EctoNucleotidase in cardiac sympathetic nerve endings modulates ATP-mediated feedback of norepinephrine release.

ATP, coreleased with norepinephrine, affects adrenergic transmission by acting on purinoceptors at sympathetic nerve endings. Ectonucleotidases terminate the actions of ATP. Previously, we had preliminary evidence for ectonucleotidase activity in cardiac sympathetic nerve terminals. Therefore, we investigated whether this ectonucleotidase might influence norepinephrine release in the heart. Sympathetic nerve endings isolated from guinea pig heart (cardiac synaptosomes) were rich in Ca(2+)-dependent ectonucleotidase activity, as measured by metabolism of exogenously added radiolabeled ATP or ADP. By its inhibitor profile, ectonucleotidase resembled ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1). Exogenous ATP elicited concentration-dependent norepinephrine release from cardiac synaptosomes (EC(50) 0.96 microM). This release was antagonized by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) and potentiated by the P2Y receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) (30 nM). Norepinephrine release promoted by ATP was also potentiated by the nucleotidase inhibitor 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156) (30 microM) and blocked by a recombinant, soluble form of human E-NTPDase1 (solCD39). In contrast, ARL67156 had no effect on norepinephrine release induced by the nonhydrolyzable analog, alpha, beta-methyleneadenosine-5'-triphosphate (alpha,beta-MeATP). Depolarization of cardiac synaptosomes with K(+) elicited release of endogenous norepinephrine. This was attenuated by PPADS and solCD39 and potentiated by MRS 2179 and ARL67156. Importantly, our results demonstrate that facilitation of ATP-induced norepinephrine release from cardiac sympathetic nerves is a composite of two autocrine components: positive, mediated by P2X receptors, and negative, mediated by P2Y receptors. Modulation of norepinephrine release by coreleased ATP is terminated by endogenous as well as exogenous ectonucleotidase. We propose that ectonucleotidase control of norepinephrine release should provide cardiac protection in hyperadrenergic states such as myocardial ischemia.     

Increased reactivity of murine mesenteric veins to adrenergic agonists: functional evidence supporting increased alpha1-adrenoceptor reserve in veins compared with arteries

These studies examined adrenergic reactivity of mesenteric arteries and veins from deoxycorticosterone acetate-salt (DOCA-salt) hypertensive and sham control mice. We measured constrictions in unpressurized arteries and veins by monitoring vessel diameter using computer-assisted video micros-copy in vitro. Veins were more sensitive than arteries to the constricting effects of norepinephrine (NE) and phenylephrine (PE), but the alpha2-agonists clonidine and UK 14,304 [5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline] did not constrict arteries or veins. Reactivity was not altered in arteries or veins from DOCA-salt mice. We next investigated the mechanism of increased venous reactivity to NE and PE by studying desensitization to maximum concentrations of NE and PE. Sham arteries desensitized to NE and PE more than DOCA-salt arteries, whereas DOCA-salt and sham veins maintained 80% of the initial NE and PE constriction. To determine whether the increased reactivity and resistance to desensitization in veins was due to a greater alpha-adrenoceptor reserve, vessels were incubated with the alkylating agent phenoxybenzamine (PBZ; 0.3, 3, 10, and 30 nM). The NE-elicited initial constriction was reduced by PBZ (3, 10, and 30 nM) in sham but only by PBZ (30 nM) in DOCA-salt veins. All doses of PBZ blocked NE responses in sham and DOCA-salt arteries. These data suggest that mesenteric veins express more alpha1-adrenoceptors than arteries, accounting for greater reactivity and resistance to desensitization compared with arteries.     

