Transmembrane Calcium Flux Research Articles

A lipid vesicle system for probing voltage‐dependent peptide–lipid interactions: Application to alamethicin channel formation

A membrane potential is shown to be established in phosphatidylcholine/cholesterol unilamellar vesicles using valinomycin in conjunction with a potassium ion gradient; this potential is monitored using the externally added fluorescent dye Safranine O. In the same system, transmembrane calcium fluxes are then detected using the (internally trapped) fluorescent dye Quin-2. The calcium-transport behavior of the channel-forming peptide alamethicin is shown to be potential dependent in this system, in contrast to calcium transport by the ionophore Br-A23187, which is unaffected by the potential. The observation of this potential-dependent behavior for alamethicin suggests that this vesicle system may be suitable for direct spectroscopic observation of the voltage-gating process.

Receptor-Operated, but Not Voltage-Operated, Calcium Channels Are Involved in Basophil Leucocyte Activation and Histamine Release

Transmembrane calcium flux is a critical step in basophil and mast cell activation and subsequent histamine release. This calcium flux is likely to take place through specialized membrane ion channels. Two types of calcium channels have been described so far: the first type is voltage operated and the second type is receptor operated. Depolarization of cell membrane by K+-rich solutions is followed by voltage-operated channel opening in excitable cells, such as smooth muscle cells. We evaluated whether high K+ extracellular concentrations can trigger basophil activation and histamine release. We found that human basophil leucocytes, showing a normal response to activating signals, such as anti-IgE antiserum and formylmethionine peptide, release no histamine when exposed to K+-rich media, alone or in combination with the K+ carrier valinomycin. These results are consistent with there being receptor-operated, but not voltage-operated, calcium channels in the basophil leucocyte plasma membrane.

Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis

Oocytes of Xenopus laevis were treated with agents which induce individual intracellular signals normally evoked during the process of meiotic maturation. Ultrastructural analysis of these oocytes allowed identification of specific second messengers that individually trigger single ultrastructural changes characteristic of the meiotic maturation process: Manipulation of intracellular cAMP levels induced changes in cortical granule position. Cytoplasmic alkalinization triggered a disruption of the annulate lamellae, a specialized organelle in the periphery of oocytes. Activation of protein kinase C caused rapid formation of a cortical endoplasmic reticulum and subsequent disruption of cortical granules. Manipulation of transmembrane calcium flux had varied results dependent upon the agent employed. Two of the treatments, Verapamil and zero external calcium, induced a reorganization in the oocyte periphery. The results indicate that these ultrastructural events are under the control of specific intracellular signals known to be elicited during meiotic maturation.

The antagonism induced by Ruthenium Red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies

Ruthenium Red, an inorganic dye which blocks transmembrane calcium (Ca) fluxes in neural tissues, reduced the capsaicin-induced release of substance P-like immunoreactivity from muscle strips of the guinea-pig urinary bladder in a concentration-dependent (30 nM - 3 μM) manner, and protected the sensory fibers from capsaicin-induced densensitization. A similar antagonism of the actions of capsaicin was observed in functional experiments (capsaicin-induced contraction of the isolated guinea-pig bladder or inhibition of twitches of the isolated rat vas deferens). In view of its established action on the depolarization-coupled entry of Ca into synaptosomes and the secretion of transmitter, we propose that Ruthenium Red could antagonize the action of capsaicin on the peripheral terminals of sensory nerves by a similar mechanism, thereby suppressing transmitter secretion and preventing the establishment of desensitization.

Role of Calcium in Prolactin-Stimulated c-mycGene Expression and Mitogenesis in NB2 Lymphoma Cells*

