Modulation of noradrenaline release in slices of rat kidney cortex through alpha 1- and alpha 2-adrenoceptors

Slices of rat kidney cortex were incubated in [3H]noradrenaline, then placed in a flow cell and subjected to electrical field stimulation. At a stimulation frequency of 5 Hz, both the alpha 2-adrenoceptor antagonist idazoxan (0.1 microM) and the alpha 1-adrenoceptor antagonist prazosin (0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity from the slice. However, neither clonidine (0.1 microM) nor methoxamine (10 microM), alpha 2- and alpha 1-agonists respectively, affected the S-I outflow of radioactivity at this stimulation frequency. At a lower stimulation frequency (1 Hz), the S-I outflow of radioactivity was not affected by idazoxan or prazosin, but was inhibited by both clonidine and methoxamine. The effect of clonidine was prevented by idazoxan (0.1 microM), but not by prazosin (0.1 microM). The effect of methoxamine was abolished by prazosin (0.1 microM), but not by idazoxan (0.1 microM). The inhibitory effect of methoxamine was not prevented by the prostaglandin synthesis inhibitor indomethacin (10 microM) or the adenosine receptor antagonist 8-phenyltheophylline (1 microM) and thus was not mediated by either prostaglandins or adenosine. The results suggest that both prejunctional alpha 1- and alpha 2-adrenoceptors are directly involved in modulation of noradrenaline release from the renal sympathetic nerves of the rat.     

Modulation of norepinephrine release through alpha 1- and alpha 2-adrenoceptors in rat isolated kidney

The aim of this study was to investigate alpha-adrenoceptor modulation of norepinephrine (NE) release from sympathetic nerves in rat isolated perfused kidney. After preincubation with [3H]NE, the renal nerves were stimulated. The stimulation-induced (S-I) outflow of radioactivity was used as an index of NE release. Clonidine (0.1 mumol/L) decreased the S-I outflow of radioactivity. This effect was abolished by the alpha 1-adrenoceptor antagonist idazoxan (0.1 mumol/L) but not by the alpha 2-adrenoceptor antagonist prazosin (0.1 mumol/L). Methoxamine (10 mumol/L) also had an inhibitory effect; this was abolished by prazosin (0.1 mumol/L), but not by idazoxan. Individually, these alpha-blocking drugs and the alpha 1-adrenoceptor antagonist corynanthine (0.3 mumol/L) enhanced S-I outflow of radioactivity. In the presence of indomethacin (10 mumol/L), the inhibitory effect of methoxamine was abolished but clonidine still inhibited S-I outflow of radioactivity. The facilitatory effect of prazosin was also unaltered by indomethacin. These results suggest the existence of inhibitory prejunctional alpha 1- and alpha 2-adrenoceptors in the kidney. The inhibitory effect of methoxamine seems to be mediated through prostaglandin inhibition of NE release. However, the evidence for inhibitory prejunctional alpha 1-adrenoceptors rests solely on the facilitatory effects of prazosin and corynanthine.     

Evidence for facilitatory and inhibitory muscarinic receptors on postganglionic sympathetic nerves in mouse isolated atria.

1. McNeil A 343 (10 microM-30 microM) enhanced the fractional stimulation-induced (S-I) outflow of radioactivity from mouse isolated atria which had been incubated with [3H]-noradrenaline. The enhancing effect of McNeil A 343 was not altered by hexamethonium (300 microM) suggesting that it was not due to an action at nicotinic receptors. It is also unlikely that McNeil A 343 enhanced the S-I outflow of radioactivity in mouse atria by blocking neuronal reuptake of noradrenaline since the effect persisted in the presence of cocaine (30 microM). 2. The facilitatory effect of McNeil A 343 on the S-I outflow of radioactivity was attenuated by atropine (0.3 microM), pirenzepine (0.2 microM or 1.0 microM), dicyclomine (1.0 microM) and methoctramine (1.0 microM) and was thus due to activation of muscarinic receptors. 3. In contrast to the effect of McNeil A 343, another muscarinic receptor agonist, carbachol (3.0 microM) significantly decreased the S-I outflow of radioactivity. The receptors through which McNeil A 343 acts to enhance the S-I outflow of radioactivity appear to be distinct from inhibitory prejunctional muscarinic receptors. The relatively M 1-selective antagonist, pirenzepine (0.2 microM), attenuated the facilitatory effect of McNeil A 343 whereas a higher concentration (1.0 microM) was required to block the inhibitory effect of carbachol. Conversely, the relatively M2-selective antagonist, methoctramine (0.1 microM), blocked the inhibitory effect of carbachol but a higher concentration of methoctramine (1.0 microM) was required to block the facilitatory effects of McNeil A 343.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the novel dopamine DA2-receptor agonist carmoxirole (EMD 45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney

