Mechanism of action of vasopressin on prostaglandin synthesis and vascular function in the isolated rat kidney: effect of calcium antagonists and calmodulin inhibitors

We have investigated the mechanism of action of arginine vasopressin (AVP) on vascular tone and renal output of prostaglandins (PGs) by examining the effect of Ca++ depletion, Ca++ antagonists and calmodulin inhibitors in the isolated Tyrode perfused rat kidney. Administration of AVP (0.027-0.27 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 (6.0 X 10(-5) M) or nimodipine (4.7 X 10(-5) M), to the perfusion fluid attenuated the renal vasoconstriction, but not the output of PGs elicited by AVP. Infusion of intracellular Ca++ antagonists, Dantrium (3.1 X 10(-5) M), TMB-8 (2.3 X 10(-6) M) or ryanodine (2 X 10(-6) M) or calmodulin inhibitors, trifluoperazine (2 X 10(-6) M) or W-7 (2 X 10(-6) M), abolished the rise in renal output of PGs produced by AVP during Ca++ depletion. Calmodulin inhibitors, which inhibited the AVP-induced release of PGs in the presence of Ca++, failed to alter the renal vasoconstrictor effect of the peptide. Administration of d(CH2)5Tyr(Me)AVP, a selective antagonist of pressor actions of AVP, abolished the renal vasoconstrictor response and release of PGs elicited by AVP. In contrast, d(CH2)5-D-ValVAVP, an antagonist of antidiuretic and to a lesser extent of pressor actions of AVP, failed to alter the renal vasoconstrictor response but attenuated the output of PGs produced by AVP. AVP antagonists did not alter the effect of angiotensin II (0.096 nmol) to cause renal vasoconstriction and enhance PG output.(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.     

Mechanism of norepinephrine release elicited by renal nerve stimulation, veratridine and potassium chloride in the isolated rat kidney

We have investigated the mechanism by which renal nerve stimulation (RNS), veratridine (Vt) and KCl promote release of norepinephrine in the isolated rat kidney perfused with Tyrode's solution and prelabeled with [3H]norepinephrine by examining the overflow of tritium elicited by these stimuli during 1) extracellular Ca++ depletion, 2) alterations in extracellular Na+ concentration and 3) administration of tetrodotoxin, amiloride, LiCl and calcium channel blockers. RNS (1-4 Hz), Vt (15-90 nmol) and KCl (150-500 mumol) produced renal vasoconstriction and enhanced the tritium overflow in a frequency- and concentration-dependent manner, respectively. Omission of Ca++ (1.8 mM) from the perfusion fluid abolished the renal vasoconstriction and the increase in tritium overflow elicited by RNA and KCl and substantially reduced that caused by Vt. Lowering the Na+ concentration in the perfusion medium (from 150 to 25 mM) reduced the overflow of tritium and the renal vasoconstriction caused by RNS (2 Hz) or Vt (45 nmol); the increase in tritium overflow in response to these stimuli was positively correlated with extracellular Na+ (25-150 mM). In contrast, KCl-induced tritium overflow was negatively correlated with extracellular Na+ concentration. Tetrodotoxin (0.3 microM) abolished the effect of RNS and Vt, but not that of KCl, to increase overflow of tritium and to produce renal vasoconstriction. Administration of amiloride (180 microM) enhanced the overflow of tritium but attenuated the associated renal vasoconstriction produced by RNS, Vt and KCl. Replacement of NaCl (75 mM) with equimolar concentration of LiCl enhanced the overflow of tritium elicited by RNS, Vt and KCl; the associated renal vasoconstriction remained unaltered. Administration of Ca++ channel blocker, flunarizine (2 microM), but not diltiazem (6 microM) or nifedipine (1.4 microM), inhibited RNS-, Vt- and KCl-induced overflow of tritium; nifedipine enhanced whereas diltiazem failed to alter tritium overflow elicited by these stimuli. The renal vasoconstriction produced by RNS, Vt and KCl was inhibited by diltiazem and nifedipine, as well as by flunarizine. These data suggest that RNS, Vt and KCl enhance release of the adrenergic transmitter by promoting influx of Ca++ into the nerve terminal, not by Na+-Ca++ exchange transport process, but rather through specific Ca++ channels that are distinct from those located in the vascular smooth muscle.     