Receptor-activated transmembrane calcium flux has been implicated as a mediator of the actions of many growth factors and hormones. We examined the effects of PRL, calcium ionophores, and calcium antagonists on 45Ca2+ flux, c-myc gene expression, and DNA synthesis in the PRL-dependent rat Nb2 lymphoma cell line. PRL had no detectable effects on 45Ca2+ uptake or efflux, and the mitogenic effects of PRL could not be reproduced by the calcium ionophore A23187 alone or in combination with the tumor-promoting phorbol ester 12-O-tetra-decanoyl-phorbol-13 acetate (TPA). PRL, but not A23187 or TPA, stimulated c-myc gene expression in quiescent Nb2 cells. Exposure to PRL for brief periods (15 min to 4 h), followed by extensive washing, resulted in a time- and dose-dependent activation of DNA synthesis measured 16 h later. This activation was not blocked by addition of excess anti-PRL antiserum after the wash steps, indicating that the observed stimulation was not due to residual PRL. Despite the marked increase in DNA synthesis, removal of PRL after 4 h prevented mitosis, suggesting that PRL may be required throughout the cell cycle for Nb2 cell proliferation. Although continuous incubation with calcium antagonists resulted in a dose-dependent inhibition of PRL-stimulated DNA synthesis, activation of DNA synthesis by brief exposure to PRL was not inhibited by the presence of EGTA, calcium channel blockers (nifedipine, cobalt chloride), or calmodulin inhibitors (trifluoperazine, N-6-aminohexyl-5-chloronaphthalene sulfonamide). PRL-stimulated c-myc expression was attenuated, but not blocked, by the calcium channel antagonists. However, the putative intracellular calcium antagonist TMB-8 inhibited both c-myc expression and DNA synthesis in a dose-dependent manner (IC50 = 16 microM). Nb2 cells were sensitive to TMB-8 throughout G1 of the cell cycle, but inhibition of DNA synthesis was greatest when TMB-8 was present during the first 3 h of mitogen presentation, indicating a block in the transition from G0 to G1 of the cell cycle. The effect of TMB-8 suggests that release of a small intracellular calcium pool may mediate the early actions of PRL in Nb2 cells.

Phorbol ester increases the dihydropyridine-sensitive calcium conductance in a vascular smooth muscle cell line.

In vascular smooth muscle, phorbol esters cause a slowly developing contraction and an associated transmembrane calcium flux, both of which are inhibited by dihydropyridine calcium channel antagonists. In the A7r5 cultured vascular cell line, we used the whole-cell voltage-clamp technique to identify voltage-dependent calcium conductances and investigate the effect of phorbol esters on that conductance having characteristic dihydropyridine sensitivity (slowly inactivating, high-threshold, "L-type"). With barium as the charge carrier, large-amplitude (100-800 pA) inward currents of two types were characterized by their kinetics and voltage dependence. With holding potential--80 mV, a rapidly inactivating, low-threshold current ("T-type") was activated by depolarizations above-40 mV and was maximal at -10 mV. With holding potential -30 mV, this component was inactivated, and a second slowly inactivating, high-threshold current was activated above -10 mV and was maximal at +10 to +20 mV. These currents are similar to the T-type and L-type currents previously described in vascular smooth muscle cells. When added to the bath, the active phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (100 nM) increased the slowly inactivating (L-type) current by 32 +/- 20% (n = 8, +/- SD). Phorbol-12,13-dibutyrate (100 nM) caused a similar effect, but the inactive phorbol, 4-alpha-phorbol (100 nM), did not. We conclude that at least two distinct calcium conductances are expressed in A7r5 vascular smooth muscle cells, and that the dihydropyridine-sensitive calcium conductance is acutely modulated by phorbol esters, presumably acting through stimulation of protein kinase C. Such modulation may play a role in increasing transmembrane calcium influx mediated by agonist-receptor interactions that lead to activation of protein kinase C and may help to sustain or amplify calcium-dependent cell responses.

The effect of calcium channel blockade on basal- and substance P-induced intestinal secretion

Calcium plays a central role in modulating many physiologic events. We have investigated the role of calcium channel blockade in the control of basal ( n = 6)- and substance P-stimulated ( n = 6) intestinal transport in the isolated perfused rabbit ileum. Twenty-centimeter segments of ileum, harvested from New Zealand rabbits, were arterially perfused at 1.5 ml/min with an oxygenated modified Krebs buffer solution containing washed human red cells (Hct = 15–20%) and 2.5 m M Ca 2+. The intestinal lumen was perfused at 2 ml/min with an isotonic solution containing 1.2 m M Ca 2+ and [ 14C]PEG as a nonabsorbable volume marker. The infusion of verapamil (1 μg/min) significantly reduced ( P < 0.05) the basal secretion of H 2O, and Cl −. Verapamil prevented the secretory effect of substance P infused at 0.25 μg/min. Intraarterial verapamil had no effect on vascular perfusion pressure. These data indicate that calcium channel blockade has significant effects on basal- and substance P-stimulated intestinal secretion and suggest that transmembrane calcium fluxes function as major determinants of basal- and secretagogue-stimulated intestinal transport.

