Neuropeptide Y stimulates renal prostaglandin synthesis in the isolated rat kidney: contribution of Ca++ and calmodulin

We have investigated the effects of neuropeptide Y (NPY) on vascular tone and renal output of prostaglandins (PGs) and the mechanism underlying these actions by examining the effects of Ca++ depletion, Ca++ channel blockers and calmodulin inhibitors in the isolated Tyrode's perfused rat kidney. Administration of NPY (0.23-2.3 nmol) into the kidney produced a dose-related renal vasoconstriction and an increase in the output of PGE2 and 6-keto-PGF1 alpha, the stable hydrolysis product of PGI2. Omission of Ca++ (1.8 mM) or addition of Ca++ channel blockers, diltiazem (60 microM) or nifedipine (1.4 microM), to the perfusion fluid abolished the effects of NPY to promote renal vasoconstriction and PG synthesis. Infusion of calmodulin inhibitors, trifluoperazine (2 microM), W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide] (20 microM) or calmidazolium (0.2 microM), attenuated the renal vasoconstriction and the increase in PG output produced by NPY (0.7 nmol). In kidneys perfused with normal Tyrode's solution, infusion of NPY, in a concentration (1.7 X 10(-8) M) that produced only a small transient increase in renal vascular tone and failed to alter the renal output of PGs, enhanced the rise in PGE2 and 6-keto-PGF1 alpha elicited by norepinephrine (0.25 nmol) but not by arginine vasopressin (0.004 nmol) or angiotensin II (0.09 nmol). The renal vasoconstriction elicited by norepinephrine and arginine vasopressin as well as by angiotensin II was enhanced by NPY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of Ca++ and calmodulin to the action of norepinephrine on renal prostaglandin synthesis and vascular tone

We have investigated the contribution of Ca++ and calmodulin to the action of norepinephrine (NE) on prostaglandin (PG) synthesis and vascular tone in the Tyrode's perfused rat kidney. Lowering the Ca++ concentration (0.6 mM) reduced and raising the Ca++ concentration (5.4 mM) enhanced the renal vasoconstriction and PG output elicited by NE. Calcium channel blockers diltiazem or nimodipine inhibited the vasoconstriction and PG output caused by NE. Ca++-free Tyrode's solution containing ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid abolished NE-induced vasoconstriction and reduced PG output by 25 to 38%. Addition of intracellular Ca++ antagonists 8-(diethylamino) octyl 3,4,5 trimethoxybenzoate, dantrolene or ryanodine to Ca++-free Tyrode's solution inhibited NE-induced PG output. Calmodulin inhibitors trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide or calmidazolium diminished PG output and the renal vasoconstriction elicited by NE in the presence and absence of Ca++. Mepacrine and indomethacin inhibited NE-induced renal vasoconstriction and PG output. Arachidonic acid-induced PG output was abolished by indomethacin but was unaltered by mepacrine, Ca++ antagonists or calmodulin inhibitors. We conclude that NE produces renal vasoconstriction by a mechanism that depends primarily on extracellular Ca++ and calmodulin, whereas NE-induced PG output depends on both extra- and intracellular Ca++ and calmodulin.     

Calcium and calmodulin antagonists binding to calmodulin and relaxation of coronary segments

Ca++ antagonist drugs (also known as Ca++ channel blockers) and the calmodulin antagonists trifluoperazine (TFP) and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] were capable of half-maximally relaxing porcine coronary segments at 1.5 X 10(-10) M for felodipine, 6.5 X 10(-8) M for verapamil, 2.6 X 10(-7) M for diltiazem, 7 X 10(-7) M for prenylamine, 7 X 10(-6) M for TFP and 45 X 10(-6) M for W-7. Their correspondent binding to calmodulin was half-maximal at 2.8 X 10(-6), 30 X 10(-6), 80 X 10(-6), 5 X 10(-7), 5.0 X 10(-6) and 11 X 10(-6) M, respectively. Only prenylamine, TFP and W-7 were capable of relaxing coronaries over the same concentration range in which they bind to calmodulin. The relaxations produced by these calmodulin antagonists and prenylamine could not be overcome by contractile agonists which release Ca++ from internal stores (histamine and serotonin), whereas the relaxations produced by felodipine, verapamil and diltiazem were readily reversed by either of these agonists. This is consistent with TFP and W-7 and to some degree prenylamine-inducing vasodilation by calmodulin antagonism and with felodipine, verapamil and diltiazem vasodilating through Ca++ antagonism of Ca++ channels at the level of the cell membrane.     

Beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart: relationship to extra- and intracellular calcium

We have investigated the contribution of extra- and intracellular Ca++ and calmodulin to beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart perfused with Krebs-Henseleit buffer. Administration of isoproterenol (100 ng) increased the output of immunoreactive 6-keto-prostaglandin F1 alpha and prostaglandin E2 as well as heart rate and developed tension; the coronary perfusion pressure was reduced. Isoproterenol-induced output of prostaglandins was positively correlated with the extracellular Ca++ concentration (0-5 mM). Infusion of the Ca++ channel blockers diltiazem (22 microM) or nifedipine (0.27 microM) inhibited isoproterenol-stimulated output of prostaglandins and the positive inotropic but not the positive chronotropic effect of the amine. Administration of the intracellular Ca++ antagonists 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (23 microM) or ryanodine (1.6 microM) reduced the outflow of prostaglandins and the positive chronotropic and inotropic effect elicited by isoproterenol. The calmodulin inhibitors trifluoperazine (50 microM) or calmidazolium (1 microM) failed to alter isoproterenol-induced output of prostaglandins; trifluoperazine but not calmidazolium reduced the developed tension and coronary perfusion pressure without altering heart rate. The prostaglandin synthesis elicited by arachidonic acid (3 micrograms) was inhibited by indomethacin but not by alterations in extracellular Ca++, Ca++ channel blockers, intracellular Ca++ antagonists or calmodulin inhibitors. These data suggest that activation of beta adrenergic receptors promotes cardiac prostaglandin synthesis and myocardial contractility by increasing the trans-sarcolemmal flux of Ca++, which releases intracellular Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin synthesis and renal vasoconstriction elicited by adrenergic stimuli are linked to activation of alpha-1 adrenergic receptors in the isolated rat kidney

The authors have investigated the effect of norepinephrine (NE) and selective alpha-1, alpha-2 and beta adrenergic receptor agonists and antagonists on prostaglandin (PG) output and vascular tone to determine the type of adrenergic receptor involved in these biological actions of NE in the isolated rat kidney perfused at a constant flow rate with Tyrode's solution. Renal arterial administration of NE (0.32-3.2 nmol) and the selective alpha-1 adrenergic receptor agonists phenylephrine (3-29.5 nmol), cirazoline (0.5-4.6 nmol) and amidephrine (4.1-41 nmol) produced dose-related increases in PG output and perfusion pressure. Administration of the selective alpha-2 adrenergic receptor agonists B-HT 933 (2-20 nmol) and guanabenz (1.7-17 nmol) into the kidney produced only small increases in PG output and perfusion pressure, whereas another selective alpha-2 adrenergic receptor agonist xylazine (1-20 nmol) failed to increase perfusion pressure or PG output. Infusion of the beta adrenergic receptor agonist isoproterenol reduced perfusion pressure, but failed to increase the output of PGs. The selective alpha-1 adrenergic receptor antagonist prazosin (2.7 X 10(-6) M) inhibited PG output and renal vasoconstriction elicited by phenylephrine, cirazoline and amidephrine, but not that caused by B-HT 933 and guanabenz. In contrast, the selective alpha-2 adrenergic receptor antagonist rauwolscine (1.3 X 10(-6) M) inhibited the small rise in PG output and perfusion pressure elicited by B-HT 933 and guanabenz, but not that caused by NE, phenylephrine, cirazoline or amidephrine. The beta adrenergic receptor antagonist propranolol (3.86 X 10(-6) M) did not alter PG output or renal vasoconstriction produced by NE or alpha-1 and alpha-2 adrenergic receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoconstrictor response induced by 5-hydroxytryptamine released from vascular adrenergic nerves by periarterial nerve stimulation

