Specific actions of gallium on norepinephrine-induced tension and associated 45Ca movements in rabbit aortic smooth muscle

Gallium ion (Ga) dose-dependently (60-360 microM) inhibited contractions induced by norepinephrine (NE, 1 microM) in rabbit aortic (and media intimal) strips, but did not affect contractions elicited with high K+ (80 mM) solution. The initial phasic portion of the NE-induced response was either unaffected or only slightly (less than 10%) reduced, but the tonic portion of the response was inhibited completely by higher concentrations (greater than or equal to 300 microM) of Ga . In resting muscles, the equilibrated (90 min) 45Ca uptake was not altered by Ga (360 microM). Also, 45Ca efflux from either high- or low-affinity Ca++ binding sites was unaltered by Ga . The effects of Ga (360 microM) on 45Ca retained after a subsequent 60-min washout at 0.5 degrees C in an isosmotic (80.8 mM) La solution were also examined. High affinity La -resistant 45Ca released by NE (1 microM) was not altered by Ga . Under conditions favoring low affinity Ca++ uptake, 45Ca retention in control and K+-treated muscles was not changed by Ga , but the additional incremental 45Ca uptake associated with NE (in the presence of high K+) was blocked. Thus, Ga appears to have a selective inhibitory action on NE-associated 45Ca uptake without affecting either resting and high K+-induced 45Ca uptake or that 45Ca fraction released by NE. This action may result from a selective blockade by Ga of receptor-linked Ca++ channels in rabbit aortic smooth muscle.     

Effects of 2-nicotinamidoethyl nitrate on agonist-sensitive Ca++ release and Ca++ entry in rabbit aorta

The effects of 2-nicotinamidoethyl nitrate (SG-75) on norepinephrine (NE)- and KCI-induced responses in rabbit aorta were quantitated, correlated with 45Ca studies and compared with the effects of nifedipine (NIF) on similar parameters. NE- and KCI-induced dose-response relationships were differentially depressed by SG-75 (NE much greater than KCI) and NIF (KCI much greater than NE). Responses to KCI were relatively insensitive to prior SG-75, yet moderately relaxed by subsequent SG-75. Conversely, NIF markedly inhibited and completely relaxed similar responses. Responses to NE were relaxed and inhibited with SG-75, but unaffected by NIF. Responses to NE in La or O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 (with and without KCI) solutions were phasic, reduced by SG-75 and insensitive to NIF. NE-dependent, Ca++-induced responses in a O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 solution (with and without KCI) were attenuated by SG-75. Equilibrated (60 min) La -resistant (residual), high apparent affinity Ca++ binding was increased 26% with SG-75 and decreased 34% with NIF, yet neither altered the rate of exchange (10 min). Rate of exchange at low apparent affinity, residual sites was increased 21% by SG-75 without altering equilibrated values, whereas NIF reduced equilibrated values 11%, without affecting rate. NE reduced, SG-75 + NE augmented and NIF + NE decreased, in an additive fashion, high apparent affinity, residual bound Ca++. Residual Ca++ binding at low apparent affinity sites was increased with 160 mM substituted KCI (380%). This increase was only partially inhibited with SG-75, and eliminated by NIF. Net Ca++ efflux was persistently slowed by SG-75 and unaltered by NIF. The primary effects of SG-75 appear to be depression of Ca++ release and inhibition of receptor-operated (potential-independent) Ca++ entry, with limited attenuation of voltage-dependent Ca++ entry. NIF primarily inhibits voltage-dependent Ca++ entry.     

