Evidence for an A2-subtype adenosine receptor on pancreatic glucagon secreting cells.

The effects of a 5'-substituted analogue of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) have been studied on glucagon secretion in vitro, using the isolated pancreas of the rat perfused in the presence of glucose (2.8 mM). NECA provoked a peak of glucagon secretion, the kinetics of which were comparable to those previously obtained with adenosine. The effect was concentration-dependent and appeared at nanomolar concentrations. The EC50 was approximately 4 X 10(-8) M. A comparison of relative potency between adenosine and NECA showed that NECA was about 800 fold more potent than adenosine in inducing glucagon secretion. Theophylline (50 microM) considerably decreased the peak of glucagon secretion induced by 1.65 microM NECA and totally suppressed the effect of 16.5 nM NECA. These results indicate the involvement of an adenosine receptor. These and other previous results (low stereoselectivity of N6-phenylisopropyladenosine) provide evidence for an adenosine receptor of the A2-subtype being involved in glucagon secretion.     

Prejunctional stimulation of cholinergic nerves in rat airway smooth muscle by an adenosine analogue

The effect of 5′-N-ethylcarboxamidoadenosine (NECA) on rat bronchial smooth muscle was examined in vitro. Both the nerve mediated muscle contraction induced by electrical stimulation and the potassium evoked release of [³H]ACh were enhanced by NECA. The apparent affinity (EC₅₀) of NECA in the contraction experiments was 0.30 ± 0.06 μM. The adenosine (ADO) receptor antagonist, 8-phenyltheophylline (8-PT), inhibited the NECA induced potentiation of both the electrical induced contraction and the potassium evoked release of [³H]ACh. The EC₅₀ and intrinsic activity of exogenous ACh were not altered in the presence of NECA (1 μM) in experiments where smooth muscle contraction were measured, indicating that NECA has a prejunctional effect and not a postjunctional effect on muscarinic receptors. The new A₂ specific ADO receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) and ADO also enhanced the nerve-mediated contraction (EC₅₀ = 35 ± 8 μM and 69 ± 20 μM, respectively). 8-PT (10 μM) and enprofylline (ENPF) (10 μM) inhibited the electrically induced contraction by 55 ± 16% and 45 ± 5% respectively. The potassium evoked release, however, was stimulated 56 ± 6% and 39 ± 7% by 50 μM 8-PT and ENPF respectively. The results provide evidence for a NECA specific ADO receptor in rat bronchi that is most likely prejunctional. Stimulation of this receptor, which may be of an A₂ receptor subtype, enhances the nerve mediated release of ACh and thereby induce contraction of the bronchial smooth muscle.     

Cholinergic mechanism of acid secretion in the dog: an in vivo and in vitro comparison

The effect of cholinergic stimulation on gastric acid secretion was examined in mongrel dogs using an in vivo and an in vitro preparation for comparison. The gastric fistula dog was used for the in vivo studies, and methacholine was infused directly into the artery supplying the fundus of the stomach to avoid systemic hemodynamic changes. Isolated parietal cells were used for the in vitro studies. In vivo, methacholine infused at 1 microgram/min was found to stimulate gastric acid secretion that was inhibited 98.5 +/- 1.4% by intravenously administered metiamide (H2 blocker) and 72 +/- 11% by intra-arterially administered glucagon. Glucagon had no effect on histamine stimulated acid secretion. In vitro, increasing concentrations of methacholine from 10(-7), 10(-6) to 10(-5) M stimulated [14C]aminopyrine uptake into parietal cells in a concentration dependent manner. This effect of methacholine was unaltered by 10(-4) M metiamide or 10(-6) M glucagon. However, atropine 10(-5) M totally blocked the stimulatory effect of methacholine. Our data suggest that the cholinergic mechanism of acid secretion is different when examined in vivo vs. in vitro even in the same species. In vivo histamine dependency has a major contribution to the cholinergic mechanism of acid secretion, whereas in vitro the interaction of the cholinergic agonist at the muscarinic receptor results in acid stimulation that does not require the presence of histamine.     

