P1-purinoceptors mediate relaxation of the bovine bronchial artery

Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine all relaxed the bronchial artery. All four purines tested were more efficacious than isoproterenol. Dipyridamole (10(-6) M) enhanced the relaxations due to adenosine and ATP while theophylline (10(-6) M) inhibited the relaxations due to adenosine and ATP. The results suggest the presence of P1-type purinoceptors in the bronchial artery of cattle.     

ATP mediates coronary vasoconstriction via P2x-purinoceptors and coronary vasodilatation via P2y-purinoceptors in the isolated perfused rat heart

The effect of ATP and its analogues on the perfusion pressure of the rat coronary vasculature and the left ventricular pressure of the isolated Langendorff perfused rat heart was examined. The response to ATP was generally biphasic, causing an increase followed by a decrease in perfusion pressure. The rank order of potency of the analogues for eliciting the vasoconstriction component was alpha,beta-methyleneATP greater than 2-methylthioATP greater than ATP, which resembles the pattern previously observed for the P2x-purinoceptor. In causing vasodilation, the rank order of antagonist potency was 2-methylthioATP greater than ATP, with alpha,beta-methyleneATP being without effect; this is a characteristic of P2y-purinoceptors. 1,3-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) antagonised some of the vasodilatory effects of ATP, showing that some of the effect is due to breakdown to adenosine. Reactive blue 2, a putative P2y-purinoceptor antagonist was found to be five times more effective at antagonising the vasodilatory responses to 2-methylthioATP than to adenosine or the vasoconstriction responses to alpha,beta-methyleneATP. We suggest that ATP exerts its biphasic effects in the coronary vasculature of the rat by interacting with P2x- and P2y-purinoceptors and partly via P1-purinoceptors after breakdown to adenosine. Reactive blue 2 was more effective at antagonising responses mediated via P2y-purinoceptors than by P2x- or P1-purinoceptors.     

Histamine-induced vasodilation and vasoconstriction in the mesenteric resistance artery of the rat

The present study was designed to examine the vascular response to histamine in rat perfused mesenteric vascular beds with active tone. In preparations with intact endothelium, perfusion of histamine (1 nM-100 microM) produced a concentration-dependent vasodilation. Histamine-induced vasodilation was attenuated by L-NAME (nitric oxide (NO) synthase inhibitor, 100 microM) and olopatadine (histamine H(1) receptor antagonist, 1 microM) but not by lafutidine (histamine H(2) receptor antagonist, 1 microM). Cold-storage denervation (4 degrees C for 72 h) of the preparation with intact endothelium attenuated the histamine-induced vasodilation. In preparations without endothelium, histamine at low concentrations (1-100 nM) produced only a small and rapid vasodilation, whereas histamine at concentrations higher than 1 muM produced triphasic vascular responses: initial sharp vasodilation followed by transient vasoconstriction and subsequent gradual vasodilation. Lafutidine abolished only the histamine-induced initial vasodilation. Olopatadine abolished the histamine-induced second vasoconstriction and third vasodilation. Cold-storage denervation of the denuded preparation abolished the histamine-induced second vasoconstriction and third vasodilation. These findings suggest that histamine induced endothelium-dependent vasodilation via endothelium histamine H(1) receptors and endothelium-independent vasodilation via smooth muscle histamine H(2) receptors. It is also suggested that the histamine-induced endothelium-independent vasoconstriction and vasodilation are mediated by histamine H(1) receptors and perivascular nerves.     