Diadenosine polyphosphates cause contraction and relaxation in isolated rat resistance arteries

The effects of diadenosine polyphosphates (APnA; n = 3-6) and adenine nucleotides on contractile reactivity of isolated rat mesenteric resistance arteries (MrA) and superior epigastric arteries (SEA), which display a dense and sparse autonomic innervation, respectively, were evaluated. All agonists examined, except adenosine and AMP, induced contractions. The rank order of potency was similar in both arteries: alpha,beta-methylene ATP (alpha,beta-meATP) > AP5A > AP6A > AP4A > ATP > ADP > AP3A. Contractions were stable during several minutes in SEA but highly transient in MrA. They were reduced after exposure to 10 microM alpha,beta-meATP and by 10 microM of the P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid. During phenylephrine (10 microM)-induced contractions, the agonists induced a further contraction in SEA. In MrA, however, further contraction was followed by marked relaxation. The rank order of relaxing potency was comparable to that of the contractile potency of agonists. Also, the relaxing effects of APnA were blunted by 10 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid and after exposure to alpha,beta-meATP. In vitro and in vivo sympathectomy with 6-hydroxydopamine and removal of the endothelium did not modify the effects of APnA in MrA. Thus, the contractile effects of APnA in resistance arteries 1) are due to a P2X purinoceptor-mediated stimulation of the smooth muscle; 2) depend on the length of the phosphate chain; and 3) are followed by endothelium-independent relaxing effects in MrA but not SEA, which may involve receptors that are similar to those mediating contraction. The regional heterogeneity of APnA effects cannot be attributed to a direct neurogenic influence.     

ATP modulates noradrenaline release by activation of inhibitory P2Y receptors and facilitatory P2X receptors in the rat vas deferens

The role of ATP on the modulation of noradrenaline release elicited by electrical stimulation (100 pulses/8 Hz) was studied in the prostatic portion of rat vas deferens preincubated with [3H]noradrenaline. In the presence of P1 antagonists, the nucleotides 2-methylthioadenosine-5'-triphosphate (2-MeSATP), 2-methylthioadenosine 5'-diphosphate (2-MeSADP), ADP, and ATP decreased electrically evoked tritium overflow up to 44%, with the following order of potency: 2-MeSATP > 2-MeSADP > ADP > or = ATP. The P2Y antagonists reactive blue 2 (RB2) and 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) increased, whereas the P2X antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) decreased evoked tritium overflow. The inhibitory effect of 2-MeSATP was antagonized by RB2 (10 microM) and by 2-MeSAMP (10 microM) but not by the selective P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179; 10 microM). When, besides P1 receptors, inhibitory P2Y receptors were blocked with RB2, alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP), beta,gamma-imidoadenosine 5'-triphosphate (beta,gamma-imidoATP), beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-meATP), 2-MeSATP, and ATP enhanced tritium overflow up to 140%, with the following order of potency: alpha,beta-meATP > 2-MeSATP = ATP = beta,gamma-meATP > or = beta,gamma-imidoATP. The facilitatory effects of alpha,beta-MeATP and beta,gamma-imidoATP were prevented by PPNDS. Under the same conditions, apyrase attenuated, whereas the ectonucleotidase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethylene 5'-triphosphate enhanced tritium overflow, an effect that was prevented by PPNDS. In the prostatic portion of the rat vas deferens, endogenous ATP exerts a dual and opposite modulation of noradrenaline release: an inhibition through activation of P2Y receptors with a pharmacological profile similar to that of the P2Y12 and P2Y13 receptors and a facilitation through activation of P2X receptors with a pharmacological profile similar to that of P2X1 and P2X3, or PX2/P2X3 receptors.     

Cysteine pairs in the third intracellular loop of the muscarinic m1 acetylcholine receptor play a role in agonist-induced internalization