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)     

Effect of noradrenaline, vasopressin and angiotensin II on renal prostaglandins in man

To examine the response of renal prostaglandins (PG) to systemic and renal vasoconstriction noradrenaline (NA), arginine vasopressin (AVP) and angiotensin II (ANG II) were each infused into eight healthy female subjects for 3 h on different days. Urinary excretion of PGE2, PGF2 alpha and 6-keto-PGF1 alpha was determined hourly. NA and ANG II stimulated excretion of PGF2 alpha significantly, but not of PGE2 or 6-keto-PGF1 alpha. AVP stimulated renal PGF2 alpha and 6-keto-PGF1 alpha significantly, but not PGE2. A weak correlation was found between urinary PGF2 alpha and diastolic blood pressure during NA and ANG II infusions, but not during AVP infusion. The release of renal PG does not appear to constitute an obligatory and concomitant response to the blood pressure rise induced by the pressor agonists. The greater response of PGF2 alpha than of PGE2 may result from a preferential direct effect on PGF2 alpha secretion or from an increased conversion of PGE2 into F2 alpha.     

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)     

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.     

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)     

Mechanism of norepinephrine release elicited by Na+-K+ adenosine triphosphatase inhibition in the isolated rat kidney: involvement of voltage-dependent Ca++ channels

The mechanism by which ouabain and Na+ depletion enhance the release of norepinephrine (NE) was investigated in the isolated rat kidney prelabeled with [3H]NE by examining the efflux of tritium elicited by these stimuli during 1) Ca++ depletion and 2) administration of tetrodotoxin, amiloride and Ca++ channel blockers. In kidneys perfused with Tyrode's solution containing low K+ solution (0.54 mM), ouabain (10(-4) M) enhanced tritium efflux markedly by about 20-fold at 30 min. Depletion of Na+ from the perfusion medium also produced an increase in tritium overflow which peaked at 20 min. Administration of tetrodotoxin (0.3 microM) inhibited the effect of ouabain, but not that of Na+ depletion, to increase tritium efflux and perfusion pressure. In contrast, amiloride (180 microM) enhanced the overflow of tritium elicited by ouabain but failed to alter that elicited by Na+ depletion. The rise in perfusion pressure caused by both stimuli was attenuated by amiloride. Omission of Ca++ (1.8 mM) from the perfusion medium inhibited the increase in tritium efflux and perfusion pressure elicited by ouabain and Na+ depletion by 80 and 65%, respectively. The Ca++ channel blockers omega-conotoxin (50 nM), diltiazem (60 microM) and flunarizine (2 microM), but not nifedipine (1.4 microM), inhibited tritium overflow elicited by ouabain. However, nifedipine, diltiazem and flunarizine, but not omega-conotoxin attenuated the tritium overflow elicited by Na+ depletion. The rise in perfusion pressure elicited by ouabain in low K+ and Na+ depletion was inhibited by these Ca++ channel blockers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine nucleotide binding proteins may modulate gating of calcium channels in vascular smooth muscle. I. Studies with fluoride