The vasoconstrictor response to 5-hydroxytryptamine (5-HT) released from vascular adrenergic nerves by periarterial nerve stimulation (PNS) was studied in the perfused mesenteric vascular bed isolated from the rat. PNS was delivered at 4 to 16 Hz, 2 msec in pulse duration for 30 sec. After treatment with 5-HT (1 and 10 microM) for 20 min, the pressor response to PNS, previously decreased by 80 to 90% with phentolamine (0.1 microM), was greatly potentiated and a frequency-dependent pressor response to PNS reappeared. However, the 5-HT treatment did not alter the pressor response to infusion of exogenous norepinephrine (0.5 and 1 nmol) previously decreased by phentolamine. This potentiation did not occur in the presence of methysergide (0.1 microM), ketanserin (0.1 microM), tetrodotoxin (0.1 microM), guanethidine (5 microM) or in Ca++-free Krebs' solution. Also, in the preparation treated with 6-hydroxydopamine, 5-HT treatment had no effect on the abolished PNS response. Either cocaine (10 microM) or fluoxetine (10 microM) but not corticosterone (10 microM) prevented the potentiation when perfused together with 5-HT. In the mesenteric vascular bed prelabeled with [3H]-5-HT, PNS evoked a frequency-dependent increase of tritium efflux, which was abolished by treatment with tetrodotoxin guanethidine or 6-hydroxydopamine and in Ca++-free Krebs' solution. These results suggest that 5-HT is taken up by vascular adrenergic nerve endings in vitro and it is released by nerve stimulation, resulting in vasoconstriction. It is also suggested that 5-HT may contribute to the maintenance of local vascular tone through this mechanism in vivo.     

Characterization of substance P-induced contractions of guinea-pig trachea

In light of current interest in substance P as a bronchoconstrictor, several pharmacologic antagonists of known mediators of anaphylaxis were tested for possible activity against this neuropeptide. Concentration-dependent contractions of the isolated guinea-pig tracheal strips to substance P (10(-8) to 10(-5) M) were elicited. These contractions were inhibited by substance P receptor antagonists, D-Arg1-D-Trp7,9-Leu11 and D-Pro2-D-Trp7,9-substance P (10(-6) to 10(-4) M). Substance P-induced contractions were not inhibited by histamine, alpha and beta adrenergic receptor antagonists or by cyclooxygenase inhibition. However, atropine enhanced contractions to substance P. Both vasoactive intestinal polypeptide (10(-7), 10(-6) and 10(-4) M) and isoproterenol (10(-7) M) were able to reverse an ongoing substance P (10(-5) M)-induced contraction. Also, at a concentration of 10(-5) M, substance P increased cyclic GMP accumulation, but had no effect on the concentration of cyclic AMP. A 15-min pretreatment with either verapamil or nifedipine (10(-8) M) had no effect on substance P-induced contractions, whereas the purported intracellular Ca++ antagonist, 8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride (10(-4) M) produced a rightward shift of a substance P concentration-response curve. A selective calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (10(-4) M) failed to affect the contraction produced by 10(-5) M substance P. When guinea-pig tracheal strips were washed and allowed to re-equilibrate in 0 Ca++ buffer, the initial maximum contractions to substance P (10(-5) M) were equal for both regular (1.8 mM) Ca++ and 0 Ca++ buffer.(ABSTRACT TRUNCATED AT 250 WORDS)     

New 1,4-dihydropyridines with vasoconstrictor action through activation of Ca++-channels

We found four new phenylcarbamoyl-substituted 1,4-dihydropyridines which produced vasoconstrictor and vasopressor effects and we further investigated the mode of vasoconstrictor action of one of the derivatives, 2-(2-pyridil)ethyl-4-(2-chlorophenyl)-2,6-dimethyl-5-phenylcarbamo yl-1, 4-dihydropyridine-3-carboxylate (YC-170). In anesthetized dogs. YC-170 (0.1-3 mg/kg i.v.) produced dose-dependent increases in blood pressure, total peripheral resistance and maximum dLVP/dt with slight reductions of heart rate and aortic blood flow. Intracoronary injection of YC-170 (10-1000 micrograms) produced not only a decrease in coronary blood flow but also an immediate reversal of the coronary vasodilation induced by nicardipine (3 micrograms). YC-170 (0.1-3 mg/kg i.v.) also produced a dose-dependent and reversible vasopressor effect in both anesthetized and pithed rats. The vasopressor response to YC-170 in pithed rats was not attenuated by either hexamethonium, prazosin, yohimbine, ketanserin or reserpine but was antagonized by either nicardipine, nifedipine, verapamil or diltiazem. In isolated rabbit aorta, YC-170 (3 X 10(-6) to 3 X 10(-5) M) induced a dose-dependent and reversible contraction which was not affected by either phentolamine, prazosin, mepyramine, ketanserin or atropine but removed by either nicardipine, verapamil or a Ca++-free medium. YC-170 (1 X 10(-6) to 3 X 10(-5) M) displaced [3H]nitrendipine binding to the canine cardiac membrane in an apparently competitive manner with a Ki value of 1.7 X 10(-6) M. The results suggest that the new phenylcarbamoyl-substituted dihydropyridines, including YC-170, exert a direct vasoconstrictor effect, which appears to be associated with an influx of Ca++ through Ca++ channels.     