The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol addiction. We investigated the effects of ATP and ethanol on spontaneous inhibitory postsynaptic currents (sIPSCs) and the spontaneous firings in the VTA dopaminergic neurons, obtained using an enzyme-free procedure. These neurons preserved some functional GABA-releasing terminals after isolation. We found that ATP (1-200 microM) either increased or decreased the frequency of sIPSCs and the activity of VTA dopaminergic neurons. The effects of ATP on sIPSC frequency inversely correlated with its effects on dopaminergic neuron activity. The ATP-induced changes in sIPSC frequency were blocked by tetrodotoxin (a sodium channel blocker) and by suramin (a nonselective P2R antagonist). Furthermore, alpha,beta-methylene ATP, a selective P2X(1) and P2X(3) receptor agonist, increased sIPSC frequency, whereas adenosine 5'-[beta-thio]diphosphate, a preferential agonist of P2Y receptors, decreased sIPSC frequency. In experiments testing the effects of ethanol (10 and 40 mM) on sIPSCs, we found that ethanol significantly attenuated ATP-induced increase and enhanced ATP-induced decrease in sIPSC frequency. Taken together, the results demonstrate that multiple subtypes of P2Rs exist on GABA-releasing terminals that make synapses on VTA dopaminergic neurons. It seems that ATP increases sIPSC frequency involving P2X(1) and/or P2X(3) receptors, and ATP decreases sIPSC frequency involving P2YRs. These findings are also consistent with the notion that P2Rs at GABA-releasing terminals on VTA dopaminergic neurons are important targets for ethanol action.     

Characterization and distribution of P2-purinoceptor subtypes in rat pulmonary vessels

To characterize P2-purinoceptors in pulmonary vessels we have examined the effects of ATP analogs on rat isolated pulmonary artery and vein in vitro. The rank order of potency for causing vasoconstriction was: alpha,beta-methylene-ATP (alpha,beta-meATP) greater than beta,gamma-methylene-ATP (beta,gamma-meATP) greater than 2-methylthio-ATP (2m.S.ATP) greater than ATP for arteries; and alpha,beta-meATP much greater than beta,gamma-meATP = 2m.S.ATP greater than ATP for veins, indicating that a P2x receptor was involved. The contractile response to these analogs was virtually abolished after desensitization of P2x-receptors by alpha,beta-meATP. Removal of the endothelial cells enhanced the contractile responses to all of the ATP analogs in both arteries and veins. The rank order of potency for vasodilatation was 2m.S.ATP much greater than ATP = beta,gamma-meATP much greater than alpha,beta-meATP for arteries and 2m.S.ATP much greater than ATP = beta-gamma-meATP, with alpha,beta-meATP being no effect for veins, indicating a P2y receptor. Pretreatment of the pulmonary arteries with the P2y-antagonist reactive blue 2 caused a rightward shift of the dose-response curves to 2m.S.ATP, ATP and beta,gamma-meATP. Reactive blue 2 was only used with the pulmonary arteries. Removal of the endothelium converted the relaxant responses to all the ATP analogs (except to ATP in pulmonary artery) to further contraction. In the pulmonary artery, the small endothelium-independent relaxation induced by ATP was abolished completely by pretreating the vessels with 100 microM theophylline (a P1-purinoceptor antagonist), suggesting that it was due to conversion of ATP to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium-independent and endothelium-dependent contractions mediated by P2X- and P2Y-purinoceptors in canine basilar arteries

Both alpha, beta-methylene ATP and beta, gamma-methylene ATP (P2X selective agonists) were shown to induce transient contraction in intact and endothelium-removed preparations of canine basilar arteries. 2-Methylthio ATP (a P2Y selective agonist) caused transient contraction of intact arteries and this response was nearly abolished by removal of the endothelium. In the presence of alpha, beta-methylene ATP (10(-6) M), the endothelium-independent contractions induced by alpha, beta-methylene ATP itself (10(-6) M) and by beta, gamma-methylene ATP (10(-5) M) were both abolished. The endothelium-dependent contraction induced by 2-methylthio ATP (10(-7) M) was not attenuated by alpha, beta-methylene ATP. The contraction induced by 2-methylthio ATP (10(-7) M) was attenuated markedly by reactive blue 2 (a P2Y antagonist) (3 x 10(-6) M), aspirin (5 x 10(-5) M), OKY-046 (thromboxane A2 synthetase inhibitor) (10(-5) M) and ONO-3708 (thromboxane A2 antagonist) (10(-8) M). However, these agents did not affect the endothelium-independent contraction induced by alpha, beta-methylene ATP (10(-6) M). Neither TMK-777 (a 5-lipoxygenase inhibitor) (10(-7) M) nor superoxide dismutase (100 U/ml) plus catalase (1,000 U/ml) affected either contraction. The present experiments demonstrate that P2X-purinoceptors mediate endothelium-dependent contraction in the canine basilar artery, and that the endothelium-derived contracting factor in this system is probably thromboxane A2.     