Inhibitory effects of amphetamine on potassium-stimulated release of [3H]dopamine from striatal slices and synaptosomes

Amphetamine, 10(-7) M or greater, evoked the release of [3H]dopamine ([3H]DA) and inhibited subsequent K+-evoked [3H]DA release from striatal synaptosomes superfused at a flow rate (1 ml/min) that prevented reuptake. Amphetamine inhibited the K+-evoked release of [3H]DA to a lesser extent in striatal slices or in synaptosomes superfused at a flow rate (0.35 ml/min) that allowed reuptake. The observed decrease in amphetamine inhibition of K+-evoked release was primarily due to amphetamine blocking [3H]DA reuptake. Interneuronal interactions may account for some of the inhibitory effects of amphetamine on K+-evoked release in the slice. Inhibition of K+-evoked release from either slices or synaptosomes was still evident when 10(-6) M amphetamine was removed from the superfusion buffer and the spontaneous release had returned to control levels. The presence of Ca++ during amphetamine exposure was required for subsequent inhibition of K+-evoked release in synaptosomes. Amphetamine in the presence of Ca++ did not affect the subsequent release of [3H]DA evoked by the Ca++ ionophore, A23187. Therefore, amphetamine inhibition of the K+-evoked release of [3H]DA cannot be explained by prior depletion of Ca++-releasable pools. Nifedipine, 1 microM, failed to block either the Ca++-dependent release of [3H]DA or the inhibition of K+-evoked release by amphetamine. However, 1 mM cobalt inhibited the Ca++-dependent release of [3H]DA by amphetamine and antagonized the inhibition of K+-evoked release after amphetamine exposure. This suggests that amphetamine may open voltage-dependent Ca++ channels sensitive to cobalt but not nifedipine. Amphetamine may desensitize these voltage-dependent Ca++ channels and inhibit their activation by K+ depolarization.     

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)     

Factors affecting rabbit mesenteric artery smooth muscle sensitivity to calcium antagonists

The sensitivity of rabbit isolated superior mesenteric artery to Ca++ antagonists was examined under various conditions. Relaxation dose-response curves for D600 or nifedipine were generated, and IC50 values were calculated. In the first series of experiments, D600 or nifedipine IC50 was found to be 20-25-fold greater for norepinephrine (NE, 5 microM) contraction than for 80 nM K+ contraction. Even when the tissues were depolarized with 80 mM K+ before NE contraction, D600 or nifedipine IC50 still remained significantly greater compared with 80 mM K+ alone and remained closer to that during NE alone. Also a protocol was designed to study NE-induced phasic contraction in EGTA-physiological salt solution (a functional indicator of intracellular Ca++ release) as well as NE-induced sustained contraction after readdition of Ca++. The effects of varying [K+]ex (0-80 nM range) on NE-induced [Ca++]i release as well as on the D600 IC50 for NE contraction was studied. Increasing [K+]ex was found to enhance NE-sensitive [Ca++]i release and lower the D600 IC50 for NE contraction. Thus, conditions causing an increase in the ability of NE to cause [Ca++]i release were associated with an increase in the sensitivity of NE contraction to D600. These data provide functional evidence that the receptor-agonist sensitive Ca++ influx process in vascular smooth muscle is not solely regulated by changes in membrane potential. Additional mechanisms, such as a modulatory role of [Ca++]i release, in this process are implicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine and adenosine triphosphate release in different ratio from guinea pig vas deferens by high potassium chloride, ouabain and monensin

Release of norepinephrine (NE) and ATP from the guinea pig vas deferens evoked by ouabain in combination with monensin or by high KCl was measured by a high-pressure liquid chromatography-ECD and luciferin-luciferase assay, respectively. Ouabain (10-100 microM) induced a concentration-dependent liberation of NE, which was enhanced by 10 microM monensin, a Na+-ionophore. The marked NE release elicited by the combined administration of both the drugs was unaffected by Ca++-removal but was reduced by lowering Na+ from the medium. This NE release in the Ca++-free medium was diminished markedly after treatment with 6-hydroxydopamine or reserpine and in low-temperature (25 degrees C) medium. This release was also decreased by ruthenium red (10-30 microM), an uptake inhibitor of Ca++ to mitochondria, and carbonyl cyanide-m-chlorophenyl hydrazone (10 microM), a metabolic inhibitor. On the other hand, 100 mM KCl caused a moderate, extracellular Ca++-dependent release of NE. ATP-outflow from the tissue evoked by 100 microM ouabain plus 10 microM monensin was almost unaltered by Ca++-removal but was inhibited by 6-hydroxydopamine or prazosin (0.3 microM), whereas release induced by high KCl was reduced by 6-hydroxydopamine and Ca++-free medium but was unaffected by prazosin. ATP/NE ratios at respective maximum effluxes evoked by 100 mM KCl and ouabain plus monensin were 6.59 and 0.22, respectively. These findings suggest that there may be more than one site of corelease for NE and ATP. Ouabain plus monensin seems to produce an extracellular Ca++-independent neuronal release of NE and ATP from the cytoplasmic and vesicular storage sites which predominantly release NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular redistribution of 45Ca in rabbit renal artery determined by electron microscopic autoradiography: changes in response to K+-induced depolarization and nitrendipine