Calcium-sensitive uptake of amino acids by human placental slices.

The placenta supplies many nutrients to the fetus, including amino acids by active transport. Although the exact regulatory mechanism is unknown, a few studies have suggested a role for calcium in amino acid transport. Therefore, we examined the relationship between calcium and amino acid uptake by term human placental slices. Calcium depletion of slices significantly reduced uptake of alpha-aminoisobutyric (AIB), which is actively transported primarily by a sodium-dependent, carrier-mediated mechanism. Impairment of AIB uptake induced by calcium depletion was reversed by repletion with calcium but not with other divalent cations. In contrast, uptake of phenylalanine, which is transported primarily by a sodium-independent mechanism, was not affected by calcium depletion. Uptake of leucine and valine, which accumulate by both sodium-dependent and independent mechanisms, was partially affected by calcium depletion. Verapamil (10 microM), an inhibitor of transmembrane calcium flux, significantly reduced AIB uptake. Trifluoperazine, a calmodulin antagonist, also inhibited AIB uptake. Analysis of AIB uptake kinetic constants for control and calcium-depleted slices showed no change in the diffusion constant, a 37% reduction in Vmax, and a 2-fold increase in Km. The results indicate that calcium may be an important factor in the cellular regulation of active transport of amino acids.

Changes in adrenergic pressor responses by calcium channel modulation in conscious dogs

The influence of the slow channel calcium entry blocker, nifedipine, and the slow channel calcium entry promoter, BAY-K 8644, on receptor-mediated hemodynamic responses was studied in chronically instrumented, conscious dogs. Following ganglionic, cholinergic, and beta-adrenergic blockade, equipressor doses of phenylephrine (0.6 microgram/kg iv), a selective alpha 1-adrenoceptor agonist, and B-HT 933 (20 micrograms/kg iv), a selective alpha 2-adrenoceptor agonist, as well as a nonadrenergic vasoconstrictor, vasopressin (0.003 IU/kg) were administered before and after infusions of nifedipine or BAY-K 8644 (0.25, 0.5, 1.0, and 2.0 micrograms X kg-1 X min-1). In doses producing minimal or no haemodynamic effects, nifedipine caused dose-related attenuation from control of phenylephrine- (from 26 +/- 3 to 8 +/- 1 mmHg), B-HT 933- (from 30 +/- 2 to 5 +/- 2 mmHg), and vasopressin- (24 +/- 1 to 2 +/- 2 mmHg) mediated changes in mean arterial pressure. In contrast, BAY-K 8644 produced dose-related potentiation from control of the same pressor responses (phenylephrine, 24 +/- 2 to 41 +/- 3 mmHg; B-HT 933, 28 +/- 3 to 46 +/- 2 mmHg; vasopressin, 25 +/- 3 to 45 +/- 5 mmHg). In addition, BAY-K 8644 produced a marked increase in the duration of the pressor response produced by all three agonists. Thus, in conscious dogs, alpha 1-, alpha 2-, and vasopressin-mediated pressor responses are strongly modulated by transmembrane calcium flux and can be influenced by dihydropyridine slow channel calcium agonists and antagonists.

Pharmacology of Opiates and Their Derivatives

Abstract: The opiates or opioids are exogenous substances which bind in a stereospecific and saturable manner to several subspecies of cell membrane receptors which are sites of action of endogenous ligands (enkephalins, B‐endorphine). The opiates, which have common structural features, are agonists or partial agonists, the potency of which generally correlates well with affinity for opiate receptors. Many of the actions of opiates are related to alteration of the release of endogenous neurotransmitters. Inhibition of intracelluar adenylate cyclase and altered transmembrane calcium fluxes also appear to be important consequences of opiate action. Most opiates share the pharmacological properties of morphine. The development of tolerance and physical dependence with chronic administration is a characteristic feature of all opiates limiting their clinical use and creating the liability for abuse. With established opiate tolerance, cessation of the drug is accompanied by a characteristic withdrawal syndrome which is suppressed by opiate administration and can also be precipitated by an opiate antagonist such as naloxone. Many of the differences between opiates are due to different pharmacokinetic properties, particularly oral bioavailability and elimination rate. The search for a non‐addictive opiate analgesic continues but is presently foiled by the inherent pharmacological properties of these drugs.