Responses to dopamine of isolated human and monkey veins compared with those of the arteries.

Isolated human gastroepiploic vein tributaries responded to dopamine only with contractions, whereas the gastroepiploic artery branches in the same region of the omentum responded with relaxations. Treatment with phentolamine converted the vein contraction to a relaxation, which was not influenced by propranolol but was abolished by droperidol. The relaxation was converted to a contraction by SCH23390 but unaffected by domperidone. Endothelium denudation abolished the relaxation caused by substance P but did not significantly alter the dopamine-induced relaxation. Dopamine increased the cyclic AMP content in the human veins. Monkey mesenteric, renal and portal veins and vena cava contracted in response to dopamine. Treatment with phentolamine converted the contraction to a slight relaxation in the mesenteric and renal veins; however, even in the presence of high concentrations of the alpha adrenoceptor antagonist, no relaxation was induced in the portal vein and vena cava partially contracted with prostaglandin F2 alpha. It is concluded that gastroepiploic veins and arteries in the human omentum respond quite differently to dopamine; the alpha adrenoceptor-mediated contraction predominates over the relaxation mediated via dopamine D1 receptor subtype in the veins, and vice versa in the arteries. Dopamine relaxes the human vein, possibly as a result of increased production of intracellular cyclic AMP by stimulation of D1 receptors. The predominant action of dopamine on alpha adrenoceptors may contribute to increasing venous return and cardiac output.     

Comparison of contractions to serotonin, carbamylcholine and prostaglandin F2 alpha in rat stomach fundus

Contractile effects of serotonin were compared to those of carbamylcholine and prostaglandin (PG) F2 alpha in an effort to characterize serotonergic receptor activation in rat stomach fundus. All three agents elicited concentration-dependent contractions of fundal strips with serotonin (EC50 = 2 X 10(-10) M) being approximately 100-fold more potent than carbamylcholine (EC50 = 2 X 10(-8) M) and PGF2, (EC50 = 10(-8) M). The calcium channel blockers, diltiazem (5 X 10(-7) to 5 X 10(-5) M) and nitrendipine (10(-8) to 10(-5) M), attenuated responses markedly to serotonin and PGF2 alpha whereas having only minimal effects on carbamylcholine-induced contractions. Neither serotonin (10(-11) to 10(-5) M) nor PGF2 alpha (10(-9) to 10(-5) M) altered [3H]inositol monophosphate generation in fundus whereas carbamylcholine (10(-6) to 10(-5) M) increased significantly [3H]inositol monophosphate with 10(-5) M eliciting an 8-fold increase. Strips of fundus contracted submaximally with either serotonin, PGF2 alpha or carbamylcholine were compared for sensitivity to relaxants. Pinacidil (10(-8) to 10(-5) M) was equipotent (EC50 = 0.1 microM) in relaxing serotonin- and PGF2 alpha-contracted tissues. In contrast, pinacidil was 10-fold less potent in relaxing contractions produced by carbamylcholine. Likewise, nitroglycerin (10(-8) to 10(-4) M) and isoproterenol (10(-12) to 10(-7) M) were at least 10-fold more potent in relaxing serotonin- and PGF2 alpha- than carbamylcholine-induced contractions. Thus, in rat fundus, contractions associated with increased phosphoinositide hydrolysis may be more resistant to relaxation than are contractions not associated with altered phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-adrenoceptors in canine mesenteric artery are predominantly 1A subtype: pharmacological and immunochemical evidence.