Redistribution of Ca++ in response to K+-induced depolarization and/or nitrendipine (10(-6) M) was studied in isolated rabbit renal arteries using 1) isometric tension measurements, 2) 45Ca uptake measurements and 3) 45Ca electron microscopic autoradiography. Renal artery rings developed a mean tension of 1.67 +/- 0.30 g in response to high K+. Preincubation with 10(-6) M nitrendipine for 1 hr inhibited 80% of the initial portion and 100% of the sustained portion of the K+-induced contraction, without affecting rest tension. Using the modified lanthanum technique, cellular uptake of 45Ca increased 25% in muscles exposed to K+-substituted solution compared to control muscles (P less than .001). Preincubation with nitrendipine for 1 hr inhibited the K+-induced increase, whereas exposure to nitrendipine alone decreased 45Ca uptake compared to control muscles (P less than .001). Electron microscopic autoradiography of control renal arteries showed that relative 45Ca activities for the plasma membrane (PM), sarcoplasmic reticulum (SR) and mitochondria were higher than the cytoplasmic 45Ca activity, whereas activities for the nucleus were similar to that for the cytoplasm. Exposure to high K+ solution substantially decreased 45Ca activities of both the PM and SR (P less than .001), but changes in relative activities of other sites were insignificant. Muscles exposed to nitrendipine alone or nitrendipine plus high K+ had SR and PM activities intermediate with values from control and high K+ groups. Thus, Ca++ is released from both the SR and PM during exposure to high K+ and this Ca++ may contribute to the Ca++ pool involved in a depolarization-induced contraction.     

Effects of ethanol on neurotransmitter release and intracellular free calcium in PC12 cells

The effect of ethanol on muscarine-stimulated release of l-[3H]norepinephrine ([3H]NE) was studied using the rat pheochromocytoma cell line, PC12. At concentrations of 25 mM and above, ethanol produced a dose-dependent inhibition of muscarine-stimulated release of [3H]NE. The inhibition of muscarine-stimulated transmitter release occurred in the absence of any detectable effect of ethanol on [3H]NE uptake or on muscarinic binding to the cells. However, ethanol produced an inhibition of muscarine-stimulated elevation of intracellular free Ca++ which corresponded with the inhibition of transmitter release. At concentrations greater than 100 mM, ethanol produced an increase in the basal release of [3H]NE. Intracellular free Ca++ also was increased by ethanol concentrations greater than 100 mM. The elevation of basal transmitter release and intracellular free Ca++ by concentrations of ethanol greater than 100 mM occurred independently of the inhibition by ethanol of muscarine-stimulated elevation of intracellular free Ca++ and transmitter secretion. These results suggest that the effects of ethanol on neurotransmitter release are associated with the effects of ethanol on intracellular free Ca++.     

Protective effect of intrarenal calcium membrane blockers before or after renal ischemia. Functional, morphological, and mitochondrial studies