Fluoride (F-), a known stimulator of G-proteins, was used to examine the relationship between G-proteins and calcium channels (CaC) in rat vascular smooth muscle (VSM). Treatment of isolated rat tail artery helical strips with F- (2.5-20 microM) produced a Ca++-dependent contraction. In the absence of added AlCl3, subthreshold NaF shifted the KCl, as well as the arginine vasopressin and norepinephrine concentration-related tension curves to the left. Nifedipine and verapamil, known CaC blockers, inhibited the NaF-related contraction. AlCl3 (20 microM), which is required for G-protein stimulation by F-, strikingly potentiated the contractile response to F-. The NaF-induced contraction was relaxed by 3-isobutyl-1-methylxanthine as well as by forskolin and by dibutyryladenosine-cyclic AMP, and the effect therefore may be independent of cAMP. 45Ca-uptake was elevated by NaF, and partially blocked by nifedipine and verapamil. NaF also inhibited the basal and forskolin-stimulated cAMP production, suggesting that F- stimulated the putative Gi in the intact VSM cells. NaF stimulated accumulation of IP in a concentration-dependent manner, indicating that F- stimulated the putative G-protein Gp which couples various receptors to hydrolysis of phosphoinositides and mobilization of Ca++. These results indicate that NaF-induced vasoconstriction is related to the opening of the CaC in the plasma membrane and perhaps a subsequent entry of the extracellular Ca++ into the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of vascular actions of prostacyclin in the rat isolated perfused mesenteric arteries

Prostacyclin (PGI2) did not alter the basal perfusion pressure in the isolated rat mesenteric arteries perfused with Krebs' solution, but produced a biphasic effect in arteries preconstricted with norepinephrine or arginine vasopressin: constriction, then prolonged dilation. Both these components of PGI2 effect were diminished in arteries denuded of their endothelia by a 10 min perfusion with distilled water or p-bromophenacyl bromide (10 microM). The present study elucidates the mechanism of these PGI2 actions. Indomethacin (0.28 microM) SQ 29548 (1 microM, thromboxane A2 receptor antagonist), saralasin (1 microM, angiotensin II receptor antagonist) or the free radical scavengers, superoxide dismutase (60 U/ml) and catalase (40 U/ml) did not inhibit the initial vasoconstriction, suggesting it was not mediated through endothelially generated thromboxane A2, angiotensin II or oxygen-derived free radicals. However, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (50 microM; Ca++ chelating agent), 8-(diethyl-amino)octyl 3,4,5-trimethoxy benzoate (10 microM; intracellular Ca++ antagonist), or neomycin (5 mM; phospholipase-C inhibitor) abolished the vasoconstriction. Ouabain (0.5 mM) did not affect the vasodilation, but perfusion with excess (50 mM) or 0 K+ Krebs' solution abolished it, suggesting this PGI2 action involves changes in membrane K+ conductance via a mechanism independent of Na+/K+ adenosine triphosphatase. Vasodilation evoked by BRL 34915 (K+ channel activator) was similarly attenuated under these conditions, but not by ouabain. Furthermore, procaine (1 mM; nonspecific K+ channel inhibitor), but not apamin (0.5 microM) or tetraethylammonium (10 mM) blocked PGI2- and BRL 34915-induced vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin and thromboxane synthesis by highly enriched rabbit proximal tubular cells in culture

Prostaglandin synthetic profiles were studied in monolayers of highly enriched rabbit renal proximal tubular cells cultured in serum-free, hormone-supplemented, defined media. The cultures were initiated from glomeruli-free cortical suspensions. Cells in culture demonstrated morphologic and functional characteristics highly suggestive of proximal tubular cells. The basal and stimulated synthesis of immunoassayable prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 in response to various agonists, as well as the effect of two cyclooxygenase inhibitors, was assessed. Under both basal and stimulated conditions, PGE2 was the major product synthesized. PGF2 alpha and 6-keto-PGF1 alpha were synthesized to a lesser extent, and TxB2 was undetectable. The basal synthesis of PGE2 and PGF2 alpha in cultured cells was found to be higher than in isolated proximal tubular fragments by sevenfold and fivefold, respectively. Exogenous arachidonate, angiotensin II, and the divalent cation ionophore A23187 stimulated all three immunoassayable prostaglandins in a dose-dependent manner. Arginine vasopressin (10(-5) mol/L) had no stimulatory effect. In Ca++-free media or in the presence of 10(-5) mol/L Ca++ channel blocker, verapamil, the stimulatory effects of angiotensin II and A23187 were ameliorated. The stimulatory effect of angiotensin II was inhibited by saralasin (10(-5) mol/L), indicating that receptor binding could mediate PGE2 synthesis. Both indomethacin and sulindac sulfide (10(-5) mol/L) reversibly inhibited PGE2 synthesis.     