We wanted to determine which alpha-adrenoceptor subtypes mediate phenylephrine (PE) contraction of dog mesenteric artery in vitro. We studied antagonisms in response to prazosin, 2-(2, 6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane, 5-methylurapidil, N-[2-(2-cyclopropyl methoxy phenoxy)ethyl]5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine HCl (RS 17053), 8-3-[4-(2-methoxyphenyl)-1-piperazinyl]propylcarbamoyl)-3-methyl-4 -oxo-22-phenyl-4H-1-benzopyran 2HCl [SB216469 (Rec 15/2739)], BMY 7378, 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]8-azaspirol++ + [4,5]decane-7,9-dione HCl, MDL 72832, and 7-chloro-2-bromo-3,4,5, 6-tetrahydro-4-methylfurol[4,3,2-ef]3-benzapine. pK(B) values for prazosin, 5-methylurapidil, MDL 72832, and RS-17053 were consistent with action on alpha(1A)-adrenoceptors but decreased with concentration. pK(B) values (9.6) for Rec 15/2739 (alpha(1L/1A)-adrenoceptor selective) were constant. Antagonism by BMY 7378, 7-chloro-2-bromo-3,4,5,6-tetrahydro-4-methylfurol[4,3, 2-ef]3-benzapine, and 8-[2-(1, 4-benzodioxan-2-ylmethylamino)ethyl]8-azaspirol[4,5]de cane-7,9-dione HCl gave pK(B) values between those expected for alpha(1A)- and alpha(1D)-adrenoceptors. Chloroethylclonidine (100 microM) shifted EC(50) values for PE rightward and decreased E(max) values but left large residual responses. After 100 microM chloroethylclonidine, either BMY 7378 (100 nM) or RS-17053 (300 nM) increased EC(50) values for PE contractions with pK(B) values like those of controls. At 6 nM, phenoxybenzamine increased the EC(50) values and reduced E(max) values; prior Rec 15/2739, but not prior BMY 7378, protected receptors against inactivation. An antibody against the alpha(1B)-adrenoceptors immunostained muscle of aorta but not mesenteric artery. We conclude that dog mesenteric artery contains alpha(1A)-adrenoceptors. Discrepancies among responses expected if only these receptors are present may result from pleiotropic functional effects at this receptor and the presence of alpha(1L)-adrenoceptors.     

The P2Y agonist UTP activates cutaneous afferent fibers

The majority of adenosine triphosphate (ATP)-induced nociceptive transduction and pain has been attributed to ionotropic P2X3 receptors. Metabotropic P2Y receptors, some of which bind pyrimidines as well as purines, have received little attention. Here we have examined the ability of P2Y receptor signaling to evoke action potential firing in functionally identified afferent fibers using the skin nerve preparation from adult mouse. The P2Y2/P2Y4 ligand UTP activated sustained action potential firing in 54% of C fibers in a concentration-dependent manner. The effect was specific for P2Y2/P2Y4 receptors, as the P2Y6 ligand UDP never activated C fibers. In comparison to C fibers, few thinly myelinated A-mechanoreceptors (AM) (12%) were activated by UTP. The majority (70-80%) of the UTP-sensitive C and Adelta fibers responded to the algogen capsaicin with a barrage of action potentials, whereas the UTP-insensitive fibers were largely unresponsive to capsaicin. Furthermore, 86% of the UTP-sensitive C fibers and 100% of the UTP-sensitive AM fibers also responded to the P2X agonist alpha,beta-methylene ATP, indicating that P2Y and P2X receptors are widely co-expressed. Surprisingly, a significant proportion (20-40%) of low threshold slowly and rapidly adapting Abeta fibers were also activated by UTP and alpha,beta-methylene ATP. These data indicate that P2Y receptors on the terminals of capsaicin-sensitive cutaneous sensory neurons effectively evoke nociceptive transmission, and support the hypothesis that UTP may be an endogenous nociceptive messenger. Furthermore, P2Y signaling may contribute to mechanotransduction in low threshold Abeta fibers under normal or pathological conditions.     