Summary The effects of the classical dopamine DA₂-receptor agonist quinpirole (LY 171555) and the recently characterized DA₂-receptor agonist, carmoxirole (EMD 45609), on neurotransmission in rat isolated kidney were investigated. After preincubation with ³H-noradrenaline, the renal nerves were electrically stimulated. The stimulation induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. Quinpirole (0.3 mol/l) inhibited S-I outflow of radioactivity and pressor responses to renal nerve stimulation (RNS) at 1 Hz. Both effects of quinpirole were blocked by the DA₂-receptor antagonist S(–)-sulpiride (10 mol/l). The ₁, ₂-adrenoceptor antagonist phentolamine (1 mol/l) did not block the inhibitory effect of quinpirole. Carmoxirole (0.003 and 0.03 mol/l) did not alter and carmoxirole (0.3 mol/l) even enhanced S-I outflow of radioactivity, however, pressor responses to RNS were markedly reduced by carmoxirole (0.003–0.3 mol/l). Pressor responses to RNS were also markedly reduced by the ₁-adrenoceptor antagonist prazosin (0.1 mol/l). Carmoxirole (0.3 mol/l), prazosin (0.1 mol/l) and phentolamine (1 mol/l) totally abolished pressor responses to exogenous noradrenaline (0.05 mol/l). In contrast, quinpirole (0.3 mol/l) did not alter pressor responses to exogenous noradrenaline (0.05 mol/l). Furthermore, carmoxirole (0.003–0.3 mol/l) markedly reduced pressor responses induced by the ₁-adrenoceptor agonist methoxamine (1 mol/l) but even the highest concentration of carmoxirole (0.3 mol/l) had no effect on pressor responses induced by bolus injections of either neuropeptide Y (1.5 ng) or angiotensin II (1 ng). Phentolamine (1 mol/l) by itself markedly enhanced S-1 outflow of radioactivity and pressor responses to RNS were virtually unchanged. In the presence of phentolamine carmoxirole (0.03 and 0.3 mol/l) and quinpirole inhibited S-I outflow of radioactivity and pressor responses to RNS. Phentolamine resistant pressor responses to RNS were also inhibited by the P_2X-receptor desensitizing agent , -methylene adenosine triphosphate (mATP, 1 mol/l), which by itself in the presence of phentolamine did not alter S-I outflow of radioactivity. The inhibitory effects of carmoxirole (0.3 mol/l) in the presence of phentolame (1mol/l) were antagonized by S(–)-sulpiride (10 mol/l). The data suggest that activation of prejunctional DA₂-receptors by quinpirole inhibits noradrenaline release and thereby reduces pressor response to RNS at 1 Hz in rat isolated kidney. Carmoxirole activates prejunctional inhibitory DA₂-receptors, but this effect is masked by simultaneous blockade of inhibitory prejunctional -adrenoceptors. Pressor responses to RNS at 1 Hz in rat isolated kidney are largely due to neuronally released noradrenaline whereas phentolamine resistant pressor responses to RNS at 1 Hz are most likely due to ATP, which is co-released with noradrenaline. Carmoxirole inhibits pressor responses to RNS at 1 Hz as well as pressor responses induced by either exogenous noradrenaline or methoxamine by blocking postjunctional ₁-adrenoceptors. In addition carmoxirole and quinpirole seem to block phentolamine resistant pressor responses by inhibiting ATP release through activation of prejunctional DA₂-receptors. Send offprint requests to L. C. Rump at the above address     

Facilitation of noradrenaline release from sympathetic nerves through activation of ACTH receptors, beta-adrenoceptors and angiotensin II receptors.