Prostaglandin synthesis elicited by cholinergic stimuli is mediated by activation of M2 muscarinic receptors in rabbit heart

This study was performed to determine the subtype of muscarinic receptors involved in the action of cholinergic stimuli on prostaglandin (PG) synthesis in the isolated rabbit heart perfused at a constant flow rate with Krebs Hanseleit buffer. Acetylcholine (ACh, 1.0-10.0 nmol), an M1 and M2 receptor agonist, and arecaidine propargyl ester (APE, 1.0-5.0 nmol), a selective M2 agonist, produced a dose-related increase in the output of 6-keto-PGF1 alpha and a decrease in heart rate, whereas 4-[m-chlorophenyl carbamoyl]-2-butynyl-trimethylammonium chloride (McN-A-343, 10 nmol-1.0 mumol), a selective M1 receptor agonist, did not alter PG output. The increase in PG output or the decrease in heart rate elicited by ACh or APE was abolished by atropine (0.1 microM), an M1 and M2 receptor antagonist, and by 11-[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6-H-pyrido-[2,3-b] [1,4]-benzodiazepine-6-one (AF-DX-116, 1.0 microM), a selective M2 antagonist, but not by pirenzepine (1.0 microM), a selective M1 antagonist. The developed tension, which was also reduced by ACh and APE, but not by McN-A-343, was minimized by AF-DX-116 and not by lower concentrations of pirenzepine that attenuated the coronary vasodilator effect of McN-A-343. Lower doses of ACh (1.0-5.0 nmol) caused coronary vasodilation, whereas higher doses of ACh (10.0 nmol) and lower as well as higher doses of APE produced a biphasic effect--an initial vasodilation followed by vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium ionophore (A23187)- and arachidonic acid-stimulated prostaglandin release from microvascular endothelial cells: effects of calcium antagonists and calmodulin inhibitors

Calcium ionophore (A23187)-stimulated prostaglandin (PG) E2 and I2 (measured as 6-keto PGF1 alpha) release from cultured rabbit coronary microvessel endothelial (RCME) cells in a time- and dose-dependent manner. A23187-stimulated PG release was reduced by the calcium channel blockers nifedipine, verapamil and diltiazem and by the intracellular calcium blocker, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate. A23187-stimulated PG release was significantly reduced by lowering the calcium concentration in the buffer to concentrations of 0.2 mM or less. A23187-stimulated 45Ca uptake was not inhibited by nifedipine (0.5 microM), diltiazem (10 micron) or verapamil (50 microM) although these same concentrations of calcium channel blockers significantly inhibited A23187-stimulated PG release. However, these concentrations of calcium blockers did inhibit K+ (10 mM)-valinomycin (5 microM)-stimulated 45Ca uptake, indicating that, although RCME cells probably have voltage-dependent calcium channels and although calcium influx via these channels is blocked by the calcium channel blockers, voltage-dependent calcium influx plays little or no role in A23187-stimulated 45Ca influx and PG release. KCl-valinomycin significantly stimulated PG release, but this increase was not significantly affected by either nifedipine (0.5 microM) or diltiazem (10 microM) despite complete inhibition of KCl-valinomycin-stimulated 45Ca influx. Verapamil (50 microM) exhibited a small but significant suppression of KCl-valinomycin-stimulated PG release. These observations most likely indicate that calcium influx by voltage-dependent calcium channels plays little or no role in the events leading to either A23187- or KCl-valinomycin-stimulated PG release. The calmodulin antagonists, trifluoperazine and calmidazolium, also reduced A23187-stimulated PG release. In vitro studies of porcine pancreatic phospholipase (PL) A2 activity suggested that the inhibitory actions of trifluoperazine, but not the calcium antagonists, may be mediated by direct inhibitory actions on PLA2. Studies with [3H]arachidonic acid (AA)- and [14C]stearic acid-prelabeled RCME cells suggested that A23187 stimulated both PLA2 and PLC activity, leading to the release of AA. Studies with exogenous AA indicated that reducing calcium availability by reducing buffer calcium concentrations resulted in an enhanced conversion of exogenous AA to PGs. RCME cells incubated in nominally calcium-free buffer exhibited a decreased rate of AA incorporation. The observed increase in AA conversion to PGs in low calcium buffer suggests that calcium may stimulate AA uptake and acylation as well as AA release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine receptor-mediated alterations in prostacyclin production and cardiac function in the isolated rabbit heart.