Heterogenous vascular effects of AP5A in different rat resistance arteries are due to heterogenous distribution of P2X and P2Y(1) purinoceptors

In the accompanying article, we showed that AP5A displayed heterogenous vasoactive effects in rat resistance arteries. It induced a stable vasoconstriction in the superior epigastric artery (SEA) and a transient vasoconstriction in the mesenteric resistance artery (MrA). In the phenylephrine-precontracted MrA AP5A induced a marked vasorelaxation. In this study the noncompetitive inhibition of the AP5A-induced vasoconstriction with pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was found to be significantly stronger in MrA than in SEA. The nonselective P2 purinoceptor antagonist suramin inhibited AP5A-induced vasoconstriction in MrA only. The vasoconstriction by the P2X purinoceptor agonist alpha,beta-methylene ATP was inhibited by with pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid and suramin similarly to that induced by AP5A. Thus, the AP5A-induced vasoconstriction is due to P2X receptor activation, but two different P2X receptors seem to be operational in the two different vessels. The AP5A-induced vasorelaxation of phenylephrine-precontracted MrA was inhibited by the P2Y(1) receptor antagonist ADP3'5'. The vasorelaxation induced by ADPbetaS (P2Y(1) agonist) also was inhibited by ADP3'5'. These findings suggest that AP5A-induced vasorelaxation of MrA is caused by P2Y(1) receptor activation. The P1 (A(2)) receptor antagonist 3, 7-dimethyl-1-propargylxanthine only slightly inhibited AP5A-induced vasorelaxation at high concentrations. Adenosine and the A(2) receptor agonist CGS21680 failed to produce significant vasorelaxation. Therefore, vasorelaxation in MrA does not involve A(2) purinoceptor activation. AP5A-induced vasorelaxation was not inhibited by Ca(2+)- or ATP-dependent K(+) channel blockade with clotrimazole, apamin, or glibenclamide. These data indicate that vasoconstriction in MrA and SEA by AP5A is due to different P2X receptors, and vasorelaxation in precontracted MrA is due to P2Y(1) receptor activation.     

Mechanisms underlying enhanced P2X receptor-mediated responses in the neuropathic pain state

P2X3 and P2X2/3 receptors in dorsal root ganglia (DRG) appear to participate in producing nociceptive responses after nerve injury. However, the mechanisms underlying the receptor-mediated nociception in the neuropathic state remain unclear. Using spared nerve injury (SNI) rats, we found that allodynic and nocifensive (flinch) behavioral responses developed after injury can be reversed by P2X receptor antagonists, indicating an involvement of P2X receptors. Immunocytochemical studies revealed that P2X3 receptors are expressed in small and medium but rarely in large DRG neurons of both normal and SNI rats. Thus, contrary to the conventional view that only large A beta cells mediate allodynia, small and medium cells are intimately involved in P2X3 receptor-mediated allodynia. Measuring ATP levels in the subcutaneous space of the rat paw, we showed that ATP release does not change after SNI. On the other hand, the P2X receptor agonist, alpha beta-methylene ATP produces 3.5-fold larger flinch responses at a 8.0-fold lower dose. Thus, sensitization of P2X3 receptors rather than a change in ATP release is responsible for the neuropathic pain behaviors. We further demonstrated that sensitization of P2X3 receptors arises from an increase in receptor function. ATP-induced P2X3 receptor-mediated currents in DRG neurons is 2.5-fold larger after SNI. The expression of P2X3 receptors on the cell membrane is significantly enhanced while the total expression of P2X3 receptors remained unchanged. Thus, the enhancement of trafficking of P2X3 receptors is likely an important mechanism contributing to the increase in receptor function after nerve injury.     

Postsynaptic alpha adrenoceptors in baboon cerebral and mesenteric arteries

Postsynaptic alpha adrenergic mechanisms were compared in cerebral and mesenteric arteries isolated from the baboon. The contractile response to norepinephrine (NE) of the cerebral artery was potent and similar to that of the mesenteric artery. The EC50 was 3.1 (2.0-5.0) X 10(-7) M for the cerebral artery and 2.6 (1.5-4.8) X 10(-7) M for the mesenteric artery. The maximum contraction expressed as a force developed/cross-sectional area did not differ between the two arteries, whereas that expressed as percentage of 30 mM KCl-induced contraction in the cerebral artery (118 +/- 9%) was less than in the mesenteric artery (145 +/- 6%). Phenylephrine produced a contraction in a manner similar to NE, although the EC50 values in both arteries were 2 to 3 times as large as those for NE. Clonidine produced a moderate contraction in the mesenteric artery (35 +/- 8% of the KCl-induced contraction) but no contraction in the cerebral artery. NE-induced contraction in the cerebral artery was inhibited more prominently by prazosin, a selective alpha-1 adrenoceptor antagonist, than that in the mesenteric artery; the pA2 value for prazosin in the cerebral artery was higher (9.70) than that in the mesenteric artery (8.95). In contrast, there was no difference in pA2 values for either phentolamine or yohimbine between the two arteries. Clonidine-induced contraction in the mesenteric artery was attenuated by prazosin rather than yohimbine at the same concentration used. Thus, it may be concluded that contractile processes related to postsynaptic alpha-1 adrenoceptor stimulation are predominantly operative in baboon cerebral and mesenteric arteries, the cerebral artery being more susceptible to the inhibitory effect of prazosin.     