1. In rabbit pulmonary artery and left atrial strips previously incubated with [3H]-noradrenaline, the active fragment of adrenocorticotropic hormone (ACTH 1-24, 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity when a cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM) was present, but not in the absence of these drugs. In rabbit pulmonary artery a facilitatory effect of ACTH 1-24 (0.1 microM) was also observed when only cocaine (30 microM) was present. 2. ACTH 1-24 (0.1 microM) did not affect the S-I outflow of radioactivity from rat atria, rat pulmonary artery or guinea-pig pulmonary artery, either in the presence or in the absence of the cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM). These results suggest that the presence of facilitatory prejunctional ACTH receptors may be restricted to rabbit sympathetic nerve endings. 3. Angiotensin II (0.01 microM), but not isoprenaline (0.1 microM) or ACTH 1-24 (0.1 microM), significantly enhanced the S-I outflow of radioactivity from rabbit pulmonary artery. In the presence of phentolamine (1 microM) to block inhibitory alpha 2-adrenoceptors, the facilitatory effect of angiotensin II (0.01 microM) was significantly enhanced, and a significant facilitatory effect of isoprenaline (0.1 microM) and of ACTH 1-24 (0.1 microM) was then revealed. These results suggest that feedback inhibition of noradrenaline release, mediated through the prejunctional alpha 2-adrenoceptor mechanism, buffers increases in noradrenaline release during activation of facilitatory prejunctional receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are sensitive to N-ethylmaleimide, but not pertussis toxin.

1. The identity of the G-proteins involved in prejunctional alpha 2-adrenoceptor signal transduction in mouse atria was examined by use of the G-protein inactivators N-ethylmaleimide and pertussis toxin. 2. The alpha 2-adrenoceptor partial agonist clonidine (0.03 microM) inhibited the electrical stimulation-induced (S-I) outflow of radioactivity from mouse atria which were incubated with [3H]-noradrenaline and stimulated at 5 Hz. The partial alpha 2-adrenoceptor agonist St 363 (10 microM) inhibited the S-I outflow of radioactivity at the lower stimulation frequency of 2.5 Hz. The inhibitory effects of these compounds were not altered in mice pretreated with pertussis toxin (1.5 micrograms, i.v.). 3. The alpha 2-adrenoceptor antagonist, idazoxan (0.1 microM), increased the S-I outflow of radioactivity from mouse atria stimulated at 5 Hz, and this effect was not altered in atria from mice pretreated with pertussis toxin. 4. The inhibitory effects of clonidine and St 363 and the facilitatory effect of idazoxan on the S-I outflow of radioactivity from mouse atria were significantly less in atria incubated with N-ethylmaleimide (NEM, 3 microM) for 60 min before the [3H]-noradrenaline incubation. 5. The results suggest that prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are NEM-sensitive, but pertussis toxin insensitive.     

Pertussis toxin differentiates between alpha 1- and alpha 2-adrenoceptor-mediated inhibition of noradrenaline release from rat kidney cortex

In slices of rat kidney cortex incubated in [3H]noradrenaline, the alpha 1-adrenoceptor agonist methoxamine (10 microM), the alpha 2-adrenoceptor agonist clonidine (0.1 microM), as well as adenosine (10 microM), inhibited the electrical stimulation-induced (S-I) outflow of radioactivity, at a stimulation frequency of 1 Hz. Prior treatment of rats with pertussis toxin (25 micrograms/kg i.v.), which abolished the negative inotropic effect of carbachol (10 microM) on isolated atria, prevented the inhibition caused by methoxamine, but not that caused by clonidine or adenosine. At a stimulation frequency of 5 Hz, the alpha 2-adrenoceptor antagonist idazoxan (0.1 microM) and the prostaglandin synthesis inhibitor indomethacin (10 microM) both facilitated the S-I outflow of radioactivity, and neither of these effects were altered by pertussis toxin. These results suggest that a pertussis toxin sensitive G-protein is involved in alpha 1-adrenoceptor inhibition of noradrenaline release, but not in alpha 2-adrenoceptor, adenosine or prostaglandin inhibition.     

THROMBOXANE A2 RECEPTOR STIMULATION SIMILARLY POTENTIATES PRESSOR RESPONSES TO 5-HYDROXYTRYPTAMINE IN PERFUSED HINDQUARTERS OF NON-DIABETIC AND ALLOXAN DIABETIC RATS

1. Dose-response curves were obtained to bolus injections of 5-hydroxytryptamine (5-HT) in Krebs'-perfused hindquarters of male Wistar rats. Vasoconstrictor responses to 5-HT (5.7-363 nmol/kg) were significantly attenuated in hindquarters of alloxan-treated 14 day diabetic rats compared with non-diabetics. 2. Infusion of the thromboxane A2 (TxA2)-mimetic U46619 (317 and 31.7, but not 3.17 nmol/L) significantly potentiated vasoconstrictor responses to 5-HT in Krebs'-perfused hindquarters of non-diabetic and diabetic rats. The degree of potentiation was similar for both groups. 3. In Krebs'-perfused hindquarters of non-diabetic rats, infusion of the alpha 1-adrenoceptor agonist methoxamine (8.96 mumol/L, which caused a rise in perfusion pressure intermediate in magnitude to that produced by infusion of 31.7 and 317 nmol/L U46619) did not significantly affect responses to bolus injections of 5-HT. 4. The same concentration of methoxamine did not cause a significant potentiation of vasoconstrictor responses to 5-HT (except for the two highest 5-HT doses, 182 and 363 nmol/kg) in hindquarters of diabetic rats. This potentiation was significantly less than that due to 317 nmol/L U46619, although there was no significant difference between the rise in basal perfusion pressures produced by these concentrations of methoxamine and U46619. 5. Infusion of the TxA2 receptor antagonist AH23848 (111 nmol/L) inhibited the potentiating effect of U46619 (317 nmol/L) on responses to 5-HT in both non-diabetic and diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endotoxin tolerance differentially alters hemodynamic responses to a thromboxane A2 mimetic and phenylephrine.