Inhibition of guinea-pig oxyntic cell function by adenosine and prostaglandins

Adenosine and prostaglandins (PGs) are known inhibitors of oxyntic cell function. Using quantitative cytochemistry of hydroxyl ion production (HIP) in guinea-pig oxyntic cells, we examined the effects of adenosine and PGs on secretagogue-stimulated HIP. Adenosine (10(-6) M) inhibited the actions of histamine (10(-14) M) and gastrin (2.5 X 10(-12) M) by 69 and 67%, respectively, but not that of dibutyryl cyclic AMP (10(-16) M) or carbachol (10(-9) M). These observations suggest that adenosine does not influence the Ca++-dependent pathway of carbachol action and that the adenosine activity precedes the generation of cyclic AMP. Adenosine and related analogs, N6-L-phenylisopropyladenosine and 5-N-ethylcarboxam-idoadenosine (10(-12) to 10(-14) M), inhibited histamine-stimulated HIP (10(-14) M) in the following order: N6-L-phenylisopropyladenosine greater than 5-N-ethylcarboxamidoadenosine greater than adenosin. The adenosine antagonist, 1,3-diethylphenylxanthine (10(-6) M), reversed the inhibitory effects of adenosine. Exogenous PGE2 (10(-6) M) also inhibited histamine- and gastrin-stimulated HIP by 65 and 55%, respectively. Indomethacin (10(-6) M) and flurbiprofen (10(-6) M), PG synthesis inhibitors, potentiated the action of histamine by 175 and 159%, respectively. Adenosine was incapable of reversing this potentiated effect. These data indicate that adenosine and related analogs are inhibitors of oxyntic cell HIP and suggest that these biological properties are mediated by binding to a cell surface receptor and thereby regulating oxyntic cell adenylate cyclase activity. The more potent properties of N6-L-phenylisopropyladenosine as compared to 5-N-ethylcarboxamidoadenosine are consistent with activity at the high-affinity surface adenosine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation by intracellular calcium pool of arginine vasopressin-induced cellular cyclic AMP production in rat renal papillary collecting tubule cells in culture.

The present study was undertaken to determine whether endoplasmic reticulum is involved in the cellular action of arginine vasopressin (AVP) in rat renal papillary collecting tubule cells in culture, using three dissimilar agents. 1 x 10(-7) M AVP increased cytosolic free Ca++ concentration ([Ca++]i) from 93.2 to 188.6 nM (P < .01). Exposure to 1 x 10(-4) M 3, 4, 5-trimethoxybenzoic acid, 8-(diethylamino) octylester hydrochloride (TMB-8), which inhibits intracellular Ca++ mobilization and blocks the function of endoplasmic reticulum, attenuated the [Ca++]i response to AVP. A significant increase in [Ca++]i in response to 1 x 10(-7) M AVP was obtained with Ca(++)-free medium containing 1 x 10(-4) M ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) (52.3 to 98.3 nM). However, when cells were preincubated with Ca(++)-free medium containing a mixture of 1 x 10(-4) M EGTA and 1 x 10(-4) M TMB-8, the 1 x 10(-7) M AVP-mobilized [Ca++]i was completely abolished. In the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine, AVP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner. Such an AVP-induced cAMP production was significantly reduced in cells exposed to Ca(++)-free medium containing 1 x 10(-4) M EGTA. After exposure to Ca(++)-free medium containing a mixture of 1 x 10(-4) M EGTA and 1 x 10(-4) M TMB-8, the cAMP response to AVP was markedly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous vasoconstrictor prostanoids: role in serotonin and vasopressin-induced coronary vasoconstriction