A1 and A2A adenosine receptor modulation of alpha 1-adrenoceptor-mediated contractility in human cultured prostatic stromal cells

1. This study investigated the possibility that adenosine receptors modulate the alpha(1)-adrenoceptor-mediated contractility of human cultured prostatic stromal cells (HCPSC). 2. The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamido-adenosine (NECA; 10 nm-10 microm), and the A(1) adenosine receptor selective agonist, cyclopentyladenosine (CPA; 10 nm-10 microm), elicited significant contractions in HCPSC, with maximum contractile responses of 18+/-3% and 17+/-2% reduction in initial cell length, respectively. 3. In the presence of a threshold concentration of phenylephrine (PE) (100 nm), CPA (1 nm-10 microm) caused contractions, with an EC(50) of 124+/-12 nm and maximum contractile response of 37+/-4%. The A(1) adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 100 nm) blocked this effect. In the presence of DPCPX (100 nm), NECA (1 nm-10 microm) inhibited contractions elicited by a submaximal concentration of PE (10 microm), with an IC(50) of 48+/-2 nm. The A(2A) adenosine receptor-selective antagonist 4-(2-[7-amino-2-[furyl][1,2,4]triazolo[2,3-alpha][1,3,5,]triazin-5-yl amino]ethyl)phenol (Zm241385 100 nm) blocked this effect. 4. In BCECF-AM (10 microm)-loaded cells, both CPA (100 pM-1 microm) and NECA (100 pm-10 microm) elicited concentration-dependent decreases in intracellular pH (pH(i)), with EC(50) values of 3.1+/-0.3 and 6.0+/-0.3 nm, respectively. The response to NECA was blocked by Zm241385 (100 nm; apparent pK(B) of 9.4+/-0.4), but not by DPCPX (100 nm). The maximum response to CPA was blocked by DPCPX (100 nm), and unaffected by Zm241385 (100 nm). 5. NECA (10 nm-10 microm) alone did not increase [(3)H]-cAMP in HCPSC. In the presence of DPCPX (100 nm), NECA (10 nm-10 microm) caused a concentration dependent increase in [(3)H]-cAMP, with an EC(50) of 1.2+/-0.1 microm. This response was inhibited by Zm241385 (100 nm). CPA (10 nm-10 microm) had no effect on cAMP, in the presence or absence of forskolin (1 microm). 6. These findings are consistent with a role for adenosine receptors in the modulation of adrenoceptor-mediated contractility in human prostate-derived cells.     

Functional characterization of the adenosine receptor mediating inhibition of peristalsis in the rat jejunum.

1. The non-selective adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), is a potent inhibitor of morphine withdrawal diarrhoea in rats. More recently we found that NECA exerts its antidiarrhoeal effect by inhibiting secretion in both the jejunum and ileum and also by inhibiting peristalsis in the ileum. The specific aim of this study was to characterize the receptor in the rat jejunum mediating inhibition of peristalsis via functional studies using a range of metabolically stable adenosine analogues based on the pharmacological criteria of relative agonist and antagonist potencies. 2. Peristalsis in the rat isolated jejunum was achieved by raising the pressure to between 7-11 cmH2O for 3 min followed by a 3 min rest period (pressure at zero). The mean rate of peristalsis during inflation was 7.3 +/- 0.1 peristaltic waves per 3 min and this rate remained consistent for up to 30 min, in 5 separate tissues. The inhibitory effects of the adenosine analogues were quantified by expressing their effects as a % reduction in the mean number of peristaltic contractions derived from the control tissues. 3. The rank order of agonist potency to reduce the rate of peristalsis was: N6-cyclopentyladenosine (CPA) > NECA > R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) > chloroadenosine (2-CADO) > S-PIA > 2-phenylaminoadenosine (CV-1808). This order complies well with the rank order of agonist potency that represents the activation of the A1 receptor subtype (CPA > R-PIA = CHA = > NECA > 2-CADO > S-PIA > CV-1808).(ABSTRACT TRUNCATED AT 250 WORDS)     

Declining catecholamine secretion in adrenal medulla on prolonged stimulation with acetylcholine

Perfused pig adrenal glands and cortex-free ox adrenal medullae were stimulated by continuous infusion of 10(-4) M acetylcholine (ACh). Secretion of adrenaline rose to a maximum in approximately 5 min but, after a further 15 min, declined to 36 +/- 19% (+/- S.D.) of maximum for pig (N = 5) and 27 +/- 10% of maximum for ox (N = 3), in spite of continued infusion of ACh. After 20 min, no further significant decline was detectable. Nevertheless, in ox medullae, oxygen consumption measured after stimulation showed no significant change relative to the pre-stimulation value, indicating that the decline in secretion did not arise from a failure of oxidative energy metabolism. In 4 pig adrenal glands subjected to a 1 hr infusion of ACh, adrenaline secreted in the last 20 min was only 52 +/- 10% of that secreted in the first 20 min but, after a 2 hr rest interval, recovered to 74 +/- 18% (P less than 0.05) in the first 20 min of a second 1 hr stimulation. In the same glands, no reproducible recovery was detectable for noradrenaline and, by the last 20 min of the second 1 hr stimulation, noradrenaline secretion had declined to 36 +/- 20% of the initial value, even though only 15% of the noradrenaline originally in the gland had been secreted. It is concluded that, while decline and recovery of adrenaline secretion may, in part, have arisen from desensitization and resensitization of the ACh receptor; the decline in noradrenaline secretion arose mainly from depletion of a readily secreted pool which was considerably smaller than the total in the gland.     

Involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of porcine coronary artery endothelial cells

This study investigated the effects of the following adenosine agonists: 5;-ethylcarboxamidoadenosine (NECA), N6-cyclopentyadenosine (CPA) 2-[p-(2-carboxyethyl)]phenylamino-5;N-ethylcarboxamidoadenosine (CGS-21680), and 2-chloroadenosine (CAD) and its antagonist, 4-(2-[7-amino-2-[2-furyl]]1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385), a selective A2A adenosine receptor antagonist, and the involvement of the K+ATP and KCa channels on the resting membrane potential (RMP) of confluent monolayers of cultured porcine coronary artery endothelial cells (PCAECs). Adenosine agonists and K+ATP channel openers (pinacidil, cromakalim) hyperpolarized cultured PCAECs. The average RMP was -32.31 +/- 1.2 mV. Adenosine agonists at 10-5 M caused a significant increase in RMP to -65.0 +/- 1.5 mV for CAD (a nonselective adenosine receptor agonist) to -75.9 +/- 1.6 mV for CGS-21680 (a selective A2A receptor agonist) and to -87.0 +/- 3.5 mV for NECA (a nonselective A1/A2A/A2B receptor agonist). Pinacidil and cromakalim at 10 microM increased the membrane potential to -76.2 +/- 1.2 mV and -75.22 +/- 0.12 mV, respectively. The hyperpolarization induced by adenosine receptor agonists and KATP openers was inhibited by an application of the K+ATP channel blocker glibenclamide (10 microM), indicating the involvement of the K+ATP channel in the adenosine-mediated hyperpolarization of PCAECs. Moreover, 1-EB10, a selective opener of the maxi-KCa channel, hyperpolarized PCAECs, and the effect of 1-EB10 was completely blocked by a selective, irreversible blocker of the high conductance KCa (maxi-K) channels (penitrem A), but it only partially blocked the effect of NECA. ZM-241385 has no effect on hyperpolarization elicited by K+ATP and KCa channel openers. However, ZM-241385 significantly blocked the hyperpolarization effect of CAD and CGS-21680. ZM-241385 partially blocked the hyperpolarizing effect of NECA, and a combination of ZM-241385 and penitrem A further blocked the hyperpolarizing effect of NECA. These results further support the involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of PCAECs.     

Acetylcholine-induced contractions in the perforating branch of the human internal mammary artery: protective role of the vascular endothelium

The effect of acetylcholine (ACh) on the isolated, nonprecontracted perforating branch of the human internal mammary artery (HIMA) was investigated. ACh induced concentration-dependent contractions of nonprecontracted rings with denuded endothelium (pEC(50) = 6.72 +/- 0.02, E(max) = 88.8% of contractions induced by phenylephrine, 10(-5) mol/l) and was without effect on arterial segments with intact endothelium. An inhibitor of nitric oxide synthase, N(G)-monomethyl-L-arginine (L-NMMA), or indometacin, a cyclooxygenase inhibitor, had no effect on acetylcholine-induced contractions of rings of the perforating branch of HIMA with denuded endothelium (pEC(50) = 6.76 +/- 0.03 and 6.62 +/- 0.05, respectively). In the presence of indometacin, ACh did not evoke contractions of arterial segments with intact endothelium. In contrast, in the same type of preparations ACh induced contractions in the presence of L-NMMA (E(max) = 34%). The muscarinic receptor antagonists atropine (no selectivity), pirenzepine (M(1)), methoctramine (M(2)), and p-fluoro-hexahydro-sila-difenidol (M(1)/M(3)) competitively antagonized the response to ACh. The pA(2) values were 9.60 +/- 0.10, 6.99 +/- 0.02, 6.37 +/- 0.17, and 8.02 +/- 0.06, respectively. In conclusion, the results obtained indicate that secretion of nitric oxide from vascular endothelium may protect the perforating branch of HIMA against the contractile effects of ACh. On the basis of differential antagonist affinity, it can be suggested that the muscarinic receptors involved in the ACh-induced contractions of the isolated perforating branch of the HIMA are predominantly of the M(3) subtype.     

Inhibition by endothelin-1 of cholinergic nerve-mediated acetylcholine release and contraction in sheep isolated trachea.