Epoxyeicosatrienoic acids mediate adenosine-induced vasodilation in rat preglomerular microvessels (PGMV) via A2A receptors

Activation of rat adenosine2A receptors (A2A R) dilates preglomerular microvessels (PGMV), an effect mediated by epoxyeicosatrienoic acids (EETs). Incubation of PGMV with a selective A2A R agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 microM), increased isolated PGMV EET levels to 7.57+/-1.53 ng mg-1 protein from 1.06+/-0.22 ng mg-1 protein in controls (P<0.05), without affecting hydroxyeicosatetraenoic acid (HETE) levels (10.8+/-0.69 vs 11.02+/-0.74 ng mg-1 protein). CGS 21680-stimulated EETs was abolished by preincubation with an A2A R antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385) (100 microM). A selective epoxygenase inhibitor, methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; 12 microM) prevented CGS 21680-induced increase in EETs, indicating inhibition of de novo synthesis of EETs. In pressurized (80 mmHg) renal arcuate arteries (110-130 microm) preconstricted with phenylephrine (20 nM), superfusion with CGS 21680 (0.01-10 microM) increased the internal diameter (i.d.) concentration-dependently; vasodilation was independent of nitric oxide and cyclooxygenase activity. CGS 21680 (10 microM) increased i.d. by 32+/-6 microm; vasodilation was prevented by inhibition of EET synthesis with MS-PPOH. Addition of 3 nM 5,6-EET, 8,9-EET and 11,12-EET increased i.d. by 53+/-9, 17+/-4 and 53+/-5 microm, respectively, whereas 14,15-EET was inactive. The responses to 5,6-EET were, however, significantly inhibited by indomethacin. We conclude that 11,12-EET is the likely mediator of A2A R-induced dilation of rat PGMV. Activation of A2A R coupled to de novo EET stimulation may represent an important mechanism in regulating preglomerular microvascular tone.British Journal of Pharmacology (2004) 141, 441-448. doi:10.1038/sj.bjp.0705640     

alpha 2-Adrenoceptors mediate sympathetic vasoconstriction in distal segments of rat tail artery

Proximal and distal segments of the tail artery were taken from normotensive Sprague-Dawley rats. Field stimulation (0.3-30 Hz) of periarterial sympathetic nerves elicited vasoconstrictor responses which were antagonized by prazosin (0.1-10 nM) to a much lesser extent in distal than in proximal segments. The selective alpha 2-adrenoceptor antagonist idazoxan (100 nM) alone had little effect in either segment; however in combination with prazosin vasoconstrictor responses were markedly reduced in distal segments. It is concluded that postjunctional alpha 2-adrenoceptors substantially mediate sympathetic vasoconstriction in distal segments of Sprague-Dawley rat tail artery and that this in vitro preparation may be a useful model for elucidating and extending data obtained in vivo in the human forearm circulation (Van Brummelen et al., 1983).     

P2-purinoceptors regulate surfactant secretion from rat isolated alveolar type II cells.

Rat isolated alveolar Type II cells were utilized to examine the effect of purine and pyrimidine analogues on secretion of pulmonary surfactant. ATP potently stimulated [3H]-phosphatidylcholine ([3H]-PC) secretion in a time- and dose-dependent manner. The effect of ATP was noted by one hour of exposure and persisted for three hours. The EC50 (concentration producing 1/2 the maximal response) for ATP-induced [3H]-PC secretion was 100 nM. ADP was also a potent secretagogue for surfactant secretion, but AMP and adenosine had no significant effect on surfactant secretion at concentrations less than or equal to 250 microM. The EC50 for ADP-induced [3H]-PC secretion was 250 nM. Other purine and pyrimidine nucleotides (ITP, GTP, CTP, TTP) were examined for their effect on [3H]-PC secretion. All purine and pyrimidine triphosphates examined significantly augmented [3H]-PC secretion, but were much less potent than ATP. The EC50s were ITP = 10 microM; GTP = 100 microM; CTP = 250 microM; TTP = 100 microM. Neither 8-phenyltheophylline (10 microM, a P1-purinoceptor antagonist), propranolol (100 microM, a beta-adrenoceptor antagonist), nor indomethacin (10 microM, a prostaglandin synthetase inhibitor) inhibited ATP-induced [3H]-PC secretion from isolated Type II cells. These data provide evidence for regulation of surfactant secretion from alveolar Type II cells by a P2-purinoceptor.     