A vasoconstrictor-induced prostacyclin (PGI2) production in a perfused rat heart was found, suggesting a mitigating role of PGI2 on coronary vasoconstriction. Treatment of the heart with cyclooxygenase inhibitors (aspirin or indomethacin) decreased PGI2 production by more than 90% and paradoxically reduced the vasoconstriction response. The attenuating effect of cyclooxygenase blockade suggested that endogenous prostanoids contribute to serotonin-, vasopressin- or U46619-induced vasoconstriction. Two prostaglandin (PG) H2/thromboxane A2 (TXA2) receptor antagonists, i.e., 13-azaprostanoic acid (13-APA) and SQ 29,548 were used to investigate putative endogenous vasoconstrictor prostanoids on the exogenously induced vasoconstriction. Retrogradely perfused (5-6 ml/min) rat hearts were rendered guiescent, yet responsive to stimuli, by local injection of lidocaine to the atrioventricular node. Changes in coronary vascular resistance (i.e., perfusion pressure at constant flow) were monitored and the cardiac effluent was collected for analysis of 6-keto PGF1 alpha (the stable metabolite of PGI2) as well as PGF2 alpha by radioimmunoassay. Three vasoconstrictors, i.e., serotonin, vasopressin and the TXA2/PGH2 analog U46619, as well as authentic PGD2, PGE2 and PGF2 alpha were infused. PGD2, PGE2 and PGF2 alpha exerted a dose-related coronary vasoconstriction, as did U46619, serotonin and vasopressin. Treatment with 13-APA (100 microM) or SQ 29,548 (100 nM) almost abolished U46619-induced vasoconstriction. The addition of PGH2/TXA2 receptor antagonists also significantly reduced the pressor effect of exogenously administered PGs, serotonin and vasopressin, with the exception that SQ 29,548 did not significantly antagonize PGE2-induced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role of calmodulin in the control of lysosomal enzyme release from human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNs) were found to contain a mean +/- S.E.M. of 21.7 +/- 7.9 ng of immunoreactive calmodulin (CaM)/10(6) PMNs, which represents 0.032 +/- 0.001% of the total cellular protein. The functional role of CaM in the control of lysosomal enzyme release from human PMNs was investigated using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (2 X 10(-5)-10(-4) M) inhibited in vitro lysosomal enzyme release from human PMNs induced by immunological (serum-treated zymosan, concanavalin A and formyl-L-methionyl-L-leucyl-L-phenylalanine) and nonimmunological (Ca++ ionophore A23187) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity for CaM, had practically no inhibitory effect on lysosomal enzyme release. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of lysosomal enzymes from PMNs. In contrast, the chloride-deficient analog, W-5, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.89; P less than .001) with their affinity for binding to CaM, supporting the concept that these agents act by binding to CaM and thereby inhibiting lysosomal enzyme release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P450 omega/omega-1 hydroxylase-derived eicosanoids contribute to endothelin(A) and endothelin(B) receptor-mediated vasoconstriction to endothelin-1 in the rat preglomerular arteriole

The preglomerular arteriole of the rat was used to evaluate the contribution of cytochrome P450-derived eicosanoids to the vasoconstrictor effect of endothelin (ET)-1 and to determine the receptors mediating the response. ET-1 (4 x 10(-11) to 2 x 10(-9) M) produced dose-dependent reductions in the intraluminal diameter of the renal arteriole ranging from 25 +/- 8 to 142 +/- 16 micrometer. BMS182874 [(5-dimethylamino)-N-(3, 4-dimethyl-5-isoxazolyl)-1-naphthalenesulfonamide; 3 microM], an ET(A) receptor antagonist, or BQ788 (N-cis-2, 6-dimethyl-piperidino-carbonyl-L-gamma-methylleucyl-D-1-methoxy carbonyl-tryptophanyl-D-norleucine; 1 microM), an ET(B) receptor antagonist, attenuated ET-1 vasoconstriction by 59 +/- 4 and 50 +/- 10%, respectively. The combined administration of both ET receptor antagonists increased inhibition of ET-1 vasoconstriction to 75 +/- 4%. 17-Octadecynoic acid (17-ODYA, 2 microM) or 12, 12-dibromododec-enoic acid (2 microM), inhibitors of 20-hydroxyeicosatetraenoic acid (20-HETE) production, attenuated ET-1-induced vasoconstriction by 50 +/- 6 and 40 +/- 3%, respectively, as did indomethacin (10 microM), an inhibitor of cyclooxygenase. Miconazole (2 microM), the epoxygenase inhibitor, was without effect. 20-HETE (10(-8) and 2 x 10(-8) M) elicited a dose-related vasoconstriction that was inhibited by 10 microM, but not 5 microM, indomethacin. The inhibition by 17-ODYA of ET-1 vasoconstriction was not greater when combined with BMS182874 or BQ788. Moreover, vasoconstriction induced by ET-3, an ET(B)-selective agonist, was inhibited by 17-ODYA. These data indicate that both ET(A) and ET(B) receptors mediate ET-1 vasoconstriction and that 20-HETE production linked to both receptors makes a major contribution to ET-1-induced renal arteriolar vasoconstriction in the rat.     

Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A

Calyculin-A (CL-A), a novel marine toxin isolated from Discodermia calyx, caused contraction in the smooth muscle of guinea pig taenia ceci and rat aorta in the presence or absence (with 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) of external Ca++ at concentrations ranging from 1 X 10(-8) to 1 X 10(-6) M. In the presence of external Ca++, the contraction induced by CL-A was accompanied by an increase in the cytosolic free Ca++ concentration [( Ca++]cyt) as measured by the fluorescence indicator fura-2. Verapamil (3 X 10(-6) M) inhibited the increase in [Ca++]cyt, but not tension development caused by CL-A. In the absence of external Ca++, CL-A still caused contraction without changing [Ca++]cyt. Thus, from studies with intact smooth muscle it was demonstrated that, in the absence of external Ca++, CL-A can induce a contraction that was not accompanied by an increase in [Ca++]cyt. In permeabilized taenia, CL-A caused contraction in the absence of Ca++ (with 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid) at concentrations similar to those required to contract intact tissue. This contraction was inhibited by the nonselective kinase inhibitors such as amiloride (1 X 10(-3) M) and K-252a (2 X 10(-5) M). Low concentrations of Ca++ (approximately 1 X 10(-6) M) augmented the CL-A-induced contraction in the permeabilized taenia. In native actomyosin prepared from chicken gizzard CL-A induced phosphorylation of the 20 kDa myosin light chain (MLC) in the absence of Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-1 adrenoceptor-induced Ca++ movements in rat aorta: antagonism by phenoxybenzamine and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

The influx of 45Ca++ produced by l-norepinephrine (3 X 10(-7) or 10(-5) M) and K+ (100 mM) in rat aorta was not significantly influenced by phenoxybenzamine (1 or 3 X 10(-8) M) present for 5 or 10 min. However, the l-norepinephrine (3 X 10(-7) or 10(-5) M)-mediated 45Ca++ efflux was markedly attenuated by this treatment. Higher concentrations of phenoxybenzamine, as well as extension of the time of exposure, impaired the 45Ca++ influx to l-norepinephrine, but not that to K+. In contrast, prazosin (10(-9)-10(-7) M), as well as N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10(-7) or 10(-6) M for 5 or 10 min), was equally effective in antagonizing the 45Ca++ in and efflux caused by l-norepinephrine. Exposure of rat aorta to 1.3 X 10(-9) M phenoxybenzamine for 30 min significantly shifted the log concentration-contractile response curve to the full alpha-1 adrenoceptor agonist, l-phenylephrine, to the right and reduced its maximum response but failed to alter the contraction to the partial agonist Sgd 101/75 (indanidine). Conversely, incubations with 2.0, 2.2 and 3.0 X 10(-8) M N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline invariably affected the contractions to Sgd 101/75 more than those to l-phenylephrine. In pithed rats, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1 or 2 mg/kg i.v., -30 min), as well as phenoxybenzamine (0.1 mg/kg i.v., -30 min), enhanced the effectiveness of nifedipine to inhibit the vasopressor responses to the alpha-1 adrenoceptor stimulant, cirazoline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that prostacyclin modulates the vascular actions of calcium in man.