The present study examined whether a pre- or postischemic infusion of verapamil (V) or a postischemic infusion of nifedipine (N), drugs which block calcium (Ca++) influx across plasma membranes, provides protection against ischemic acute renal failure (ARF) in dogs. Renal hemodynamics and excretory function were examined 1 h (initiation phase) and 24 h (maintenance phase) after a 40-min intrarenal infusion of norepinephrine (NE). In each case, the uninfused contralateral kidney served as control. Four groups were studied: (a) dogs receiving NE alone; (b) dogs receiving an intrarenal infusion of V for 30 min before NE (V + NE); (c) dogs in which intrarenal V was infused for 2 h, beginning immediately after completion of NE infusion (NE + V); and (d) dogs in which intrarenal N was infused for 2 h, beginning immediately after completion of NE infusion (NE + N). Glomerular filtration rate (GFR) in the NE kidneys, as assessed by inulin clearance, at 1 and 24 h averaged 2.4 +/- 1.1 and 5.0 +/- 2.0 ml/min, respectively, as compared with control kidney GFRs of 28.0 +/- 3.5 and 43.8 +/- 5.0 ml/min, respectively (both at least P less than 0.01). In the V + NE group, GFR at 1 and 24 h averaged 15.0 +/- 5.5 and 31.0 +/- 4.5 ml/min, respectively, both at least P less than 0.05 as compared with values from NE kidneys. GFRs in the NE + V group averaged 15.0 +/- 2.4 and 16.3 +/- 3.6 ml/min at 1 and 24 h, both at least P less than 0.02 as compared with values from NE kidneys. GFR in the NE + N group averaged 18.6 +/- 6.0 ml/min at 24 h (P less than 0.05 as compared with GFRs in the NE kidneys). In addition, function of cortical mitochondria (Mito) was examined at the end of the 40-min NE infusion and after 1 and 24 h of reperfusion in the NE alone and NE + V groups. Mito respiration, assessed by acceptor control ratios, was reduced at each period in the NE alone kidneys. After 24 h, these Mito had accumulated Ca++ and exhibited reduced Ca++ uptake and increased Ca++ release rates. Mito from NE + V kidneys respired normally, did not accumulate Ca++, and exhibited no alterations in Ca++ uptake or release. Light and electron microscopy also demonstrated morphological protection of V against tubular necrosis and cell injury. Mito from the NE + N kidneys also respired normally and did not accumulate significant amounts of Ca++. The results of the present studies therefore demonstrated that chemically dissimilar calcium entry blockers exert substantial functional, cellular, and morphological protection against experimental ischemic ARF. These findings are compatible with the hypothesis that increased cytosolic Ca++ is critically important in the maintenance of renal vasoconstriction and the development of cellular necrosis with subsequent tubular obstruction in NE-induced ischemic ARF. V or N may provide protection against renal injury by retarding any increase in cytosolic Ca++ in renal vasculature and epithelium.     

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)     

Relationship between 45Ca movements, different calcium components and responses to acetylcholine and potassium in tracheal smooth muscle

Correlations between tension responses elicited with acetylcholine (ACh) and high K+ and corresponding alterations in Ca++ mobilization were obtained in rabbit and canine tracheal smooth muscle. Removal of Ca++ or preincubation with D-600 (50 microM) inhibited responses to K+ (50 or 80 mM) and low ACh (89 nM) and had only a small effect on responses to high ACh (8.9 microM). Conversely, solutions containing Sr++ instead of Ca++ inhibited responses to both concentrations of ACh to a greater degree than were those to K+. Washout of slow component 45Ca into a O-Ca solution was more rapid in rabbit trachea than reported previously for rabbit aorta. Washout of tracheal smooth muscle into an 80.8 mM La -substituted solution at 0.5 degrees C removed superficial (La -accessible) 45Ca and blocked both 45Ca uptake and most 45Ca efflux. D-600, which had no significant effect on control 45Ca uptake in rabbit aortic smooth muscle, decreased 45Ca uptake by 33% in rabbit tracheal smooth muscle. The uptake of 45Ca from the Ca++ binding sites with low affinity for Ca++ was increased by 80 mM K+, 50 mM K+ or 8.9 microM ACh, and the accumulation of Ca++ from the Ca++ binding sites with high affinity for 45Ca was inhibited by Sr++. The stronger effect of either Ca++ removal or D-600 on responses to K+ and the correspondingly greater effect of Sr++ on responses to ACh indicate that different Ca++ stores are present in tracheal smooth muscle. These Ca++ components appear to be qualitatively similar to those present in aortic smooth muscle but they differ quantitatively and are not as readily dissociated as are aortic Ca++ components.     

Mechanism of inhibitory effects of azelastine on smooth muscle contraction.