Repeated sublethal doses of endotoxin render rats tolerant to lethal doses of endotoxin and reduce thromboxane (Tx) A2 synthesis. Endotoxin-tolerant rats are also more resistant to lethal doses of U46619, a stable TxA2 mimetic. These observations raised the possibility that tolerance may alter hemodynamic responses to TxA2 via one or more mechanisms. Mean arterial pressure (MAP) responses to i.v. injections of the stable TxA2 mimetic U46619 at doses ranging from 0.17 to 8.4 micrograms/kg were determined. Despite an initial lower systemic vascular resistance in tolerant rats compared to control rats (2.4 +/- 0.3 vs 3.1 +/- 0.2 mm Hg/ml/min/100 g of body weight, respectively, p less than 0.05), the maximum pressor response to U46619 was significantly greater (p less than 0.05) at the higher doses of U46619 in tolerant rats compared to control rats. Tolerant and control rats also exhibited qualitatively different changes in MAP in response to U46619. Control rats manifested an initial hypotensive response (15 s) not observed in tolerant rats. In contrast, tolerant rats exhibited no depressor response, but a higher peak pressor response to U46619 than that seen in controls. Since prostaglandins may modulate vascular responses to U46619, subsequent studies were conducted in indomethacin-pretreated or essential fatty acid (EFA) deficient rats that were depleted of arachidonic acid substrate. Either indomethacin or EFA deficiency significantly prevented the initial hypotensive response in control rats, suggesting that prostaglandins may mediate this response to U46619. In additional studies, the MAP response in tolerant and control rats to the alpha 1-adrenergic agonist phenylephrine was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Facilitation of noradrenaline release from sympathetic nerves in rat anococcygeus muscle by activation of prejunctional beta-adrenoceptors and angiotensin receptors.

1. Isolated preparations of rat anococcygeus muscle were incubated with [3H]-noradrenaline and the efflux of radioactivity induced by stimulation of intramural sympathetic nerves was used as a measure of release of transmitter noradrenaline. Isometric contractile responses were also measured. 2. Angiotensin I (0.03 microM) and angiotensin II (0.03 microM) produced non-sustained contractile responses and enhanced the stimulation-induced (S-I) effluxes of radioactivity as well as the contractile responses to electrical stimulation. These effects were blocked by the angiotensin II receptor antagonist saralasin (0.03 microM), and the effect of angiotensin I, but not angiotensin II, was blocked by the angiotensin converting enzyme inhibitor captopril (0.1 microm). 3. The findings indicate that there are both pre- and postjunctional receptors for angiotensin II and that angiotensin I is converted to angiotensin II in the anococcygeus muscle preparation. 4. Isoprenaline (0.1 microM) slightly enhanced the S-I efflux of radioactivity, and produced a greater enhancement after neuronal uptake blockade with desipramine (0.03 microm) and alpha-adrenoceptor blockade with phentolamine (1 microM). 5. The facilitatory effect of isoprenaline on S-I efflux of radioactivity was abolished by propranolol (0.3 microM), but was not affected by low concentrations of saralasin (0.03 microM) or captopril (0.1 microM) which abolished the effect of angiotensin I. The findings suggest that isoprenaline acts directly on prejunctional beta-adrenoceptors to enhance S-I noradrenaline release, rather than indirectly by releasing angiotensin II from within the tissue. Higher concentrations of saralasin (0.1 microM) or captopril (5 microM) did block the facilitatory effect of isoprenaline. The significance of this finding is not clear.     

Alpha 2-autoreceptor subclassification in rat isolated kidney by use of short trains of electrical stimulation.