The subtype of adenosine receptor linked to cardiac prostacyclin (PGI2) synthesis, measured as immunoreactive 6-keto-PGF1 alpha, was investigated in the rabbit heart perfused with Krebs' buffer at 20 ml/min. Adenosine (6.4-50 nmol) decreased 6-keto-PGF1 alpha synthesis, coronary perfusion pressure (PP) and myocardial contractility (dp/dt max), whereas higher doses (200 nmol) increased 6-keto-PGF1 alpha output and decreased PP, heart rate (HR) and dp/dt max. Injections (3.2-50 nmol) or infusion (0.6 microM) of A1 receptor agonist 1-deaza,2-chloro,N6 cyclopentyladenosine increased 6-keto-PGF1 alpha production and decreased HR and PP without affecting dp/dt max. 1-Deaza,2-chloro,N6 cyclopentyladenosine 100 to 200 nmol produced similar effects as lower doses except that it decreased transiently PP and reduced dp/dt max. 1,3-Dipropyl,8-cyclopentylxanthine (0.06 microM) prevented the effects of 1-deaza,2-chloro,N6 cyclopentyladenosine (50 mumol) and adenosine (10 microM) to increase 6-keto-PGF1 alpha output and decrease HR and minimized the decrease in dp/dt max. A2 receptor agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido-ade nos ine (1.6-12.5 nmol) or 0.6 microM decreased 6-keto-PGF1 alpha output, PP and dp/dt max without changes in HR. 3,7-Dimethyl-1-propargylxanthine prevented 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine-induced decrease in 6-keto-PGF1 alpha output, PP and dp/dt max; HR was not altered by this agent. These data suggest that stimulation of A2 receptors reduce cardiac PGI2 synthesis and PP, but activation of A1 adenosine receptors increased PGI2 synthesis, produced vasoconstriction and decreased HR.(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.     

Acute effects of antidiuretic hormone on urinary prostaglandin excretion

Although it is well established that chronic treatment with antidiuretic hormone increases renal prostaglandin (PG) excretion, the effects of short-term infusions are controversial. Therefore, in the present study the effect of acute administration of arginine vasopressin on urinary PG excretion was investigated in conscious Brattleboro rats and in water-diuresing Long-Evans rats. Water balance was kept constant during arginine vasopressin infusion. Arginine vasopressin caused a significant, dose-related and reversible increase in urinary PG excretion within 20 min in both models. Similar results were obtained during the infusion of 1-deamino-8-D-arginine vasopressin in the Brattleboro rat. Normalization of the hydropenia of Brattleboro rats by infusion of large amounts of hypotonic fluid failed to elevate urinary PG excretion. These results give no support to the hypothesis that the acute enhancement of urinary PG excretion by vasopressin is mediated through either vasoconstriction or volume retention or induction of cyclooxygenase, but rather they indicate that antidiuretic hormone can increase renal PG synthesis through a more direct mechanism in vivo.     