1. The relative roles of ETA and ETB receptor activation on cholinergic nerve-mediated contraction and acetylcholine (ACh) release were examined in sheep isolated tracheal smooth muscle. 2. Electrical field stimulation (EFS; 90 V, 0.5 ms duration, 1 Hz, 10 s train) applied to sheep isolated tracheal smooth muscle strips induced monophasic contractile responses that were abolished by either 1 microM tetrodotoxin or 0.1 microM atropine, but were insensitive to 10 microM hexamethonium and 100 microM L-NAME. Thus, EFS-induced contractions resulted from the spasmogenic actions of ACh released from parasympathetic, postganglionic nerves. 3. As expected, sheep isolated tracheal smooth muscle preparations did not contract in response to the ETB receptor-selective agonist, sarafotoxin S6c (0.1-100 nM). However, sarafotoxin S6c caused a concentration-dependent and transient inhibition of EFS-induced contractions. The inhibitory effect induced by a maximally effective concentration of sarafotoxin S6c (10 nM; 72.1 +/- 5.7%, n = 6) was abolished in the presence of the ETB receptor-selective antagonist BQ-788 (1 microM). Contractile responses to exogenously administered ACh (10 nM-0.3 mM) were not inhibited by sarafotoxin S6c (1 or 10 nM; n = 7). 4. In contrast to sarafotoxin S6c, endothelin-1 induced marked contractions in sheep isolated tracheal smooth muscle. These contractions were inhibited by BQ-123, consistent with an ETA receptor-mediated response. In the presence of BQ-123 (3 microM), endothelin-1 produced a concentration-dependent inhibition of EFS-induced contractions (30 nM endothelin-1, 68.9 +/- 10.2% inhibition, n = 5). These responses were inhibited by 1 microM BQ-788, indicative of an ETB receptor-mediated process. Endothelin-1 was about 3 fold less potent than sarafotoxin S6c. 5. EFS (90 V, 0.5 ms duration, 1 Hz, 15 min train) induced the release of endogenous ACh (1.94 +/- 0.28 pmol mg-1 tissue, n = 12), as assayed by h.p.l.c. with electrochemical detection. EFS-induced release of ACh was inhibited to a similar extent by 100 nM endothelin-1 (47 +/- 4%, n = 9) and 10 nM sarafotoxin S6c (46 +/- 9%, n = 3). These effects of endothelin-1 on ACh release were inhibited by 1 microM BQ-788 alone (n = 4), by BQ-788 in the presence of 3 microM BQ-123 (n = 4), but not by 3 microM BQ-123 alone (n = 5). 6. In summary, sheep isolated tracheal smooth muscle contains two anatomically and functionally distinct endothelin receptor populations. ETA receptors located on airway smooth muscle mediate contraction, whereas ETB receptors appear to exist on cholinergic nerves that innervate tracheal smooth muscle cells and mediate inhibition of ACh release. The inhibitory effect of ETB receptor stimulation on cholinergic neurotransmission is in stark contrast to the enhancing effects hitherto described in the airways.     

Developmental changes in endothelium-dependent pulmonary vasodilatation in pigs.

1. We compared in vitro endothelium-dependent vasorelaxant responses to acetylcholine (ACh) and the endothelium-independent vasodilator response to sodium nitroprusside (SNP) in prostaglandin F2 alpha (PGF2 alpha)-precontracted muscular pulmonary arteries (PA) from pigs aged 5 min to 2 h (neonatal), 3-10 days, 3-8 weeks and adults. 2. In the pulmonary artery (PA) rings from neonatal animals, the vasodilator response to ACh was negligible. However, responses to ACh were present in all PA rings from older animals, being greatest at 3-10 days and then decreasing with age (P less than 0.001, ANOVA). ACh (30 microM) induced a 1 +/- 1%, 92 +/- 9%, 62 +/- 5% and 51 +/- 6% reduction of the PGF2 alpha-generated tension in neonatal, 3-10 days, 3-8 weeks and adult groups, respectively. 3. The relaxant response to SNP was present in the PA rings from all age groups and increased with age (P less than 0.001, ANOVA). SNP (1 microM)-induced relaxation was 55 +/- 9%, 73 +/- 7%, 97 +/- 5% and 93 +/- 6% in neonatal, 3-10 days, 3-8 week and adult groups, respectively. 4. Removal of the vascular endothelium abolished the relaxant response to ACh but had no effect on the response to SNP in any groups. 5. NG-monomethyl-L-arginine (30 microM), a nitric oxide synthesis inhibitor, inhibited the response to ACh but not to SNP. The lipoxygenase inhibitor, nordihydroguaiaretic acid, had no significant effect on responses to ACh or SNP in any group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of protease inhibitors and calmodulin inhibitors on Ca2+ influx and catecholamine secretion in isolated bovine adrenal medullary cells