Ontogeny of P2-purinoceptors in the longitudinal muscle and muscularis mucosae of the rat isolated duodenum

1. The ontogeny of P2-purinoceptors in the longitudinal muscle and the muscularis mucosae of the rat isolated duodenum was investigated by use of functional assays in tissues from neonatal animals. The degradation of purinoceptor agonists by the rat duodenum muscularis mucosae was also investigated.
2. In the rat duodenum muscularis mucosae adenosine 5′-(α,β-methylene)triphosphonate (AMPCPP), adenosine 5′-triphosphate (ATP), uridine 5′-triphosphate (UTP) and 2-methylthioadenosine 5′-triphosphate (2-Me-S-ATP) all caused a contraction from day 10 to day 40, day 10 being the earliest age it could be tested. The potency order of agonists above day 25 was AMPCPP>ATP=UTP>2-Me-S-ATP and this is similar to the potency order previously obtained for the adult tissue. However, in the neonatal tissues below day 20, 2-Me-S-ATP was the most potent agonist and at days 10 and 15 the order was 2-Me-S-ATP>AMPCPP>ATP=UTP.
3. In the rat duodenum muscularis mucosae desensitization was observed with AMPCPP at day 30 but not at day 15. At day 30, cross-desensitization was also observed between AMPCPP and 2-Me-S-ATP but not between AMPCPP and ATP or UTP, whereas no cross-desensitization was observed at day 15 with AMPCPP and any of the agonists. At day 15 and below AMPCPP and 2-Me-S-ATP may therefore both activate P2Y-receptors (2-Me-S-ATP>AMPCPP, no desensitization with AMPCPP) whereas above day 20 the agonists activate P2X-receptors (AMPCPP>2-Me-S-ATP, desensitization with AMPCPP) which is similar to the adult tissue. Since ATP and UTP were equipotent in the muscularis mucosae and as no cross-desensitization was observed with AMPCPP and UTP or ATP at days 15 or 30, it is likely that ATP and UTP both activate P2U-receptors throughout the ages, as in the adult.
4. The potency of all the agonists in causing contraction in the rat duodenum muscularis mucosae decreased with age. The potency of AMPCPP and 2-Me-S-ATP in causing contractions was highest in the neonates before day 25, and reached values not significantly different from adult by day 30, and the potency of ATP and UTP causing contractions in this tissue was also highest in the neonates at days 10 and 15, and reached values not significantly different from adult by day 20. This suggests either that the receptor populations mediating contraction are highest in the neonates below day 20 or that the agonists are degraded by the muscularis mucosae to a greater extent after day 20.
5. In the rat duodenum muscularis mucosae the degradation of ATP, UTP, 2-Me-S-ATP and AMPCPP was followed by high pressure liquid chromatography at days 15 and 30. ATP was degraded to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP) and inosine with no adenosine being detected, 2-Me-S-ATP was degraded to 2-methylthioadenosine 5′-diphosphate (2-Me-S-ADP), 2-methylthioadenosine 5′-monophosphate (2-Me-S-AMP) and 2-methylthioadenosine (2-Me-S-adenosine), and UTP was degraded to uridine 5′-diphosphate (UDP), uridine 5′-monophosphate (UMP) and uridine. The rate of degradation of these agonists was much faster at day 30 than at day 15, probably due to the increase in the size of the tissue. AMPCPP was also degraded with adenosine 5′-(α,β-methylene)diphosphonate (AMPCP) being detected at both ages. However, at day 30 the rate of degradation of AMPCPP was much slower than for ATP, UTP or 2-Me-S-ATP.
6. In the rat duodenum longitudinal muscle 2-Me-S-ATP and AMPCPP both caused a relaxation with a potency order of 2-Me-S-ATP>AMPCPP, suggesting the activation of P2Y-receptors, as previously found for the adult tissue. Weak relaxations were observed to both the agonists at day 15 (the earliest age it could be studied), and the potency of the agonists reached values not significantly different from adult tissues by day 25.
7. Overall, these results suggest that in the neonatal rat duodenum longitudinal muscle there are P2Y-receptors mediating relaxation and that the receptor population is fully developed by day 25. In the neonatal rat duodenum muscularis mucosae before day 20 there are P2Y-receptors mediating contraction, while after day 20 P2X-receptors mediate this effect. P2U-receptors also mediate contraction throughout the ages. The results also indicate that the ectonucleotidase activity in the rat duodenum muscularis mucosae increases with age, and the potency of agonists in the rat duodenum may therefore reflect both the number and nature of the receptors involved and the activity of the ectonucleotidases in the tissue.