Increases in extracellular calcium (Ca++) can alter vascular tone, and thus may result in increased blood pressure (Bp) and reduced renal blood flow (RBF). Ca++ can stimulate prostaglandin E2 (PGE2) and/or prostacyclin (PGI2) release in vitro, which may modulate Ca++ vascular effects. However, in man, the effect of Ca++ on PG release is not known. To study this, 14 volunteers received low-dose (2 mg/kg Ca++ gluconate) or high-dose (8 mg/kg) Ca++ infusions. The low-dose Ca++ infusion did not alter systemic or renal hemodynamics, but selectively stimulated PGI2, as reflected by the stable metabolite 6-keto-PGF1 alpha in urine (159 +/- 21-244 +/- 30 ng/g creatinine, P less than 0.02). The same Ca++ infusion given during cyclooxygenase blockade with indomethacin or ibuprofen was not associated with a rise in PGI2 and produced a rise in Bp and fall in RBF. However, sulindac, reported to be a weaker renal PG inhibitor, did not prevent the Ca++ -induced PGI2 stimulation (129 +/- 33-283 +/- 90, P less than 0.02), and RBF was maintained despite similar increases in Bp. The high-dose Ca++ infusion produced an increase in mean Bp without a change in cardiac output, and stimulated urinary 6-keto-PGF1 alpha to values greater than that produced by the 2-mg/kg Ca++ dose (330 +/- 45 vs. 244 +/- 30, P less than 0.05). In contrast, urinary PGE2 levels did not change. A Ca++ blocker, nifedipine, alone had no effect on Bp or urinary 6-keto-PGF1 alpha levels, but completely prevented the Ca++ -induced rise in Bp and 6-keto-PGF1 alpha excretion (158 +/- 30 vs. 182 +/- 38, P greater than 0.2). However, the rise in 6-keto-PGF1 alpha was not altered by the alpha 1 antagonist prazosin (159 +/- 21-258 +/- 23, P less than 0.02), suggesting that calcium entry and not alpha 1 receptor activation mediates Ca++ pressor and PGI2 stimulatory effects. These data indicate a new vascular regulatory system in which PGI2 modulates the systemic and renal vascular actions of calcium in man.     

Effect of protein kinase C inhibitors on the actions of phorbol esters on vascular tone and adrenergic transmission in the isolated rat kidney

The effect of protein kinase C (PKC) inhibitors 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), polymyxin B (PMB), D-sphingosine (SPH), sangivamycin (SNG) and staurosporin (ST) on the action of PKC activators phorbol 12,13-dibutyrate (PDBu) and 12-o-tetradecanoylphorbol-13-acetate (TPA), on adrenergic neuroeffector events was investigated to determine the contribution of PKC in adrenergic transmission in the rat kidney. Infusion of TPA (5 x 10(-6) mM) or PDBu (6 x 10(-6) mM) produced renal vasoconstriction and enhanced the overflow of tritium elicited by periarterial renal nerve stimulation (RNS) (2 Hz) in the isolated rat kidney perfused with Tyrode's solution and prelabeled with [3H]norepinephrine. H-7 (2.7 x 10(-3) mM) and ST (2 x 10(-5) mM) did not alter RNS-induced overflow of tritium but attenuated the vasoconstrictor response to RNS and exogenous NE. PMB (1 x 10(-8) mM) and SPH (3.3 x 10(-4) mM) but not SNG (3.3 x 10(-3) mM) attenuated the RNS-induced overflow of tritium but increased the basal renal vascular tone and enhanced the vasoconstrictor response to RNS and exogenous NE. H-7, PMB, SPH, SNG or ST failed to alter the effects of PDBu to increase basal vascular tone and the overflow of tritium and the increase in renal vasoconstriction to RNS. PMB at 1 x 10(-9) mM but not at 1 x 10(-8) mM and SPH (3.3 x 10(-4) mM) but not H-7, SNG or ST inhibited the effect of TPA to increase the overflow of tritium. The effect of TPA on the vasoconstrictor response to RNS or to increase basal vascular tone was not altered by PKC inhibitors. These data suggest that in the rat kidney, PKC is either resistant to the actions of H-7, PMB, SPH, SNG and ST, or PDBu and TPA produce renal vasoconstriction and facilitate adrenergic transmission by a mechanism unrelated to PKC activation.     

Mode of vasorelaxing action of 5-[3-[[2-(3,4-dimethoxyphenyl)-ethyl]amino]-1-oxopropyl]-2,3,4,5- tetrahydro-1,5-benzothiazepine fumarate (KT-362), a new intracellular calcium antagonist