Propafenone modulates potassium channel activities of vascular smooth muscle from rat portal veins

We have studied the effects of the class Ic antiarrhythmic propafenone on K+ currents in freshly isolated smooth muscle cells from rat portal veins and on the spontaneous contractions in whole tissues. Under Ca2+-free conditions, when cells were clamped at -80 mV (whole-cell configuration) depolarizing steps from -80 to +50 mV induced a family of K+ currents (I(Ktotal)) that mainly comprised the delayed rectifier current [I(K(V))], whereas when held at -10 mV only small-amplitude, noninactivating, currents (I(NI)) were recorded. Propafenone (10 microM) markedly inhibited I(Ktotal), but at potentials positive to +30 mV it also induced a noisy outwardly rectifying current [I(BK(Ca))] that was abolished by iberiotoxin (0.1 microM). Inhibition of I(Ktotal) by propafenone was concentration-dependent (EC50 = 0.059 +/- 0.009 microM). Propafenone also inhibited the transient outward current [I(K(A))] and ATP-sensitive potassium current [I(K(ATP))] induced by levcromakalim (10 microM). Inhibition of I(K(V)), I(K(A)), and I(K(ATP)) by propafenone was voltage-independent. In Ca(2+)-containing conditions propafenone inhibited I(K(V)) and I(BK(Ca)) and immediately abolished spontaneous outward transient K+ currents. In whole veins, propafenone behaved as the K(V) inhibitor 4-aminopyridine, increasing the amplitude and duration of spontaneous contractions. Propafenone also inhibited the inhibitory effects of the K(ATP) channel opener levcromakalim on spontaneous contractions. These results indicate that in vascular smooth muscle cells, propafenone inhibits K(V), K(A), BK(Ca), and K(ATP) channels. These actions correlated with its effects on mechanical activity in whole portal veins.     

Enhanced thromboxane receptor-mediated responses and impaired endothelium-dependent relaxation in human corpus cavernosum from diabetic impotent men: role of protein kinase C activity

We have evaluated the influence of protein kinase C (PKC) activity on penile smooth muscle tone in tissues from diabetic and nondiabetic men with erectile dysfunction. Human corpus cavernosum (HCC) strips were obtained from impotent diabetic and nondiabetic men at the time of penile prosthesis implantation and studied in organ chambers. Contractility responses to a prostanoid precursor, to prostanoids, and to the endothelium-dependent vasodilator acetylcholine were studied. Arachidonic acid (AA; 100 microM) caused cyclooxygenase-dependent relaxation of HCC. This relaxation was impaired in diabetic tissues and normalized by blocking thromboxane (TP) receptors with 20 nM [1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548). Diabetes did not affect prostaglandin (PG)E(1)-induced relaxation, but it reduced relaxation induced by the PGE(1) metabolite PGE(0). This effect was related to an interaction of PGE(0) with TP receptors. Diabetic tissues had reduced endothelium-dependent relaxation, which was partially improved by SQ29548 and completely normalized by the PKC inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X; 1 microM). In HCC from nondiabetic patients, treatment with the PKC activator phorbol-12,13-dibutyrate (0.3 microM) significantly attenuated endothelium-dependent relaxation, an effect prevented by coadministration of GF109203X. Tissues from diabetic patients had enhanced sensitivity to the contractile effects of the TP receptor agonist 9,11-dideoxy-9alpha,11alpha-epoxymethano PGF(2alpha) (U46619) (EC(50) = 0.65 +/- 0.42 and 6.01 +/- 2.28 nM in diabetic and nondiabetic patients, respectively). Inhibition of PKC with 1 microM GF109203X, prevented diabetes-induced hypersensitivity to U46619-induced contractions (EC(50) = 8.55 +/- 3.12 microM). Overactivity of PKC in diabetes is responsible for enhanced contraction and reduced endothelium-dependent relaxation of HCC smooth muscle. Such alterations can result in erectile dysfunction.     