     

Contribution of P1-(A2b subtype) and P2-purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed.

1. The direct vascular effects of adenosine and ATP were compared in the isolated and perfused mesenteric arterial bed of the rat. The actions of analogues of adenosine and ATP were also examined. 2. In preparations at basal tone, adenosine lacked vasoconstrictor actions, while ATP elicited dose-dependent vasoconstrictor responses. When the tone of preparations was raised by adding methoxamine to the perfusate, adenosine and its stable analogue, 2-chloroadenosine (2-CADO) elicited dose-dependent vasodilation. The A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) was active at lower doses than adenosine, while the A2a-selective agonist, CGS 21680 and the selective A1 agonist, N6-cyclopentyladenosine (CPA) failed to induce vasodilatation. ATP and its analogue, 2-methylthio ATP, elicited dose-dependent vasodilatation at doses 400 fold lower than adenosine. 3. Vasodilator responses to adenosine and 2-CADO were sensitive to antagonism by 1 microM 8-sulphophenyltheophylline (8-SPT) and were unaffected by inhibition of nitric oxide synthase by N omega-nitro-L-arginine methyl ester (L-NAME). In contrast, vasodilator responses to ATP were not sensitive to antagonism by 8-SPT and were almost abolished by L-NAME treatment. 4. These results indicate that in the rat mesenteric arterial bed, while both adenosine and ATP participate in the purinergic control of vascular tone, adenosine appears to be a weaker vasodilator than ATP and lacks vasoconstrictor action. A2b adenosine receptors account for the adenosine-induced vasodilatation which is independent of the production of nitric oxide.     

Presence of P2-purinoceptors in the rat pineal gland.

1. The effects of noradrenaline, ATP, adenylyl-imidodiphosphate (AMP-PNP), adenosine, alpha,beta-methylene-ATP and the P2-purinoceptor antagonist, suramin on N'-acetyl-5-hydroxytryptamine production were studied in cultured denervated rat pineal glands. 2. Noradrenaline (3 nM-1 microM) increased N'-acetyl-5-hydroxytryptamine production as measured both in the gland and the culture medium. 3. In noradrenaline (10 nM)-stimulated pineal glands, ATP (0.03 nM-1 mM) or AMP-PNP (0.1 microM-1 mM) increased N'-acetyl-5-hydroxytryptamine production in a concentration-dependent manner. 4. Alpha,beta-Methylene-ATP at the concentration of 0.1 mM, but not 3 microM, attenuated the enhancement by ATP (0.1 mM) of noradrenaline (10 nM)-induced N'-acetyl-5-hydroxytryptamine production. 5. Suramin (0.1 mM) blocked the potentiating effect of ATP (0.1 mM), but not the potentiating effect of adenosine (0.1 mM) in glands incubated with noradrenaline (10 nM). 6. These findings suggest that the rat pineal gland possesses P2-purinoceptors which when stimulated potentiate the effect of noradrenaline but do not, by themselves, induce an increase in N'-acetyl-5-hydroxytryptamine production.     

Adrenergic nerves mediate acetylcholine-induced endothelium-independent vasodilation in the rat mesenteric resistance artery.