Effects on calmodulin of bepridil, an antianginal agent

Using biopharmacological techniques, we determined the effect on calmodulin of bepridil, a Ca++ channel blocker. We used two Ca++/calmodulin-dependent enzymes, Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain and myosin light chain kinase from chicken gizzard. Bepridil inhibited the calmodulin-induced activation of Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase and the concentration of this drug producing 50% inhibition (IC50) of this enzyme was 8 microM. There was no significant effect on unactivated Ca++/calmodulin-dependent cyclic nucleotide phosphodiesterase (in the absence of Ca++-calmodulin), up to a concentration of 100 microM. Bepridil inhibited specifically Ca++/calmodulin-dependent phosphorylation of chicken gizzard myosin light chain with an IC50 value of 18 microM. Moreover, this agent produced a marked displacement of [3H]N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, an antagonist that interacts selectively with calmodulin. The influence of bepridil on the dose-response curves of mesenteric arterial strips for CaCl2, norepinephrine and serotonin differed from the influence seen with nifedipine, another Ca++ blocker. Bepridil (100 microM) suppressed the Ca++-induced contraction of saponin-skinned mesenteric arteries and calmodulin (26 microM) reversed partly the relaxant effect of this agent. These results suggest that the effect of bepridil on the cardiovascular system is due not only to its Ca++ channel blocking action but also to a calmodulin antagonistic action.     

Influence of protein kinase C activators on vascular tone and adrenergic neuroeffector events in the isolated rat kidney

To investigate if protein kinase C (PKC) activation is involved in mediating or modulating adrenergic transmission, we have investigated the effect of phorbol esters, PKC activators, on the release of adrenergic transmitter elicited by periarterial renal nerve stimulation (RNS) in the isolated rat kidney perfused with Tyrode's solution and prelabeled with [3H]norepinephrine. Infusion of 12-o-tetra-decanoyl-phorbol 13-acetate (TPA) at 5 x 10(-7) mM produced renal vasoconstriction and a rise in basal perfusion pressure without any consistent effect on the rise in tritium efflux or the perfusion pressure elicited by RNS. Higher concentrations of TPA (5 x 10(-6) to 5 x 10(-5) mM) increased both the basal as well as RNS-induced efflux of tritium; the basal perfusion pressure was increased so dramatically that the effect of RNS to raise perfusion pressure was reduced compared to that observed in the vehicle group. Infusion of phorbol-12,13-dibutyrate at 6 x 10(-6) mM reduced the basal but increased the RNS-induced efflux of tritium; the basal as well as the rise in perfusion pressure caused by RNS was increased. Phorbol-13-monoacetate that does not activate PKC failed to alter the basal or the increase in tritium efflux and perfusion pressure elicited by RNS. The rise in perfusion pressure produced by TPA or phorbol-12,13-dibutyrate was reduced by nifedipine (1.4 x 10(-6) mM) but it was abolished by omission of Ca++ from the perfusion medium. These data suggest that PKC activation with phorbol esters produces renal vasoconstriction by promoting influx of extracellular Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-activating factor is a potent stimulus for renal prostaglandin synthesis: possible significance in unilateral ureteral obstruction

Platelet-activating factor (PAF), a lipid with potent platelet-stimulating and hypotensive properties, has been shown to stimulate the release of prostaglandins (PGs) from a number of cell types. It is produced by a variety of inflammatory cells and the renal medulla. However, no studies to date have examined the effect of PAF on the intact kidney. We, therefore, studied the effect of bolus injections of PAF on the isolated perfused rabbit kidney subjected to aseptic ureteral ligation for 72 hr. Intrarenal resistance and release of PGs by both the hydronephrotic (HNK) and contralateral kidney (CLK) were quantified. Intrarenal administration of PAF causes a dose-dependent stimulation of the release of PGE2, thromboxane B2 (TxB2) and the PGI2 metabolite, 6-keto-PGF1 alpha from the HNK and CLK. The magnitude of the release from the HNK is much greater than that for the CLK. A 100-ng bolus injection of PAF into the HNK results in the release of 1561.0 +/- 312.0 and 117.7 +/- 38.2 ng of PGE2 and TxB2, respectively, whereas administration of this dose to the CLK causes 291.0 +/- 35.0 ng of PGE2 and 19.0 +/- 4.2 ng of TxB2 to be released. Renal vascular resistance (RVR) is increased by PAF in the HNK. Product identity was confirmed using selective inhibitors and bioassay. These data show that PAF is a potent stimulus for renal PG release and that this release may have vascular consequences.