1 Rat kidneys were perfused with Krebs-Henseleit solution and incubated with [3H]-noradrenaline. The renal nerves were electrically stimulated at either 1 Hz for 30 s or 100 Hz for 0.06 s. The stimulation induced (S-I) outflow of radioactivity was taken as an index of endogenous noradrenaline release. 2 At a frequency of 1 Hz for 30 s the alpha-adrenoceptor antagonists BRL 44408 (0.01, 0.1 microM) and imiloxan (0.1, 1.0 microM) enhanced S-I outflow of radioactivity. However, at a frequency of 100 Hz for 0.06 s the alpha-adrenoceptor antagonists, idazoxan (0.1, 1.0 microM), imiloxan (0.1, 1.0 microM), BRL 44408 (0.1, 1.0 microM), BRL 41992 (0.1, 1.0 microM) and prazosin (0.01 microM) failed to enhance S-I outflow of radioactivity. 3 Thus, the rat isolated kidney stimulated at 100 Hz for 0.06 s, avoids autoinhibition by endogenous noradrenaline and alpha-adrenoceptor antagonist affinities (pKB) at the prejunctional alpha-autoreceptor were estimated without disturbance by the endogenous activator. 4 The alpha 2-adrenoceptor agonist, clonidine, inhibited the S-I outflow of radioactivity with a maximum of 90% and an EC50 of 7.2 nM. 5 All alpha-adrenoceptor antagonists used caused parallel shifts of the concentration-response curve for clonidine to the right. The rank order of potencies was: rauwolscine (alpha 2A/B) > idazoxan (alpha 2A/B) > phentolamine (alpha 2A/B) > WB 4101 (alpha 2A) > BRL 44408 (alpha 2A) > BRL 41992 (alpha 2B) > prazosin (alpha 2B) = imiloxan (alpha 2B).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostanoid-induced contraction of human bronchial smooth muscle is mediated by TP-receptors

1. A range of naturally-occurring prostaglandins sulprostone, 16,16-dimethyl prostaglandin E2 (DME2) and the thromboxane A2 (TXA2)-mimetic, 11 alpha,9 alpha-epoxymethano prostaglandin H2 (U-46619) have been tested for contractile agonist activity on human isolated bronchial smooth muscle. 2. Prostaglandin D2 (PGD2), PGF2 alpha, 9 alpha,11 beta-PGF2 (11 beta-PGF2) and U-46619 all caused concentration-related contractions. U46619 was at least 300 fold more potent than the other prostanoids with a mean EC50 of 12 nM. Sulprostone caused contraction only at the highest concentration tested (30 microM). PGE2 and PGI2 caused relaxations at low concentrations, and only caused contractile responses at high concentrations (greater than or equal to 10 microM). In contrast, DME2 caused small contractions at low concentrations but relaxation at the highest concentration tested (30 microM). 3. The rank order of contractile agonist potency was: U-46619 much greater than 11 beta-PGF2 congruent to PGF2 alpha greater than PGD2 greater than PGE2 greater than PGI2 congruent to sulprostone congruent to DME2. 4. The TP-receptor blocking drug, AH23848 (1 microM) antagonized the contractile effects of U-46619, PGD2, PGF2 alpha and 11 beta-PGF2, but had no effect against contractions to carbachol. In a single experiment, a pA2 of 8.3 (slope = 1.2) was obtained for AH23848 against U-46619. 5. In most preparations, administration of AH23848 (1 microM) to human bronchus resulted in small, transient contractile responses. 6. The results obtained with both the agonists and the antagonist, AH23848 are therefore consistent with prostanoid-induced contractions of human bronchial smooth muscle being mediated by TP-receptors.     

Effect of phorbol ester and pertussis toxin on the enhancement of noradrenaline release by angiotensin II in mouse atria.

1. Mouse atria were incubated with [3H]-noradrenaline, and the outflow of radioactivity due to electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (0.01 and 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity. 2. Phorbol 12-myristate 13-acetate (0.001, 0.03, 0.1 and 1.0 microM), a protein kinase C activating phorbol ester, significantly enhanced the S-I outflow of radioactivity. When angiotensin II (0.1 microM) was present with the concentration of phorbol 12-myristate 13-acetate that was maximally effective in increasing the S-I outflow (0.1 microM), the enhancement of S-I outflow produced by angiotensin II was maintained. 3. Polymyxin B (70 microM), an inhibitor of protein kinase C, significantly inhibited the S-I outflow. Polymyxin B also inhibited the enhancement of the S-I outflow produced by angiotensin II (0.1 microM). 4. In another series of experiments mice were injected with pertussis toxin (1.5 micrograms per mouse), 4 days before their atria were removed. The effectiveness of pertussis toxin pretreatment was determined indirectly using carbachol. Carbachol caused a concentration-dependent fall in both the rate and force of beating of isolated spontaneously beating atria from mice pretreated with vehicle. This effect of carbachol was not seen with atria from mice pretreated with pertussis toxin. 5. Pertussis toxin pretreatment did not alter the enhancement of the S-I outflow of radioactivity produced by angiotensin II (0.01 and 0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the actions of U-46619, a prostaglandin H2-analogue, with those of prostaglandin H2 and thromboxane A2 on some isolated smooth muscle preparations.