Mechanisms underlying acetylcholine-induced endothelium-independent vasodilation were studied in the rat mesenteric vascular bed isolated from Wistar rats. In preparations without endothelium, and contracted by perfusion with Krebs solution containing methoxamine (2-7 microM), perfusion of acetylcholine (1-100 microM) for 1 min produced a concentration-dependent vasodilation. Denervation of denuded preparations by cold storage (4 degrees C for 72 h) abolished the acetylcholine-induced vasodilation; 10 and 100 nM atropine abolished 1 and 10 microM acetylcholine-induced vasodilation, but it inhibited only 20% of vasodilation by 100 microM acetylcholine. The acetylcholine-induced atropine-resistant vasodilation was inhibited by 10 and 100 microM hexamethonium, 5 microM guanethidine, 50 microM bretylium, in vitro 6-hydroxydopamine (2 mM for 20 min, twice), 1 microM capsaicin and 0.5 microM calcitonin gene-related peptide (CGRP)-(8-37) (CGRP receptor antagonist). These findings suggest that the acetylcholine-induced endothelium-independent nicotinic vasodilation requires the presence of intact adrenergic nerves, and is mediated by endogenous CGRP released from CGRP-containing nerves.     

Chemical removal of the endothelium by saponin in the isolated dog femoral artery

Chemical removal of the endothelium by saponin in the isolated dog femoral artery was investigated by comparing the relaxant responses to endothelium-dependent and -independent vasodilators of saponin-treated rings with the responses of non-treated rings. Saponin treatment was done by incubating rings with Krebs-Henseleit solution containing 0.1, 0.3 or 1 mg/ml of saponin for 45 min at 37 degrees C. In non-treated rings, acetylcholine (10(-8)-3 X 10(-6) M) caused a concentration-dependent relaxation of rings precontracted with prostaglandin F2 alpha (3 X 10(-6) M). The acetylcholine-induced relaxation was reduced in rings pretreated with 0.1 mg/ml of saponin and almost abolished with 0.3 or 1 mg/ml. Prostaglandin F2 alpha-induced contraction was suppressed weakly by treatment with 0.3 mg/ml and markedly with 1 mg/ml saponin. The treatment with 0.3 mg/ml saponin markedly reduced relaxations caused by substance P (10(-9)-3 X 10(-8) M) and by Ca2+-ionophore A23187 (10(-6) M). Relaxant responses of saponin-treated rings to nitroglycerin and to nitroprusside were almost identical with those of non-treated rings. These results showing selective suppression by saponin of the endothelium-dependent relaxation suggest that saponin removes the endothelial cells from the intimal surface of the artery, and this was confirmed by electron microscopy. The endothelium removing method with saponin seems to be useful as a pharmacological tool for vascular investigations.     

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.     

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.

     

P2-purinoceptors of two subtypes in the rabbit mesenteric artery: reactive blue 2 selectively inhibits responses mediated via the P2y-but not the P2x-purinoceptor.

alpha,beta-Methylene ATP and ATP both produced concentration-dependent contractions of the isolated mesenteric artery of the rabbit that were not inhibited by reactive blue 2. In preparations where the tone had been raised with noradrenaline, ATP and 2-methylthio ATP, but not alpha,beta-methylene ATP, produced relaxations of the vessel. These relaxations were inhibited in the presence of reactive blue 2. Reactive blue 2 did not inhibit the contractions to noradrenaline, and only slightly inhibited relaxations to adenosine and acetylcholine. The rank order of potency of purine nucleotide analogues in contracting the vessel was: alpha,beta-methylene ATP greater than beta,gamma-methylene ATP = 2-methylthio ATP greater than ATP, and in relaxing the vessel at raised tone was: 2-methylthio ATP greater than ATP greater than beta,gamma-methylene ATP greater than alpha,beta-methylene ATP. It is concluded from this study that in the isolated mesenteric artery of the rabbit, purine nucleotides act via P2y-purinoceptors to cause the muscle to relax and via P2x-purinoceptors to cause the muscle to contract. The results also suggest that reactive blue 2 selectively inhibits responses mediated via the P2y-purinoceptor, at least within a limited concentration range.     

Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery

1. The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves.
2. Vasopressin (10⁻¹⁰–10⁻⁷ M) caused concentration-dependent contractions (pD₂=8.36±0.09). The V₁-receptor antagonist d(CH₂)₅Tyr(Me)AVP (10⁻⁹–10⁻⁸ M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA₂ value of 9.83 with a slope of 1.10±0.14.
3. Vasopressin (3×10 ⁻¹⁰ and 10⁻⁹ M) caused concentration-dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration-response curve for noradrenaline. The V₁-receptor antagonist induced concentration-dependent inhibitions of potentiation induced by vasopressin. The selective V₁-receptor agonist [Phe^*, Orn⁸]-vasotocin (3×10 ⁻¹⁰ and 10⁻⁹ M) induced potentiation of electrical stimulation-evoked responses which was also inhibited in the presence of the V₁ antagonist (10⁻⁸ M). In contrast, the V₂-receptor agonist deamino-8-D-arginine vasopressin (desmopressin 10⁻⁸–10⁻⁷ M) did not modify the electrical stimulation-induced responses and the V₂-receptor antagonist [d(CH₂)₅, D-Ile^*, Ile⁴, Arg⁸]-vasopressin (10⁻⁸–10⁻⁷ M) did not affect the potentiation evoked by vasopressin.
4. In artery rings contracted by 10⁻⁶ M noradrenaline in the presence of 10⁻⁶ M guanethidine and 10⁻⁶ M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency-dependent relaxations which were unaffected by 10⁻⁹ M vasopressin but abolished by 10⁻⁶ M tetrodotoxin.
5. Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl₂ to KCl depolarized vessels. The augmenting effects were inhibited by the V₁ antagonist.
6. In the presence of the calcium antagonist nifedipine (10⁻⁶ M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl.
7. The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V₁ receptor stimulation which brings about an increase in calcium entry through dihydropyridine-sensitive calcium channels.

     

Mechanisms of the dilator action of Danshen (Salvia miltiorrhiza) on rat isolated femoral artery

This study investigates the actions of Danshen crude extract (Salvia miltiorrhiza) on rat isolated femoral artery rings precontracted with phenylephrine. Low concentrations of Danshen (10 to 30 microg/mL) enhanced the phenylephrine-precontracted tone by a maximum of 31.20+/-2.71%. At concentrations 100 microg/mL or above, Danshen relaxed the precontracted tone, with full relaxation obtained at 1 mg/mL. Involvement of endothelium-dependant mechanisms in the dilator effect of Danshen was investigated by pretreatment of the artery rings with a cyclooxygenase inhibitor flurbiprofen (10 microM), a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), a muscarinic receptor antagonist atropine (100 nM), and by mechanical removal of the endothelium; none of these procedures produced a significant change on the Danshen-induced effect. Involvement of endothelium-independent mechanisms was investigated in endothelium-denuded artery rings pretreated with a histamine H2 receptor antagonist cimetidine (10 microM), a beta-adrenoceptor antagonist propranolol (100 nM), an adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 microM), a guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), and a potassium channel inhibitor tetraethylammonium (TEA, 10 and 100 mM); only TEA was effective in partially inhibiting the Danshen-induced effect. These findings suggest the dilator action of Danshen on rat femoral artery was mediated in part by the opening of TEA-sensitive K+ channels in the smooth muscle cells. Muscarinic receptors, histamine receptors, beta-adrenoceptors, endothelium-derived relaxant factors, adenylyl cyclase, and guanylyl cyclase-dependent pathways did not play a role in its vasodilatory effect.     