Alpha adrenergic receptor subtypes in human, monkey and dog cerebral arteries

In helical strips of human and monkey cerebral arteries, norepinephrine produced a greater contraction than that in dog cerebral arteries. In monkey cerebral arteries, phenylephrine and norepinephrine produced a similar magnitude of maximum contractions, although the ED50 value of phenylephrine was approximately 5.6 times greater than that of norepinephrine. Clonidine (up to 10(-5) M) did not produce contractions. Dog cerebral arteries responded to phenylephrine in high concentrations with a greater contraction than that induced by norepinephrine and to clonidine with significant contractions. Contractions induced by norepinephrine of human and monkey cerebral arteries were attenuated by low concentrations of prazosin but were not influenced by yohimbine in concentrations up to 10(-8) M. In contrast, norepinephrine-induced contractions of dog cerebral arteries were attenuated by yohimbine but were unaffected by prazosin. It appears that norepinephrine-induced contractions are mediated by alpha-2 adrenoceptors in dog cerebral arteries and by alpha-1 receptors in human and monkey cerebral arteries as well as monkey and dog mesenteric arteries. The relative unresponsiveness of monkey and dog cerebral arteries to adrenergic nerve stimulation may not be explained by a paucity of alpha adrenoceptors in neuroeffector junction.     

Decreased vasopressin responsiveness in vasodilatory septic shock-like conditions

OBJECTIVE: To determine the effect of vasodilatory septic shock-like conditions on vasoconstricting responses to vasopressin and norepinephrine in isolated resistance arteries. DESIGN: Prospective, randomized animal study. SETTING: University research laboratory. SUBJECTS: Male adult Sprague-Dawley rats. INTERVENTIONS: Small mesenteric arteries (outside diameter, 50-150 microm) were cannulated and studied in vitro under physiologic conditions. A vasodilatory septic shock-like state was produced by treatment with the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP), and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Vasoconstricting concentration-response relationships were determined for norepinephrine and vasopressin before and after application of SNAP or SNAP+ IBMX. Synergism between low-dose vasopressin and norepinephrine and between low-dose norepinephrine and vasopressin was determined before and after SNAP or SNAP+IBMX. MAIN RESULTS: Norepinephrine and vasopressin produced concentration-dependent contractions (half-maximal effective concentration [EC(50)] = 2.5 microM and 3.9 nM, respectively) that were significantly inhibited by 1 microM SNAP (EC(50) = 3.6 microM and 8.1 nM, respectively) or 100 microM SNAP + 10 microM IBMX (EC(50) = 10 microM and 8.2 nM, respectively). Low-dose vasopressin significantly increased the responsiveness to norepinephrine (EC50 = 0.5 microM) just as a low-dose norepinephrine significantly enhanced the vasopressin response (EC(50) = 2.3 nM). The synergistic effects of low-dose vasopressin and norepinephrine, or low-dose norepinephrine and vasopressin, were also significantly inhibited by 1 microM SNAP (EC(50) = 2.5 microM and 4.2 nM, respectively) or 100 microM SNAP + 10 microM IBMX (EC(50) = 9 microM and 8.4 nM, respectively). CONCLUSIONS: Vasoconstriction produced by vasopressin or norepinephrine, and the synergistic vasoconstriction produced by the combinations, was inhibited in vasodilatory septic shock-like conditions. Thus, in addition to the well-described vasopressin deficiency in vasodilatory septic shock, these studies indicate that decreased vasopressin responsiveness further contributes to a state of relative vasopressin insufficiency in this condition.