1 The actions of the prostaglandin H2 (PGH2) analogue, U-46619, have been compared with those of PGH2 on thromboxane A2 (TxA2) on a range of isolated smooth muscle preparations in a superfusion cascade system. 2 U-46619 was a potent agonist on guinea-pig lung strip, dog saphenous vein and rat and rabbit aortae. In contrast, U-46619 was weak or inactive on guinea-pig ileum and fundic strip, cat trachea and dog and cat iris sphincter muscles, preparations on which either PGE2 or PGF2 alpha was the most potent agonist studied. 3 PGH2 was active on all of the preparations and displayed little selectivity. On some of the preparations, the actions of PGH2 may have been mediated indirectly by conversion to other prostanoids. 4 In contrast, TxA2 displayed the same pattern of selectivity as U-46619, being a potent agonist on the lung strip and vascular preparations but weak or inactive on the others. 5 It is suggested that U-46619 is a selective TxA2-mimetic and that it should therefore be a valuable tool in the study of the actions of TxA2.     

Sumatriptan (GR43175) inhibits cyclic-AMP accumulation in dog isolated saphenous vein.

Sumatriptan (GR43175) contracts rings of dog isolated saphenous vein by an action at 5-HT1-like receptors. We have now examined the effects of sumatriptan on prostaglandin E2(PGE2)-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in this tissue. Sumatriptan and 5-hydroxytryptamine (5-HT) produced a concentration-dependent inhibition of PGE2-stimulated cyclic AMP accumulation (EC50 values of 250 nM and 80 nM respectively), responses that were mimicked by 5-carboxamidotryptamine but not by U-46619 or methoxamine. The response to sumatriptan (1 microM) was antagonised by methiothepin (1 microM), but not by metergoline (0.1 microM), spiperone (1 microM) or ondansetron (GR38032, 1 microM). These results suggest that 5-HT1-like receptors which mediate contraction of the dog isolated saphenous vein are negatively coupled to adenylate cyclase in this preparation.     

Actions of the novel thromboxane A2 receptor antagonist sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11- dihydrodibenz[b,e]oxepine-1-carboxylate monohydrate on smooth muscle preparations.

The effects of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)- ethylidene]-6,11-dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate, CAS 127166-41-0) on smooth muscle preparations were examined. In isolated guinea-pig aorta, KW-3635 competitively inhibited the U-46619 (a thromboxane mimetic) induced contractions (pA2 = 7.74), the effect being more potent than those of sulotroban and daltroban. In canine saphenous vein, KW-3635 also antagonized the U-46619-induced contraction (pA2 = 8.11). In this preparation, solutroban and daltroban, but not KW-3635, exhibited intrinsic agonistic action. KW-3635, even at a high concentration of 10(-5) mol/l did not affect the norepinephrine- or KCl-induced contractions of guinea-pig or rat aorta, prostaglandin (PG)E2- or PGF2 alpha-induced contractions of guinea-pig ileum nor the PGE2-induced contraction of rat fundus. KW-3635 at concentrations higher than its thromboxane A2- (TxA2-)antagonistic one, non-competitively inhibited the PGF2 alpha-induced contractions of guinea-pig aorta (pD2' = 6.23), as was the case with daltroban. The inhibitory effect of KW-3635 (3 x 10(-6) mol/l) on U-46619-induced contractions of guinea-pig aorta persisted for longer than 2 h following washout of the tissue, whereas that of daltroban (10(-5) mol/l completely disappeared at 1 h after the washout. In anesthetized guinea-pigs, KW-3635 at doses of 10 to 1000 micrograms/kg (i.v.) inhibited U-46619 (1 microgram/kg i.v.)-induced pressor responses in a dose-dependent manner. The effect of KW-3635 (0.1 to 1 mg/kg i.v.) persisted for longer than 3 h. These results demonstrate that KW-3635 is a potent and specific TxA2 antagonist without agonistic action in vascular smooth muscles. KW-3635 is considered to be a promising candidate for the treatment of patients with disorders mediated via TxA2.     