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.     

Local anesthetics differentiate dihydropyridine calcium antagonist binding sites in rat brain and cardiac membranes

Local anesthetics were used to probe differences in the binding of [3H]nitrendipine to dihydropyridine calcium antagonist binding sites on rat brain and cardiac membranes. Local anesthetics inhibited [3H]nitrendipine binding to brain and cardiac membranes with the rank order of potency, dibucaine = proadifen much greater than tetracaine greater than meproadifen greater than RAC-109 (S) greater than RAC-109 (R) greater than benzocaine. Lidocaine, procaine, piperocaine and bupivacaine produced either a small potentiation or inhibition of [3H]nitrendipine binding. Dibucaine inhibited [3H]nitrendipine binding to brain membranes (IC50, 4.9 +/- 0.5 microM) by increasing the Kd, whereas in cardiac membranes (IC50, 8.5 +/- 0.9 microM) it both increased the Kd and decreased the maximum binding site capacity of [3H]nitrendipine. The potency of dibucaine to inhibit [3H]nitrendipine binding was reduced in both tissues by monovalent (Li+ greater than Na+ = K+ = Rb+; EC50, 40-50 mM) and divalent (Ca++, Mg++ and Mn++; EC50, 10-50 microM) cations. These cations reduced the effect of dibucaine on the Kd of [3H]nitrendipine in brain and on the maximum binding site capacity of [3H]nitrendipine in cardiac membranes. Inhibition of [3H]nitrendipine binding by dibucaine was best described by high (2 microM) and low (50 microM) affinity sites. The apparent affinities of these sites, but not the fractional occupancies, were similar in brain and cardiac membranes. Na+ modulated the occupancies of these sites in brain, but not in cardiac membranes, whereas Ca++ inhibited occupancy of the high affinity site in both tissues. The effects of Li+ were similar to those of Ca++. These findings indicate that brain and cardiac dihydropyridine calcium antagonist binding sites are coupled to different allosteric effectors or exist in a different membrane environment.     

o-Isothiocyanate dihydropyridine (oNCS-DHP), a long-acting, reversible inhibitor of the Ca++ channel

The binding characteristics and pharmacological properties of o-isothiocyanate dihydropyridine [oNCS-DHP; 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyanatophenyl)-1, 4-dihydropyridine] were investigated in guinea pig heart and ileum. [3H]oNCS-DHP bound to a single population of high-affinity sites (Bmax = 107 fmol/mg of protein and Kd = 0.99 nM) in cardiac membranes, with a specificity characteristic of dihydropyridine receptors. After incubation of membranes with the tracer (0.5 nM), addition of excess nifedipine (1 microM) caused a dissociation of [3H]oNCS-DHP from its binding site. The reversibility of [3H]oNCS-DHP binding was confirmed by the lack of affinity labeling of cardiac membranes as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. oNCS-DHP inhibited the inward Ca++ current of isolated guinea pig cardiac myocytes as determined in voltage-clamp experiments. In isolated perfused guinea pig hearts, oNCS-DHP caused a concentration-dependent increase in coronary artery flow and a decrease in left ventricular pressure. The effects of the highest concentration (0.3 microM) were still near maximal after a 1-h washout. Suppression of K+ depolarization-induced contractures of isolated ileal longitudinal muscle strips by oNCS-DHP remained maximal even after 5 h of washout. In all of the three biological test systems investigated, the Ca++ channel activator Bay K 8644 caused a complete and rapid reversal of the inhibitory effects of oNCS-DHP. Thus, it can be concluded that oNCS-DHP does not bind irreversibly to Ca++ channel dihydropyridine receptors in guinea pig heart and ileum. However, the o-isothiocyanatophenyl substituent on the dihydropyridine molecule confers upon the compound a very long duration of Ca++ channel blocking activity.     