Acetylcholine (ACh) (10(-4) M) induced Ca2+ influx (21.2 nmol/mg cell protein/3 min) and catecholamine (CA) secretion (11.5% of total CA content) in isolated bovine adrenal medullary cells. Both excess K+ (56 mM KCl) and a calcium ionophore (A-23187: 10(-5) M) also induced CA secretion (9.7 and 7.5%, respectively, of total CA content). Protease inhibitors [gabexate mesilate (FOY), TLCK], a synthetic substrate of protease (TAME) and calmodulin inhibitors (W-7, trifluoperazine, promethazine, chlorpromazine) inhibited Ca2+ influx and CA secretion induced by ACh. Protease inhibitors and a substrate of protease had no effect on CA secretion induced by excess K+, and a high concentration of calmodulin inhibitors was required to inhibit excess K+-induced CA secretion. CA secretion induced by A-23187 was not inhibited by these inhibitors and the substrate.     

Effects of amino acids, glucagon, insulin and acetylcholine on cyclic nucleotide metabolism and amylase secretion in isolated mouse pancreatic fragments

The effects of amino acids, exogenous islet hormones and acetylcholine on cyclic nucleotide metabolism and amylase secretion in the isolated mouse pancreas have been investigated. The changes in levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) were measured at different times during exposure of pancreatic fragments to amino acids (L-alanine and L-arginine), islet hormones (insulin and glucagon) or acetylcholine (ACh). L-Alanine (1-20 mM) evoked a transient increase in cyclic AMP concentration accompanied by an initial decrease and subsequent increase in the tissue concentration of cyclic GMP. L-Arginine (1-20 mM) induced a complex triphasic change in cyclic AMP concentrations involving an initial rise and a delayed sustained elevation. The changes in levels of cyclic GMP increased only transiently. The effects of insulin (10(-6) M) and to some extent glucagon (5 X 10(-7) M) resembled those seen with L-arginine. The effects of amino acids and islet hormones were all dose-dependent. ACh (10(-7) M) elicited a marked reduction in cyclic AMP concentration and this was accompanied by a concomitant increase in the level of cyclic GMP. The amino acids and the islet hormones had no significant effect on amylase secretion whereas ACh, of course, evoked a large increase in amylase output. The results with the amino acids and islet hormones reveal a clear dissociation between cyclic nucleotide changes and amylase secretion and further suggest that the marked reciprocal changes in cyclic AMP and cyclic GMP concentrations may constitute an important physiological role for the cyclic nucleotides to regulate amino acid transport in the pancreas.     

Differences between the vasorelaxant activity of adenosine-receptor agonists on guinea-pig isolated aorta precontracted with noradrenaline or phenylephrine

The relaxant effect of adenosine and 5'-(N-ethylcarboxamido)adenosine (NECA) against alpha-adrenoceptor-mediated contractile tone in guinea-pig isolated aortic rings has been examined to determine if this A2B-receptor-mediated relaxation was dependent upon the contracting agent, and whether the contractions were dependent upon intracellular or extracellular calcium. Relaxation responses were consistently greater for aortic rings pre-contracted with phenylephrine (3x10(-6) M) than for rings pre-contracted with noradrenaline (3x10(-6) M). Maximum inhibition by NECA was significantly greater for phenylephrine-contracted aortae than for noradrenaline-contracted (81.9+/-2.8% compared with 25.0+/-1.5%). These differences persisted in the presence of beta- and alpha2-adrenoceptor blockade and could not, therefore, be attributed to stimulation of these receptors by noradrenaline. The ratio of the contractions obtained before and in the presence of adenosine or NECA was compared with the control ratio obtained before and after vehicle. Experiments were performed both in the presence of normal calcium levels and under calcium-free conditions. In normal-calcium medium, NECA inhibited phenylephrine-induced contractions (test ratio, 76.7+/-3.9%; control ratio, 133.1+/-9.8%) to a greater extent than noradrenaline-induced contractions (108.4+/-4.1 and 123.4+/-4.9%); adenosine similarly inhibited phenylephrine-induced contractions more than those induced by noradrenaline. Under calcium-free conditions, adenosine (36.7+/-11.9 and 110.7+/-26.6%) and NECA (55.2+/-9.1 and 87.1+/-14.9%) were only effective against phenylephrine-induced contractions. This suggests that activation of the A2B-receptor by these agonists inhibited intracellular mobilization of calcium for phenylephrine-induced contractions only. The effects on extracellular calcium influx were examined for phenylephrine- and noradrenaline-induced contractions in normal-calcium medium but in the presence of ryanodine to prevent intracellular calcium mobilization. NECA inhibited phenylephrine-induced contractions (77.3+/-12.4 and 111.4+/-9.3%), presumably by interfering with influx of calcium through receptor-operated calcium channels. In contrast, NECA failed to reduce noradrenaline-induced contractions (121.5+/-10.7 and 122.4+/-11.6%), suggesting that the effect on noradrenaline is predominantly via interaction with intracellular calcium. Adenosine was consistently a more effective relaxant than NECA, possibly because of an additional intracellular component of the response. We conclude that adenosine receptor agonists inhibit phenylephrine-induced contractions of guinea-pig aorta more selectively than noradrenaline-induced contractions. A2B-receptor stimulation might reveal a fundamental difference between the modes of contraction elicited by these two alpha-adrenoceptor agonists.     