Cysteinyl leukotrienes and leukotriene B mediate vasoconstriction to arginine vasopressin in rat basilar artery

Arginine vasopressin (AVP) has been reported to be involved in the development of cerebral vasospasm after haemorrhage and cerebral oedema following ischaemia. Endogenously produced 5-lipoxygenase metabolites are able to contract isolated endothelium-preserved arterial strips and modulate vascular permeability. The present study addresses the role of 5-lipoxygenase and its products, namely cysteinyl leukotrienes (CysLTs) and leukotriene (LT) B4, in the contraction induced by AVP in rat basilar artery. Contractile responses to LTD4, LTC4, LTB4 or AVP were assessed in spiral preparations of rat endothelium-intact basilar artery. Contractions to AVP were determined in the absence or presence of 5-lipoxygenase inhibitors or CysLT1 or BLT receptor antagonists. Contractile responses to leukotrienes and AVP are expressed as a percentage of the contraction induced by 80 mmol/L KCl. Leukotriene D4, LTC4 and LTB4 acted as vasoconstrictor agents in rat basilar artery, causing contractions (all at concentrations of 1 micromol/L) of 42 +/- 13, 54 +/- 15 and 25 +/- 6% of the response to 80 mmol/L KCl, respectively. A concentration-response curve was constructed for AVP over the range 1 pmol/L to 10 nmol/L and an EC50 value of 0.19 +/- 0.02 nmol/L (n = 30) was determined. The presence of the 5-lipoxygenase inhibitors ZM 230487 (10 nmol/L and 0.1 and 1 micromol/L) and AA 861 (1, 3, 10, and 30 micromol/L), the CysLT1 receptor antagonist MK 571 (3, 10 and 30 micromol/L) or the BLT receptor antagonists CP 105696 and LY 255283 (3, 10 and 30 micromol/L for both) in the organ bath significantly attenuated the contractions induced by AVP in rat basilar artery (P < 0.05). The experimental results of the present study provide the first evidence for the involvement of CysLTs and LTB4 in the in vitro constriction induced by AVP in rat basilar artery. In the context of previously reported involvement of AVP in the development of cerebral vasospasm and oedema, the present study draws attention to the potential role played by the 5-lipoxygenase pathway in these pathological processes.     

Postjunctional α2C-adrenoceptors mediate vasoconstriction in rat tail artery: influence of precontraction and temperature on vasoreactivity

The isolated rat tail artery (RTA) represents an in vitro model of the cutaneous circulation. We have characterised the postjunctional α₂-adrenoceptor subtype mediating vasoconstriction to the α₂-adrenoceptor (α₂-AR) agonist UK14304 in RTA. In non-precontracted arterial rings at 32°C, a physiological temperature for the RTA, UK14304 elicited only slight contractions which were markedly enhanced after precontraction with serotonin (5-HT; 10–50 nM). Under the condition of elevated vascular tone, the contractile UK14304 response was competitively antagonised by MK912 (pA₂?=?10.05?±?0.07), rauwolscine (pA₂?=?8.82?±?0.06), yohimbine (pA₂?=?8.45?±?0.04), WB4101 (pA₂?=?8.05?±?0.05), BRL44408 (pA₂?=?7.20?±?0.04), ARC239 (pA₂?=?6.90?±?0.05) and prazosin (pA₂?=?6.80?±?0.05). Schild regressions were linear and had slopes of unity. Affinities (pA₂) for MK912, rauwolscine, yohimbine and WB41104 were in the same range as binding data (pK_D) for these drugs at α_2C-ARs of rat cerebral cortex. In addition, the presence of α_2C-ARs was confirmed by Western blotting. In experiments to study the influence of temperature on vasoreactivity, UK14304-induced contractions did not differ at 37°C, 32°C or 27°C and were similarly blocked by rauwolscine (apparent pA₂?=?8.73–8.90). After rapid cooling (from 37°C to 27°C), the maximal UK14304 response was enhanced only in precontracted arteries; antagonism by rauwolscine was the same before and after cooling (apparent pA₂?=?8.80–8.90). The enhancement of the maximal UK14304 response was abolished by rewarming to 37°C. It is concluded that α_2C-ARs predominantly mediated vasoconstriction in RTAs at any temperature tested. Since α_2C-ARs may be involved in Raynaud’s phenomenon, the isolated RTA represents a convenient in vitro bioassay to test novel compounds for the treatment of this syndrome.     