Calcium channel blockers in systemic hypertension.

Calcium channel blockers selectively inhibit transmembrane flux of calcium in excitable tissue. Their ability to block calcium-mediated electromechanical coupling in contractile tissue produces peripheral vasodilation and has led to their clinical use in patients with systemic hypertension. The drugs appear to be well tolerated and have excellent safety profiles. Sustained-release preparations allow the drugs to be used once or twice daily. The calcium channel blockers will play an important role in the management of patients with mild, moderate, and severe hypertension when used as monotherapy or when combined with other antihypertensive medications. They can also be used in patients with concomitant diseases, such as angina pectoris and arrhythmia.

Antagonism between (–)‐N6‐phenylisopropyl‐adenosine and the calcium channel facilitator Bay K 8644, on guinea‐pig isolated atria

Antagonism between (-)-N6-phenylisopropyladenosine (PIA) and the dihydropyridine calcium channel facilitator Bay K 8644 was investigated in guinea-pig spontaneously beating or electrically driven isolated atria, taken from normal and from reserpine-treated animals. PIA (3-100 nM) produced a dose-dependent decrease in contractile tension and frequency in spontaneously beating atria being more effective in reserpinized preparations. Bay K 8644 (5-200 nM) produced an increase in contractile tension in both normal and reserpinized atria. In electrically driven left atria the positive inotropic effect of Bay K 8644 was similar to that in spontaneously beating preparations. The positive chronotropic effect of Bay K 8644 was slight and variable. PIA produced a rightward parallel shift of the concentration-response curves for the positive inotropic effects of Bay K 8644 in all experimental conditions. In spontaneously beating atria from normal guinea-pigs, the Schild regression plot was linear and its slope near to unity; pA2 of PIA 8.63 +/- 0.05 (IC50 2.35 +/- 0.25 nM). In electrically driven atria the antagonism by PIA of the effects of Bay K 8644 was apparently competitive, and the IC50 of PIA was 18.6 +/- 0.4 nM. PIA antagonized the positive chronotropic effect of Bay K 8644 in spontaneously beating preparations, both from normal and from reserpine-treated animals. Carbachol did not modify the positive inotropic effects of Bay K 8644. These data indicate that PIA may interact with Bay K 8644 at the level of the slow calcium channels, and may decrease the transmembrane calcium flux into the cell.

Acute effect of calcium and insulin on hyperfiltration of early diabetes.

We examined the effect of calcium administration on renal hyperfiltration in streptozotocin-treated diabetic rats. Rats were studied 7-10 days after streptozotocin injection. Intrarenal infusion of CaSO4 in Ringer's solution had no effect on the hyperfiltration of the diabetic kidney. Infusion of insulin in a dose that did not effect hyperglycemia also had no effect on the hyperfiltration. However, when insulin and calcium were infused together, a rapid decrease in glomerular filtration rate, single-nephron filtration rate, glomerular hydraulic pressure, and renal plasma flow occurred. The contralateral control kidney was unaffected. Verapamil infusion had no significant effect in untreated diabetic rats, but immediately reversed the vasoconstriction induced by insulin plus calcium. Similar intrarenal insulin and calcium infusions had no effect in euvolemic or chronically salt-loaded nondiabetic rats. The observations indicate that renal vascular cells (probably preglomerular) are hyperresponsive to calcium in early insulin-dependent diabetes mellitus and that this response requires insulin. We suggest that decreased renal vascular tone in early insulin-dependent diabetes mellitus may be due in part to defective transmembrane calcium flux across vascular smooth muscle cells. Insulin appears to be required for calcium entry or mobilization, to initiate renal vascular smooth muscle contraction in diabetes.