EXAMINATION OF ADENOSINE RECEPTOR-MEDIATED RELAXATION OF THE PIG CORONARY ARTERY

1. The adenosine receptors mediating relaxation of porcine isolated left anterior descending coronary arteries (LAD) and the effects of the level and type of preconstriction on the responses to adenosine analogues were examined in the present study. 2. Relaxation responses to the non-selective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA) were endothelium independent. N-Ethylcarboxamidoadenosine, GR 79236 (A1 receptor selective) and 8-cyclopentyl-1,3-dipropylxanthine (CGS 21680) (A2A receptor selective) produced full relaxation in LAD precontracted to 50% of the response to potassium depolarization with the thromboxane receptor agonist U46619. The order of potency was CGS 21680 = NECA > GR 79236, consistent with that defining the A2A receptor subtype. 3. 3,7-Dimethyl-1-propargylxanthine (DMPX; A2 receptor selective) competitively antagonized NECA and CGS 21680 with pKB values of 4.95 +/- 0.09 and 5.06 +/- 0.22, respectively. The A1 receptor selective antagonist 1,3-[3H]-dipropyl-8-cyclopentylxanthine (DPCPX) had no effect on NECA relaxation, even in the presence of DMPX. 4. The sensitivity to relaxation by NECA was dependent on the precontracting agent. Arteries precontracted with endothelin (ET)-1 were most sensitive to NECA, U46619-precontracted arteries were intermediate and KCl-precontracted arteries were least sensitive. 5. The potency of NECA was reduced when the preconstriction level was increased from 50 to 90% of maximum in U46619-precontracted arteries (pEC50 7.94 +/- 0.12 and 7.35 +/- 0.04, respectively) and, in KCl-precontracted arteries, both the potency and maximum effect of NECA were reduced when the preconstriction level increased from 50 to 80% of maximum (pEC50 7.52 +/- 0.13 and 6.91 +/- 0.26, respectively; maximum responses 82.5 +/- 10.2 and 23.9 +/- 3.6%, respectively, of the preconstricted tone). Relaxation responses to NECA were independent of the level of precontraction in ET-1-precontracted arteries. 6. In porcine LAD, relaxation responses to adenosine analogues were endothelium independent and were mediated via A2A adenosine receptors. Responses to NECA were dependent on both the level and type of preconstriction.     

Cyclo-oxygenase inhibitors antagonize indirectly evoked contractions of the guinea-pig isolated ileum by inhibiting acetylcholine release

The effects of indomethacin, sodium meclofenamate and ketoprofen on the contractile responses of the guinea-pig isolated ileum to directly and indirectly evoked stimuli were investigated. The effects of the cyclo-oxygenase inhibitors on acetylcholine (ACh) release from plexus containing longitudinal muscle strips were also studied. The cyclo-oxygenase inhibitors reduced contractile responses to transmural stimulation (TMS) and nicotine at concentrations which had no effect on ACh-induced contractions. In whole ileum preparations (WIP) indomethacin and ketoprofen (40 micrograms ml-1) reduced TMS responses by 17 +/- 1.8% and 12 +/- 1.8% (n = 6), respectively (30 min incubation). In longitudinal muscle strips (LMS) in which Auerbach's plexus is exposed, indomethacin and ketoprofen (1 microgram ml-1) reduced TMS responses by 28 +/- 2.3% and 34 +/- 2.7% (n = 6), respectively (10 min incubation). Thus the cyclo-oxygenase inhibitors were up to 80 times more effective in LMS than in WIP. The drugs were similarly more effective in blocking nicotine contractions in LMS than in WIP. The cyclo-oxygenase inhibitors reduced basal and stimulated ACh release from LMS. For example, indomethacin (1 microgram ml-1) reduced stimulated ACh release by 35% after 10 min incubation. The percentage inhibition increased to 79% after 40 min incubation (n = 6). Prostaglandin E2 (PGE2) (0.1-2.5 ng ml-1) restored the contractile responses and ACh release depressed by the cyclo-oxygenase inhibitors but not the contractile responses depressed by atropine. PGF2 alpha had no effect on mechanical responses or ACh release depressed by the cyclo-oxygenase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of RP 73401, a novel,potent and selective phosphodiesterase type 4 inhibitor,on contractility of human,isolated bronchial muscle.

