returned to the baseline levels after a few minutes. The magnitude of the contractile response to NANC nerve stimulation was reduced after exposure to the drug. The inhibitory effect was related to the drug concentration; at 4 microM the nerve-mediated contraction was abolished and frequently converted to a relaxation. 3. Adenosine 5'-triphosphate (ATP, 100 microM), bovine neurotensin (2.5 nM) and K+-rich solutions (30 nM and 60 nM) all produced a transient contraction of the isolated rectum preparation. The exposure to alpha,beta-Me ATP (0.2 and 4 microM) also rendered the preparation less sensitive to these stimulant substances. 4. Alpha,beta-Me ATP (0.2 and 4 microM) caused a membrane depolarization in cells of the longitudinal muscle strip. The depolarization reached a peak within 2-3 min after application and then decayed to a steady level that was still more positive than the baseline level. The electrotonic potentials were reduced in amplitude to 44 +/- 8% (n = 7) of the normal amplitude if measured at the peak depolarization produced with 0.2 microM alpha,beta-Me ATP, and to 62 +/- 10% (n = 7) if measured at the steady-state depolarization. With 4 microM, the corresponding percentages were 33 +/- 7% (n = 8) and 55 +/- 7% (n = 8), indicating a decrease in membrane resistance. 5. The e.j.ps in response to field stimulation of the intramural nerves and Remak's nerve stimulation were decreased in amplitude and duration during exposure to alpha,beta-Me ATP (0.2 and 4 microM). 6. The smooth muscle cells regained normal membrane resistance and sensitivity to ATP on washout of alpha,beta-Me ATP (4 microM) more rapidly than the responses to NANC nerve stimulation. 7. It can be argued from the results that the suppression by alpha,beta-Me ATP of the contraction and e.j.p. evoked by NANC nerve stimulation in the chicken rectum, unlike the mammalian preparation described previously, is due mainly to a change in the electrical properties of the membrane of the smooth muscle cells, rather than being due, or only partly due, to desensitization of the purine receptor.     

Actions of ATP and alpha, beta-methylene ATP on neuromuscular transmission and smooth muscle membrane of the rabbit and guinea-pig mesenteric arteries.

In the rabbit mesenteric artery, adenosine triphosphate (ATP), showed two actions on the membrane potential of muscle cells: low concentrations (1-10 microM) hyperpolarized and high concentrations (greater than or equal to 50 microM) depolarized the membrane. Both changes in the potential were accompanied by increases in ionic conductance. In the rabbit mesenteric artery, alpha, beta-methylene ATP (MeATP), (greater than or equal to 30 nM) depolarized the muscle membrane at a lower concentration than ATP (greater than or equal to 50 microM), and increased the ionic conductance of the membrane. The depolarization induced by ATP was prevented by low concentrations of MeATP, but the hyperpolarization was retained. Furthermore, the hyperpolarization was not affected by theophylline (10 microM). In the guinea-pig mesenteric artery, ATP and MeATP depolarized and increased the ionic conductance of muscle membrane, but to depolarize the membrane, higher concentrations of both agents were required, compared to those in the rabbit mesenteric artery. In the mesenteric arteries from both species, perivascular nerve stimulation evoked excitatory junction potentials (e.j.ps). In both tissues, MeATP inhibited the amplitude of e.j.ps at lower concentrations than did ATP, and both agents had more potent inhibitory actions on rabbit than on guinea-pig. The inhibition of e.j.p. induced by low concentrations of these agents showed no relationship to depolarization, but the inhibition induced by high concentrations was paralleled by depolarization and increase in ionic conductance of the membrane. In the rabbit mesenteric artery, overflow of noradrenaline (NA) and its metabolite (3,4-dihydroxyphenylglycol; DOPEG) produced by perivascular nerve stimulation was examined. ATP (0.1 mM) but not MeATP (0.1 microM) reduced the overflow of NA, whereas both agents had no effect on the overflow of DOPEG. Exogenously applied high concentrations of NA (greater than or equal to 3 microM) depolarized the muscle membrane in both species. These NA-induced depolarizations were not affected by treatment with ATP or MeATP. It is concluded that, in the rabbit mesenteric artery, ATP is more likely to be involved in generation of e.j.ps than is NA. A similar interpretation in the guinea-pig mesenteric artery is complicated by the depolarization produced by high concentrations of ATP or MeATP.     

Contractile responses of smooth muscle strips from rat and guinea-pig urinary bladder to transmural stimulation: effects of atropine and alpha,beta-methylene ATP.

1. Strength-duration curves for threshold mechanical responses to single transmural stimuli were identical for rat and guinea-pig detrusor. In both species atropine had no effect on the curves, but the curves were shifted to the right by nerve blockade with tetrodotoxin (TTX), and by blockade of P2-purinoceptors with alpha,beta-methylene ATP (alpha,beta-MeATP). 2. With short duration pulses of 50 V and less, the responses were nerve-mediated. Increase in either the strength or duration of the stimulus caused direct muscle stimulation, resistant to blockade with atropine, TTX and alpha-beta-MeATP. 3. The shape of the contractile response to a single nerve stimulus varied from tissue to tissue. The responses could be mono-, bi-, or multiphasic. Bi- or multiphasic responses were normally seen in tissues which were spontaneously active. The multiphasic nature of the response was enhanced by factors which increased the excitability of the cells and was reduced by factors which decreased the excitability. 4. The frequency-response curves in the rat are similar to those previously obtained in the guinea-pig. Atropine suppresses the high frequency response by 25%, with little effect at low frequencies, whereas desensitization of P2-purinoceptors with alpha,beta-MeATP suppresses the responses maximally at low frequencies but still by 75% at high frequencies. A combination of both drugs eliminates the nerve-mediated responses. 5. It is concluded that the response to a single nerve stimulus is mediated by a non-cholinergic transmitter, through activation of P2-purinoceptors. The possibility that simultaneous release of acetylcholine can modify the excitability of cells and thus the configuration of the response to a single stimulus is discussed.     

Effects of reduced calcium ion concentration and of diltiazem on vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in rat isolated tail artery.

In isolated, perfused proximal segments of Sprague-Dawley rat tail artery, idazoxan (100 nmol l-1) displaced the concentration-response curve to noradrenaline (NA) to the right. The log shift of the NA concentration-response curve was greater at lower concentrations than at higher concentrations of NA. Idazoxan (100 nmol l-1) had no effect on responses to electrical stimulation. Prazosin (10 nmol l-1) displaced the concentration-response curve to NA to the right as well as markedly reducing responses to sympathetic nerve stimulation. The concentration-response curve to NA, obtained after reducing the concentration of calcium ions in the Krebs solution from 2.5 to 0.6 mmol l-1, was significantly displaced to the right. Responses to sympathetic nerve stimulation were not affected by this reduction in the concentration of calcium ions. Diltiazem (1 and 10 mumol l-1) significantly displaced the concentration-response curve to NA to the right but had no effect on sympathetic nerve stimulation. These in vitro results in peripheral arterial smooth muscle confirm the findings of previous in vivo studies which suggest that alpha2-adrenoceptors contribute to the vasoconstrictor responses elicited by alpha-adrenoceptor agonists and that these responses but not those mediated by alpha1-adrenoceptors are dependent on extracellular calcium.