In rabbit aorta, pretreatment with KT-362 (KT; 10(-6) and 10(-5) M) inhibited contractile responses to norepinephrine (NE; 3 X 10(-9)-10(-5) M) and methoxamine (10(-7)-10(-4) M) but failed to affect responses to potassium (10-70 mM). KT (10(-5) M) partially inhibited Ca++-induced contractions in K+-depolarized aorta pre-equilibrated in a Ca++-free medium. After incubation of tissues for 30 min in a Ca++-free medium containing EGTA (0.2 mM), residual responses to NE and methoxamine were inhibited by KT (10(-6)-10(-4) M) and nitroglycerin (10(-5) M), but not by nifedipine, verapamil or diltiazem (all 10(-5) M). The inhibitory action of a combined treatment with KT and nitroglycerin (both 10(-5) M) on the residual response to NE was also much greater than that of either agent alone. In a Ca++-free medium, the residual caffeine-induced contraction of rabbit iliac artery was inhibited by KT (10(-5)-10(-4) M) but not by nifedipine (10(-5) M). The inhibitory action of KT on the residual responses to methoxamine and caffeine in a Ca+-free medium was much greater than that of nitroglycerin. In a Ca++-free medium with low EGTA (0.01 mM), D600 (10(-5) M) and NE (3 X 10(-7) M), the addition of Ca++ (2 mM) resulted in a tonic contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inhibition of Na+/K(+)-adenosine triphosphatase on vascular action of vasopressin.

The present study was undertaken to examine the cellular interaction between a Na+/K(+)-ATPase inhibitor, ouabain, and arginine vasopressin (AVP) in rat vascular smooth muscle cells (VSMC) in culture. Preincubation with 10(-5) M ouabain for 60 min increased basal cytosolic free Ca2+ [( Ca2+]i) concentration and intracellular 45Ca2+ uptake. Ouabain, however, did not affect basal 45Ca2+ efflux or AVP-stimulated 45Ca2+ efflux. As assessed by cell shape change, preincubation with 10(-5) M ouabain for 60 min also enhanced the sustained cellular contractile effect of a submaximal (10(-8) M AVP, 21.5% vs. 30.5%, P less than 0.01) but not maximal dose of 10(-6) M AVP. Preincubation with 10(-5) M ouabain for 60 min did not change AVP-induced V1-specific surface receptor binding or AVP-induced inositol phosphate production but did however potentiate the mobilization of [Ca2+]i induced by a submaximal (10(-8) M AVP, 301 vs. 385 nM, P less than 0.01) but not a maximal dose of AVP. These effects of ouabain on the mobilization of [Ca2+]i were abolished by incubation in Ca2(+)-free buffer or 5 X 10(-5) M verapamil. Ouabain (10(-5) M) also enhanced the sustained cellular contractile effect of a direct protein kinase C activator, phorbol 12-myristate 13-acetate. The present results therefore indicate that the inhibition of Na+/K(+)-ATPase may enhance the vascular action of AVP, and perhaps other vasoconstrictors, by increasing the AVP-induced mobilization of [Ca2+]i and by potentiating the activity of protein kinase C stimulated by AVP through enhancing basal and AVP-stimulated cellular Ca2+ uptake.     

Human vascular vasopressin receptors: analysis with selective vasopressin receptor antagonists

The vascular activity of arginine vasopressin (AVP) and selective AVP receptor antagonists was investigated in isolated arterial ring segments from human superior mesenteric arteries. AVP elicited a potent and concentration-dependent contraction in human mesenteric arterial rings with an EC50 value of 2.01 X 10(-9) M. The presence or absence of the vascular endothelium did not affect significantly AVP-induced contraction. AVP induced slight, although significant, tachyphylaxis in human mesenteric arteries. The selective vascular (V1) receptor antagonist [d(CH2)5 1Tyr(Me)2]AVP (SK&F 100273) shifted the concentration-response curves for AVP-induced vascular contraction to the right in a parallel manner (KB = 2.23 X 10(-9) M). A mixed V1/V2 receptor antagonist, [d(CH2)5 1D-Tyr(Et)2Val4desGly9]AVP (SK&F 101926), was also a potent antagonist of AVP-mediated vascular contraction; however, inhibition was marked by a nonparallel shift of the concentration-response curves with depression of maximum contraction. Furthermore, a relatively renal (V2) selective receptor antagonist [d(CH2)5 1D-Ile2Val4]AVP (SK&F 101485) was approximately 100-fold less potent at inhibiting AVP-induced vascular contraction (KB = 1.37 X 10(-7) M). These studies illustrate for the first time the in vitro effects of selective vasopressin receptor antagonists in isolated human blood vessels. Studies of other blood vessels and the design of therapeutically useful antagonists should proceed with the hypothesis that the vasopressin receptors mediating vascular contraction in human mesenteric arteries are of the V1 subtype.