1. The aim of this study was to investigate the smooth muscle relaxant effects of the novel, selective phosphodiesterase (PDE) type 4 inhibitor, RP 73401 in comparison with the classical PDE 4 inhibitor, rolipram, the non-selective PDE inhibitor, theophylline and the beta-adrenoceptor agonist, isoprenaline on the human, isolated bronchus. 2. At resting tone, the rank order of potency (pD2) for the relaxants was RP 73401 > or = rolipram > or = isoprenaline >> theophylline. In terms of maximum relaxation produced (Emax) the PDE 4-selective inhibitors were similar, but the maximal effects (70-75% of theophylline, 3 mM) were lower than that observed with isoprenaline (98% of theophylline, 3 mM) or theophylline itself (100%). 3. On the human isolated bronchus pre-contracted with acetylcholine (ACh, 0.1 or 1.0 mM), the rank order of potency remained the same. The maximal responses to RP 73401 and rolipram were however markedly reduced (Emax 39.9-46.6%) compared with isoprenaline (Emax 79-85%). 4. In tissues pre-contracted with ACh (0.1 mM), RP 73401 and rolipram (10(-9)-10(-7) M) significantly and concentration-dependently increased tissue sensitivity to isoprenaline. RP 73401 and rolipram were similar in potency. Both selective PDE 4 inhibitors also significantly increased the maximal relaxant effects of isoprenaline. These effects were not observed with the PDE 3 inhibitor, siguazodan. 5. In terms of retention by tissues (an index of duration of action), the onset of action of RP 73401 (2.11 +/- 0.53 min) and rolipram (1.70 +/- 0.45 min) was significantly slower than that of isoprenaline (0.33 +/- 0.06 min) or theophylline (1.17 +/- 0.25 min). The retention of RP 73401 (89.0 +/- 21.9 min) on the human isolated bronchial tissues after washing was however dramatically longer than that of rolipram (18.3 +/- 4.5 min), theophylline (3.43 +/- 0.58 min) or isoprenaline (2.81 +/- 0.31 min). 6. These data indicate that RP 73401 is a potent and long acting relaxant of human bronchial muscle in vitro. RP 73401 is more potent than the classical PDE 4-selective inhibitor rolipram and the non-selective PDE inhibitor theophylline and is retained in bronchial tissue for a much longer period of time.     

Effect of quipazine, a serotonin-like drug, on striatal cholinergic interneurones.

Quipazine (30 mg/kg i.p., 60 min), a serotonin-like drug increased ACh levels in the striatum (37%) but was without effect on the transmitter content in the hippocampus and the parietal cortex of the rat. Added in vitro(10(-5) M) or injected in vivo, quipazine did not affect choline acetylase and cholinesterase activities in striatal tissue. The drug effect on striatal ACh levels did not appear to be related to an interaction with dopamine metabolism. Indeed quipazine still increased striatal ACh levels after degeneration of the dopaminergic neurons had been induced by local injection of 6-OH-DA. p-Chlorophenylalanine (PCPA) pretreatment (300 mg/kg, 48 and 24 h before the experiment) definitely prevented the quipazine effect on ACh levels. This result suggested that the drug may partially act by its interference with 5-HT metabolism. 5-Methoxy-N,N-dimethyltryptamine (10 mg/kg, i.p., 30 min), a serotonergic agonist, induced a weak but significant increase in ACh levels. These data provide some preliminary evidence for the existence of an inhibitory control of the cholinergic interneurones by the serotonergic neurones projecting to the striatum. However, the lack of effect of 5-hydroxytryptophan (100 mg/kg i.p.), PCPA (2 x 300 mg/kg i.p.) and of Lilly 110 140 (10 mg/kg i.p.) and chlorimipramine (10 mg/kg i.p.), two potent inhibitors of 5-HT uptake, on striatal ACh levels indicate that further experiments are required to retain this hypothesis.     

Regional and endothelial differences in cyclosporine attenuation of adenosine receptor-mediated vasorelaxations