Sources of activator calcium for extrinsic vascular tone and nimodipine inhibition of that tone in proximal vs. distal rabbit ear arteries

The contractile responses of three artery segments of diminishing diameter in the rabbit ear (i.e., unstretched lumen diameter approximately 300 mu in central ear artery, unstretched lumen diameter approximately 150 mu in main side branch off the central ear artery and unstretched lumen diameter approximately 75 mu in terminal branch off the main side branch) to high K+, norepinephrine (NE) and 5-hydroxytryptamine (5-HT) were tested before and after their incubation in Ca++-free physiologic salt solution for times varying from 3 to 60 min. The time course of reduction of the contractile responses to K+ with Ca++-free conditions in all classes of vessels could be represented by monoexponential curves that were not significantly different from each other. The contractile response of all the ear arteries to NE and 5-HT was biphasic. The first rapid transient phase (phase I) was more resistant to change upon the removal of exogenous Ca++ than the second usually equilibrium-like component (phase II), which was dramatically and rapidly reduced (but not necessarily eliminated) by this procedure. The extent of decline upon Ca++ removal in most instances was greater for 5-HT than for NE. The rate of falloff of both phases of contraction to NE and 5-HT was faster in the smaller compared with the larger arteries. These results suggest that, as vessels in the rabbit ear arterial tree get smaller, the contribution of a tightly bound or intracellular Ca++ pool to both phases of amine-induced contraction becomes smaller, but this contribution is greater to the transient compared with the equilibrium phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of calcium channel inhibition by phenytoin: comparison with classical calcium channel antagonists

The mechanism of calcium channel antagonism by phenytoin was studied by comparing the effects of phenytoin and classical calcium channel antagonists on K+-stimulated 45Ca uptake and [3H]nitrendipine binding in the PC12 pheochromocytoma cell line. Inhibition of K+-stimulated 45Ca uptake occurred at clinically relevant concentrations of phenytoin (IC50 = 9.6 +/- 2.1 microM) and was not significantly modified by Na channel blockade with tetrodotoxin, K channel blockade with tetraethylammonium or depolarization with carbachol rather than K+. Phenytoin, verapamil and diltiazem inhibited 45Ca uptake with Hill coefficients of less than 0.7, whereas values for nimodipine and flunarizine were close to 1.0. Phenytoin inhibited binding of the dihydropyridine Ca channel antagonist [3H]nitrendipine to PC12 membranes (Ki = 31 +/- 3 microM) by decreasing binding affinity, with no change in the maximal number of binding sites. Phenytoin and nimodipine reduced [3H]nitrendipine binding without altering the first-order rate constant for dissociation; this rate was increased by verapamil and flunarizine and decreased by diltiazem. Diltiazem enhanced inhibition of [3H]nitrendipine binding by phenytoin, reversed inhibition by verapamil and flunarizine and had no effect on inhibition by nimodipine. These findings suggest that phenytoin and classical Ca channel antagonists inhibit voltage-gated Ca++ flux by distinct but functionally linked mechanisms.     

Intracellular Ca++ antagonist, HA1004: pharmacological properties different from those of nicardipine

Pharmacological properties of N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a newly synthesized intracellular Ca++ antagonist, were studied by comparing its cardiovascular actions with those of nicardipine, a Ca++ entry blocker. Both i.a. and i.v. injections of HA1004 produced a dose dependent increase in vertebral, coronary and renal blood flow, respectively, in pentobarbital-anesthetized dogs. Continuous infusion of atropine, propranolol or aminophylline exerted no significant effect on the HA1004-induced vasodilation. A Ca++ entry blocker, nicardipine, increased vertebral and coronary blood flow after injections but did not produce an increase in renal blood flow, thereby suggesting notable differences between the cardiovascular effects of HA1004 and the Ca++ entry antagonist, nicardipine. HA1004 inhibited KCl, phenylephrine and prostaglandin F2 alpha-induced contraction of isolated canine renal arterial preparations, in a dose-dependent fashion, whereas nicardipine was much less effective in blocking the contraction of the preparations induced by phenylephrine and prostaglandin F2 alpha. HA1004 inhibited the phenylephrine-induced contraction of the renal artery in the absence of Ca++. HA1004 neither depressed the amplitude nor the duration of the slow action potentials of isolated guinea-pig hearts. These results suggest that HA1004 is a Ca++ antagonist of different class from Ca++ entry blockers such as nicardipine.     