Noradrenaline and extracellular nucleotide cotransmission involves activation of vasoconstrictive P2X(1,3)- and P2Y6-like receptors in mouse perfused kidney

Nucleotides like ATP and UTP act as potent extracellular signalling molecules. Released from sympathetic nerve endings as cotransmitters of noradrenaline or paracrine from nonexcitatory cells, they activate specific receptors (ion-gated P2X(1-7) and G-protein-coupled P2Y(1,2,4,6,11-15)). Which of these subtypes, however, are able to modulate vasoconstriction in the kidney is unclear. Wild-type- and P2Y4-receptor-deficient mice kidneys were isolated and perfused with Krebs-Henseleit solution. Pressor responses to renal nerve stimulations (RNS) and added drugs were recorded. Release of endogenous noradrenaline was measured by HPLC. RNS (1-15 Hz) induced a frequency-dependent increase in the perfusion pressor (14.2+/-5.1-67.3+/-6.9 mmHg) and noradrenaline release (1.4+/-0.3-24.2+/-3.4 ng g(-1) kidney). Pressor responses to RNS were not (1-2 Hz) or only partially (5-15 Hz) blocked by the alpha-adrenoceptor antagonist phentolamine (1 microM). Combination of phentolamine and the P2-receptor blocker PPADS (5 microM) prevented RNS-induced pressor responses. The P2X(1,3)-receptor selective antagonist NF279 (10 microM) reduced RNS-induced pressor responses in a frequency-dependent manner. Perfusion of ATP, ADP, UTP, UDP and alpha,beta-meATP concentration dependently increased perfusion pressor with the following rank order of potency alpha,beta-meATP>ADP approximately ATP approximately UDP > or = UTP. NF279 (10 microM) reduced alpha,beta-meATP- (0.1 microM) (21.7+/-3.9% of control) but not UTP- (0.3 microM) (102.6+/-15.3% of control) induced pressor responses. No differences in nucleotide-induced effects were detected among wild-type and P2Y4-receptor knockout mice. Continuous perfusion of alpha,beta-meATP (0.01 microM) potentiated UTP-, UDP- and ATP-gamma S-induced pressor responses. Neuronally and paracrine-released nucleotides evoked renal vasoconstriction by activation of P2X(1,3)- and P2Y6-like receptors in mice. Pretreatment with the P2X(1,3)-receptor agonist alpha,beta-meATP potentiated P2Y6-like receptor-mediated vasoconstrictions.     

Antagonism of prostanoid-induced vascular contraction by 13-azaprostanoic acid (13-APA)

Isometric contractions in vitro of helically cut strips of canine mesenteric arteries (O.D. = 0.6-1 mm) were recorded in the presence of 10 microM indomethacin. PGF2 alpha PGE2, U-46619 (TXA2 mimetic), norepinephrine, 5-hydroxytryptamine, and KCl produced dose-related contractions. The EC50 values of the prostanoids in the control period were: PGF2 alpha, 7.3 +/- 0.7 X 10(-7) M; PGE2, 2.1 +/- 0.2 X 10(-6) M; and U-46619, 4.1 +/- 1.0 X 10(-10) M. Exposure to 13-azaprostanoic acid (13-APA), 5 X 10(-5) M and 2 X 10(-4) M, for 20 min caused parallel and dose-related shifts to the right of the dose-response curves generated by all three prostanoids without affecting the contractile responses to KCl, norepinephrine, or 5-hydroxytryptamine or relaxation induced by PGI2. 13-APA, at the concentrations used, had no agonist activity. Small contractions (10-15%) seen on the addition of 13-APA to the baths were found to be related to the alcohol content of the solvent. Dose ratios (with/without antagonist) at the EC50 level were found to be: in the presence of 5 X 10(-5) M 13-APA PGF2 alpha, 2.7 +/- 0.3; PGE2, 3.5 +/- 0.9; U-46619, 5.2 +/- 0.9; in the presence of 2 X 10(-4) M 13-APA PGF2 alpha, 17.1 +/- 3; PGE2, greater than 50; and U-46619, 23.3 +/- 5.7. Thus, 13-APA is a specific antagonist of these prostanoids with no agonist activity of its own.     

Prostaglandin action, release and inactivation by rat isolated perfused mesenteric blood vessels.