The present study investigated the acute effects of the immunosuppressant drug cyclosporine A on vasorelaxations evoked via activation of adenosine receptors in the phenylephrine-preconstricted rat perfused kidney and isolated aorta. The roles of endothelial relaxing factors in this interaction were also evaluated. The adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA; kidney, 6 x 10(-9)-1 x 10(-7) mol; aorta, 1 x 10(-9)-1 x 10(-5) M) elicited dose-dependent vasorelaxations. In the perfused kidney, NECA responses were similarly and significantly attenuated by N-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) or tetraethylammonium (K channel blocker) versus no effect for diclophenac (cyclooxygenase inhibitor). NECA relaxations in the aorta were reduced by the three inhibitors; the reduction in the response evoked by the highest dose of NECA (1 x 10(-5) M) amounted to 37.7 +/- 2.0% (L-NAME), 19.8 +/- 1.7% (tetraethylammonium), and 29.4 +/- 1.1% (diclophenac). A combination of the three inhibitors almost abolished NECA relaxations in the two preparations. Cyclosporine (2 microM) reduced NECA relaxations in the two preparations. In the aorta, cyclosporine attenuation of NECA responses was significantly reduced after exposure to L-NAME or diclophenac but not tetraethyl-ammonium, suggesting selective involvement of nitric oxide and vasodilator prostanoids in the interaction. In contrast, the cyclosporine attenuation of NECA responses in the kidney was reduced by L-NAME or tetraethylammonium. L-arginine, a nitric oxide substrate, partially restored NECA relaxations in cyclosporine-treated preparations. These findings demonstrate that cyclosporine attenuates endothelium-dependent vasorelaxations elicited via activation of adenosine receptors and highlight the interesting possibility that the relative contribution of the endothelial relaxing factors to cyclosporine-NECA interaction is largely region dependent.     

Subtype-specific actions of beta-amyloid peptides on recombinant human neuronal nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4) expressed in Xenopus laevis oocytes

Two-electrode voltage-clamp electrophysiology has been used to study the actions of two amyloid peptides (Abeta(1-42), Abeta(1-40)) on alpha7, alpha4beta2 and alpha3beta4 recombinant human neuronal nicotinic acetylcholine receptors (nicotinic AChRs), heterologously expressed in Xenopus laevis oocytes.The application of Abeta(1-42) or Abeta(1-40) (1 pM-100 nM) for 5 s does not directly activate expressed human alpha7, alpha4beta2 or alpha3beta4 nicotinic AChRs.Abeta(1-42) and Abeta(1-40) are antagonists of alpha7 nicotinic AChRs. For example, 10 nM Abeta(1-42) and Abeta(1-40) both reduced the peak amplitude of currents recorded (3 mM ACh) to 48+/-5 and 45+/-10% (respectively) of control currents recorded in the absence of peptide. In both the cases the effect is sustained throughout a 30 min peptide application and is poorly reversible.Abeta(1-42) and Abeta(1-40) (10 nM) enhance currents recorded in response to ACh (3 mM) from oocytes expressing alpha4beta2 nicotinic AChRs by 195+/-40 and 195+/-41% respectively. This effect is transient, reaching a peak after 3 min and returning to control values after a 24 min application of 10 nM Abeta(1-42). We observe an enhancement of 157+/-22% of control ACh-evoked current amplitude in response to 100 nM Abeta(1-42) recorded from oocytes expressing alpha4beta2 nicotinic AChRs.Abeta(1-42) and Abeta(1-40) (10 nM) were without antagonist actions on the responses of alpha3beta4 nicotinic AChRs to ACh (1 nM-3 mM).     

Adenosine induces cyclic-AMP formation and inhibits endothelin-1 production/secretion in guinea-pig tracheal epithelial cells through A(2B) adenosine receptors

1. The adenosine receptor subtype mediating adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin-1 (ET-1) secretion were studied in primary cultures of tracheal epithelial cells. 2. Adenosine analogues showed the following rank order of potency (pD(2) value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5'-N-ethylcarboxyamidoadenosine (NECA, A(1)/A(2A)/A(2B), pD(2): 5.44+/-0.16)>adenosine (ADO, non selective, pD(2): 4.99+/-0. 09; 71+/-9% of NECA response) >/=2-Cl-adenosine (2CADO, non selective, pD(2): 4.72+/-0.14; 65+/-9% of NECA response)>CGS21680 (A(2A); inactive at up to 100 microM). 3. Cyclic AMP formation stimulated by NECA in guinea-pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA(2) value): Xanthine amine congeners (XAC, A(2A)/A(2B), 7.89+/-0.22)>CGS15943 (A(2A)/A(2B), 7.24+/-0. 26)>ZM241385 (A(2A), 6.69+/-0.14)>DPCPX (A(1), 6.51+/-0. 14)>3n-propylxanthine (weak A(2B), 4.30+/-0.10). This rank order of potency is typical for A(2B)-adenosine receptor. 4. Adenosine decreased basal and LPS-stimulated irET production in a concentration-dependent manner. Moreover, NECA but not CGS21680 inhibited LPS-induced irET production. 5. The inhibitory effect of NECA on LPS-induced irET production was reversed by XAC (pA(2)=8.84+/-0. 12) and DPCPX (pA(2)=8.10+/-0.22). 6. These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea-pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A(2B) subtype. This adenosine receptor may be involved in the regulation of the level of ET-1 production/secretion by guinea-pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.