Electrophysiologic and biochemical studies on the putative Ca++ channel blocker MDL 12,330A in an endocrine cell

MDL 12,330A is a molecule structurally unrelated to other organic Ca++ channel ligands that may alter Ca++ channel function. Using whole cell patch clamp, [3H]PN200-110 binding and 45Ca++ uptake studies, we examined the effects of this compound on voltage-dependent Ca++ channels in rat anterior pituitary cells. At a concentration of 10(-5) M, MDL 12,330A showed little effect on outward K+ current, Na+ current or low-threshold Ca++ current in this cell line. At 10(-6) M, MDL 12,330A reversibly inhibited slow Ca++ current in a voltage-dependent manner. 45Ca++ uptake was also blocked by this compound at 10(-6) M, whereas [3H]PN200-110 binding was stimulated at concentrations of 10(-7) to 10(-6) M. The results are consistent with an interaction of MDL 12,330A with slow Ca++ channels at a site allosterically linked to the 1,4-dihydropyridine binding site.     

Inositol 1,4,5-trisphosphate may regulate rat brain Cai++ by inhibiting membrane bound Na(+)-Ca++ exchanger.

The role of inositol 1,4,5-trisphosphate (1,4,5-IP3) in regulating cytosolic Ca++ by stimulating Ca++ release from intracellular organelles is well established. However, other modes of intracellular Ca++ regulation by 1,4,5-IP3 have not been determined. To determine if 1,4,5-IP3 may regulate cell cytosolic Ca++ by acting on plasma membrane bound Na(+)-Ca++ exchanger, we investigated Ca++ transport in synaptosomes using 45Ca++ as tracer. In the presence of either an inhibitor of voltage gated Na+ channels (tetrodotoxin) or the K+ ionophore (valinomycin), Ca++ uptake was significantly inhibited (P less than 0.05) by 1,4,5-IP3 in a concentration dependent manner, with half-maximal inhibition occurring at submicromolar concentrations between 10(-9) M and 10(-10) M 1,4,5-IP3. Similarly, Ca++ efflux by the exchanger was significantly inhibited 40% by 1,4,5-IP3. The inhibitory effect of 1,4,5-IP3 on the Na(+)-Ca++ exchanger was observed in the presence of Ca++ channel blockers, and in vesicles pretreated with caffeine to deplete the 1,4,5-IP3-sensitive stores of Ca++. These results suggest that during signal transduction in brain, 1,4,5-IP3 may increase cytosolic [Ca++] in part by inhibiting the Na(+)-Ca++ exchanger and thus, Ca++ efflux from cell.     

Veratrine-induced decrease of (Na+ + K+)-adenosine triphosphatase activity in rat brain slices

The effect of membrane excitability on (Na+ + K+)-adenosine triphosphatase (ATPase) was studied in rat brain slices. The treatment of the brain cortical slices with veratrine for more than 10 min caused a significant decrease of the (Na+ + K+)-ATPase activity. The similar inhibition of the enzyme by veratrine was observed in the hippocampus and hypothalamus, and the veratrine treatment did not affect the sensitivity of the cortical enzyme for ouabain inhibition. These findings suggest that two isozymes of (Na+ + K+)-ATPase are equally inhibited by the treatment. Veratrine inhibited the partial reactions such as Na+-dependent phosphorylation and K+-stimulated phosphatase as well as the specific binding of [3H]ouabain. Agents which increase intracellular Na+ concentration also inhibited the enzyme activity. The effects of veratrine were blocked by Na+-free medium or tetrodotoxin. Low Na+ medium decreased the enzyme activity, and the effect was blocked by amiloride or Ca++-free medium, indicating the involvement of Na+/Ca++ exchange in the inhibition. The decreased activity induced by low Na+ or high K+ medium was restored to the normal level by the subsequent incubation in normal medium. The inhibitory effect of veratrine was dependent on external Ca++, and was blocked by addition of W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide]. A23187 also decreased (Na+ + K+)-ATPase activity in the slices. High Mg++ medium blocked the effect of veratrine but not that of monensin which was not dependent on external Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)