1 The following experiments were undertaken to confirm that prostaglandin is necessary for noradrenaline to exert its full vasoconstrictor effect in rat mesenteric blood vessels. Prostaglandin release and inactivation were also studied. 2 The cyclo-oxygenase inhibitor, 5, 8, 11, 14-eicosatetraynoic acid caused a significant depression of the concentration-effect curve to noradrenaline. As with indomethacin, responses were restored to control levels by prostaglandin E2 (PGE2) but PGE2 did not restore responses to noradrenaline depressed by papaverine. 3 PGE2-like activity was released from tissues at rest, equivalent to 50 +/- 20 pg PGE2/min. The substance was probably a stable prostaglandin since activity remained on acidifying and extracting into chloroform. The increase in release stimulated by noradrenaline was reduced below resting values by indomethacin. 4 There was a net loss of 7 +/- 1 and 1 +/- 0.2 ng PGE2/min from tissues perfused with 40 and 4 ng/min PGE2 respectively. No uptake occurred at lower PGE2 perfusion rates. 5 When indomethacin was used to depress responses to noradrenaline 15(S)-15-methyl PGE2 methyl ester was 12 times more potent than PGE2 in restoring responses to control values. The cyclic endoperoxide analogue U-46619 caused only partial restoration of indomethacin-depressed responses to noradrenaline but increased perfusion pressure at 2 ng/ml and above. 6 The results confirm that endogenous prostaglandin release, possible of PGE2, is obligatory to the full vasoconstrictor effect of noradrenaline. Noradrenaline increases the amount of prostaglandin released which may be taken up and inactivated by 15-hydroxy prostaglandin dehydrogenase or beta-oxidase. U-46619 may mimic both PGE2 and thromboxane A2.     

Angiotensin II receptors involved in the enhancement of noradrenergic transmission in the caudal artery of the spontaneously hypertensive rat.

1. The effects of the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD 123319, on actions of angiotensin II in isolated caudal arteries of spontaneously hypertensive (SH) and age-matched normotensive (Wistar-Kyoto) rats were compared. 2. Angiotensin II (0.1-3 microM) produced concentration-dependent increases in perfusion pressure in artery preparations from both SH and Wistar-Kyoto (WKY) rats, the maximal increase in the SH rat being significantly greater than the increase in WKY rats. The increase in perfusion pressure in preparations from both strains of rats was prevented by losartan (0.1 microM) and unaffected by PD 123319 (0.1 microM), indicating that the vasoconstrictor action of angiotensin II is subserved by AT1 receptors. 3. Angiotensin II (0.1-3 microM) produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of caudal artery from both SH and WKY rats, in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The maximum enhancement of S-I efflux produced by angiotensin II (1 microM) was significantly greater in artery preparations from WKY rats than in preparations from SH rats, whereas the maximum enhancement of stimulation-evoked vasoconstrictor responses was greater in preparations from SH rats than in those from WKY rats. 4. In artery preparations from both WKY and SH rats, the AT1 angiotensin II receptor antagonist, losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of both S-I efflux and vasoconstrictor responses by 1 microM angiotensin II. 5. The combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced both the S-I efflux and stimulation-evoked vasoconstrictor response in caudal artery preparations from WKY rats, whereas 0.1 microM angiotensin alone was ineffective. The AT2 receptor antagonist PD 123319 (0.01 and 0.1 microM) prevented the enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses by the combination of angiotensin II and losartan. 6. In contrast to findings in WKY preparations and those previously obtained for arteries from another normotensive strain (Sprague-Dawley), in artery preparations from SH rats there was no synergistic interaction between losartan and angiotensin II. Rather, combinations of 0.1 microM angiotensin II and PD 123319 (both 0.01 and 0.1 microM) enhanced S-I [3H]-noradrenaline efflux, whereas 0.1 microM angiotensin II alone was without effect. Moreover, losartan (0.1 microM) prevented the enhancement of S-I efflux by the combination of angiotensin II and PD 123319. 7. The present findings indicate that in the caudal artery of WKY and SH rats, and as previously found in Sprague-Dawley preparations, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 8. As previously suggested for Sprague-Dawley caudal artery preparations, the synergistic prejunctional interaction of losartan and 0.1 microM angiotensin II in caudal artery preparations from WKY rats may be due to either the unmasking by losartan of a latent population of angiotensin II receptors subserving facilitation of transmitter noradrenaline release, or blockade by losartan of an inhibitory action of angiotensin II on transmitter release. 9. The synergistic interaction of PD 123319 and 0.1 microM angiotensin II in caudal arteries of SH rats may also be explained by either of the mechanisms proposed for the normotensive strains, but the involvement of different receptor subtypes would need to be postulated for each of the proposed mechanisms.