Studies on the Mechanisms by Which Tumor Promoters Stimulate the Growth of Primary Neonatal Rat Hepatocytes

A single exposure to a low concentration (10(-10) mol/L) of several tumor promoters, namely 12-O-tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), nafenopin, saccharin, teleocidin, benzoyl peroxide, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT), lindane, clofibrate, and melittin significantly stimulated DNA synthesis of neonatal rat hepatocytes in 4-day-old primary cultures. These cultures were kept in low-calcium (0.01 mmol/L) HiWoBa2000 synthetic medium, thereby evoking a neoplastic phenotype in otherwise normal (i.e., non-initiated) cells. The simultaneous addition of a single dose of alpha-tocopherol (10(-4) mol/L) or selenous acid (10(-5) mol/L), just as that of exogenous superoxide dismutase (SOD) (4), together with each of the above agents fully suppressed the stimulation of hepatocytic DNA synthesis by the xenobiotics. Hence, these findings strengthen the view that superoxide anions (or some other oxidizing compounds) act as the common mediators of the mitogenic effects of various tumor promoters in hepatocytes. Inhibition kinetics studies, in which TPA in a single dose (10(-10) mol/L) was used as the paradigmatic compound together with several kinds of inhibitors of its activity showed that the early mitogenic effects of TPA, i.e., the commitment of quiescent (G0) hepatocytes and the reentry into active cycling of hepatocytes spontaneously poised at the G1/S boundary, required oxidizing compounds, arachidonate metabolism derivatives, and plasmalemmal calcium-binding sites and transmembrane calcium fluxes. Instead, a later TPAs effect, the flow into DNA synthesis of hepatocytes previously committed to cycle, was shown to be controlled by retinoid-modulable activities, by some product(s) of the lipoxygenase pathway, and again by plasmalemmal calcium-binding sites and transmembrane calcium fluxes. Such results reveal that in the neonatal rat hepatocyte the ability to answer to a single mitogenic stimulus and the metabolic pathways by which this answer is enacted depend upon the mitotic cycle setting of the hepatocytes at the moment of the experimental treatment.

Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes.

We evaluated CD2 (E rosette) and CD3 (T3)-triggered activation of resting lymphocytes by measuring the intracellular free calcium concentration ([Ca2+]i) of individual cells. The [Ca2+]i of indo-1-loaded cells was measured by flow cytometry and responses were correlated with cell surface phenotype. Stimulation with anti-CD3 antibody caused an increase in [Ca2+]i in greater than 90% of CD3+ cells within 1 min, and furthermore, the response was restricted to cells bearing the CD3 marker. In contrast, stimulation of cells with anti-CD2 antibodies produced a biphasic response pattern with an early component in CD3- cells and a late component in CD3+ cells. Thus, the CD2 response does not require cell surface expression of CD3. In addition, stimulation of a single CD2 epitope was sufficient for activation of CD3- cells, whereas stimulation of two CD2 epitopes was required for activation of CD3+ cells. Both the CD2 and CD3 responses were diminished in magnitude and duration by EGTA. However, approximately 50% of T cells still had a brief response in the presence of EGTA, indicating that the increased [Ca2+]i results in part from intracellular calcium mobilization, and furthermore demonstrates that extracellular calcium is required for a full and sustained response. Our results support the concept that CD2 represents the trigger for a distinct pathway of activation both for T cells that express the CD3 molecular complex and for large granular lymphocytes that do not.

Protein binding properties of some histamine H 2 antagonists: A 1H nuclear magnetic resonance study of antihistamine-calmodulin interactions

Oxmetidine (SK & F 92994) is a potent histamine H 2 antagonist, which, however, also demonstrates cardiac effects consistent with its inhibiting transmembrane calcium fluxes. 1H nuclear magnetic resonance has been used to show that oxmetidine binds to a single site on the regulatory calcium-binding protein, calmodulin. Binding requires the presence of at least two equivalents of calcium per mol protein, is characterized by fast exchange behaviour and a dissociation constant of about 4 mM and is not affected by the presence of trifluoperazine. Protein-induced spectral changes and a limited study of structure-affinity relationships suggest the importance of the drug imidazole and benzyldioxymethylene groups in determining the strength of the interaction. Drug-induced perturbations in the spectrum of calmodulin indicate that the binding site is in the C-terminal half of the protein, and involves a hydrophobic area containing His-107, Met-144, Met-145 and possibly Phe-89, Phe-141, and calcium binding site III.