Characterization of P1-purinoceptors on rat isolated duodenum longitudinal muscle and muscularis mucosae.

1. P1-purinoceptors mediating relaxation of the rat duodenum longitudinal muscle and contraction of the rat duodenum muscularis mucosae were characterized by the use of adenosine and its analogues, 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyl-adenosine (CPA), N6-(phenylisopropyl)adenosine (R-PIA), 2-chloroadenosine (2-CADO) and 2-p-((carboxyethyl)phenethylamino)-5'-carboxamidoadenosine (CGS21680), as well as the P1-purinoceptor antagonist 8-phenyltheophylline (8-PT) and the A1-selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). 2. In the rat duodenum longitudinal muscle, the order of potency of the adenosine agonists was CPA > NECA > adenosine > CGS21680. DPCPX antagonized responses to CPA and NECA at a concentration of 1 nM suggesting that they are acting at A1 receptors. A Schild plot versus CPA gave a slope near to unity (slope = 0.955) and a pA2 of 9.8 confirming that CPA was acting via A1 receptors. Schild analysis for DPCPX versus NECA, however, gave a slope of 0.674 suggesting that NECA was acting on both A1 and A2 receptors. CGS21680, a selective A2a agonist, was much less potent than adenosine suggesting that the A2 receptors are of the A2b subtype. 3. In the rat duodenum muscularis mucosae, the order of potency of the adenosine agonists was NECA > or = R-PIA = CPA > 2-CADO > adenosine, and DPCPX antagonized responses to CPA and NECA at a concentration of 1 microM. CGS21680, at a concentration of 10 microM, had no effect on this tissue. This suggests the presence of A2 receptors in this tissue and that they are of the A2b subtype. 4. These results are in agreement with previous studies in the whole duodenum showing the presence of A1 and A2b receptors causing relaxation, and this shows that the longitudinal muscle dominates the response of the whole tissue. In addition, a contractile A2b receptor has been revealed on the muscularis mucosae, the first time this subtype has been reported to elicit an excitatory response in a smooth muscle preparation.     

Desensitization of the P2-purinoceptors on the rat colon muscularis mucosae.

1. Adenosine 5''-triphosphate (ATP) and adenosine have been shown to contract the rat colon muscularis mucosae, and the receptors at which they act have been classified as P2Y and A1 respectively. Uridine 5''-triphosphate (UTP) also contracts this tissue, and desensitization was used to investigate the receptors by which it acts, in the light of recent suggestions that specific pyrimidinoceptors may exist for UTP, or that nucleotide receptors may exist which are responsive to both ATP and UTP but not to some ATP analogues such as 2-methylthioadenosine 5''-triphosphate (2-MeSATP). 2. ATP, UTP and adenosine each contracted the rat colon muscularis mucosae in a concentration-dependent manner over the concentration range 0.3-300 microM, although maximal responses to ATP and UTP were not obtained. ATP was approximately 4 times as potent as UTP and approximately equipotent with adenosine although the maximal response to adenosine appeared to be less than that to ATP or UTP. 3. Desensitization of the tissue with ATP (200 microM) given immediately before each concentration of the agonists reduced subsequent contractions induced by ATP itself and also by UTP, but did not reduce contractions induced by adenosine. Desensitization of the tissues with UTP (200 microM) also reduced contractions induced by ATP and UTP but not by adenosine, whereas desensitization with adenosine (200 microM) reduced contractions induced by adenosine itself but not by ATP or UTP. 4. Desensitization of the tissue with 2-MeSATP (200 microM), which is a more potent agonist than ATP at P2Y-purinoceptors, greatly reduced the responses to ATP and to UTP, but had no effect on responses induced by adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)