Calcium Channel Blocker Nisoldipine Limits Ischemic Damage in Rat Kidney

The effects of the calcium channel blocker nisoldipine on renal function after 60 min. normothermic ischemia and contralateral nephrectomy were studied in male Wistar rats. Nisoldipine (300ppm) was given in a standard diet as well as one hour prior to ischemia (10mg./kg. orally). Survival, serum urea, serum creatinine, urine volume and creatinine clearance were used to test the effectiveness of the drug. Nisoldipine treatment resulted in the survival of all animals (compared to 66.6 per cent in the untreated group) and improved immediate and long term (14 days) renal function. The drug given post ischemia only was not effective, suggesting that nisoldipine must be present in the kidney during ischemia. The beneficial effects of the drug in postischemic acute renal failure may be attributed in part to effects on postischemic renal hemodynamics. Additional direct effects on ischemic renal epithelial cells, presumably by inhibiting transmembrane calcium fluxes, cannot be excluded.

Inhibition of macrophage activation by calcium channel blockers and calmodulin antagonists

The biochemical mechanisms by which macrophages become activated to the tumoricidal state are poorly understood. To investigate the role of calcium in this process, the effect of calcium channel blockers and calmodulin antagonists on the acquisition of tumoricidal properties by macrophages activated by a number of different agents was examined. Activation of thioglycollate-stimulated C57BL/6 mouse peritoneal macrophages by macrophage activation factor (MAF) plus LPS, IFN-γ plus LPS or the calcium ionophore, A23187, was inhibited in a doseependent fashion by the calcium channel blockers nifedipine and verapamil. These agents blocked the influx of 45Ca into macrophages activated by MAF plus LPS. Macrophage activation was also inhibited by chlorpromazine, W-7, and calmidazolium at concentrations known to perturb calmodulin function. The data suggest that activation of macrophages to the tumoricidal state is a calcium-dependent process involving the participation of calcium-regulated biochemical reactions whose activities can be modulated by pharmacological agents that frustrate transmembrane calcium fluxes and/or inhibit calmodulin function.

Antigen-dependent increase in cytosolic free calcium in specific human T-lymphocyte clones.

Calcium has been implicated as an intracellular messenger in the cellular response to various external stimuli. Exposure of lymphocytes to various mitogens and lectins results in rapid transmembrane calcium fluxes and increased cytoplasmic calcium concentrations ([Ca2+]i). It is not clear, however, whether the mechanisms by which these non-physiological stimuli activate cells are related to those involved in antigen-specific activation. We have now used antigen-specific T-cell clones to study changes in [Ca2+]i associated with specific activation and show here that these cells respond specifically in the presence of antigen and antigen-presenting cells (APC) with increased [Ca2+]i and that this increased [Ca2+]i shows the same genetic restrictions as are seen in the proliferation assay. The kinetics of the [Ca2+]i response to antigen indicate that antigen undergoes a time-dependent processing step as a prerequisite for recognition by T cells, as has been shown for T-cell proliferative responses, but that the [Ca2+]i response to processed antigen is extremely rapid. The close correlation between changes in [Ca2+]i and cell activation resulting in proliferation suggests that Ca2+ may act as an intracellular messenger in antigen-specific responses.

Ca antagonists do not alter aldosterone secretion during established Na depletion

Conscious sheep whose sole adrenal gland had been transplanted to the neck to allow access to the adrenal vasculature were used to study the effect of calcium antagonists on aldosterone secretion. All animals were sodium depleted by uncompensated loss of parotid saliva. The drugs EDTA, verapamil, methoxyverapamil, nisoldipine, lanthanum, propylmethylenedioxyindene, and ryanodine were infused on separate occasions in three or four increasing dose levels. All infusions were made to produce known concentrations directly into the adrenal arterial blood supply. None of these infusions had any significant effect on aldosterone secretion rate, cortisol secretion rate, and little or no effect on plasma [Na], [K], or blood pressure. At high infusion rates some agents (verapamil, methoxyverapamil) caused tachycardia. In contrast, angiotensin II stimulation of aldosterone secretion is inhibited by both verapamil and nisoldipine. The data demonstrate that the sustained elevation of aldosterone secretion caused by sodium depletion is not dependent on a sustained alteration in transmembrane calcium flux. Furthermore, if circulating angiotensin II is the primary stimulus to aldosterone during sodium depletion, its mechanism of action appears to switch to one which is not dependent on calcium alone.