Reduction Of Extracellular Calcium Research Articles

Coronary flow stimulates auricular-ventricular transmission in the isolated perfused guinea pig heart.

In the heart in situ coronary flow stimulates oxygen consumption, glycolytic flux, myocardial contractility, and the release of bioactive substances. Studies have indicated that the coronary flow-enhanced contraction is similar to a hormonelike effect because the enhanced contraction results from an elevation in intracellular free calcium. In fact, if extracellular calcium is raised sufficiently, the contraction amplitude rises and remains constant and independent of coronary flow. We hypothesized that coronary flow could also stimulate other calcium-dependent cardiac functions such as auricular-ventricular (A-V) transmission. This hypothesis was tested in isolated guinea pig hearts perfused at constant flow. Our results show that increases in coronary flow (6-25 ml/min range) decrease the A-V delay solely as a result of reduced propagation time in the A-V node and not in atrial or ventricular propagation. When coronary vascular resistance was altered by dilation (nitroglycerin, bradykinin, nitroprusside, and adenosine) or by constriction (angiotensin II), this dromotropic effect of flow remained the same despite wide changes in perfusing pressure. Also, this dromotropic effect of flow was not altered by energy-altering substrates in the perfusate or by perfusion of adenosine receptor blockers. Furthermore, the effectiveness of flow as a dromotropic stimulus varied inversely with changes in calcium entry caused either by elevation or reduction of extracellular calcium. In addition, enhanced viscosity of the perfusing medium amplifies the positive dromotropic effect of flow. These results suggest that coronary flow is a stimulus that exerts a positive dromotropic effect mediated by shear stress.

Osmolar changes regulate the paracellular permeability of cultured human cervical epithelium.

Extracellular nucleotides induce a biphasic change in the transepithelial electrical conductance (GT) of human cervical cells grown on filters: a rapid increase (phase I) followed by a sustained decrease (phase II). To probe the involvement of the intercellular space, its magnitude was varied by manipulating cell volume through changes in extracellular osmolarity. Under baseline conditions [GT = 115 mS/cm2 (approximately 9 omega.cm2)] and during phase II, hypertonic challenges resulted in an increase in GT (0.98% .mosmol-1.l-1 and 0.73%.mosmol-1.l-1, respectively). However, a hypertonic challenge during phase I decreased GT (-0.16%.mosmol-1.l-1). Hypotonic challenges decreased GT during baseline, phase I, and phase II conditions by -1%.mosmol-1.l-1. Similar trends were observed with regard to pyranine permeability. Reduction of extracellular calcium increased GT, abrogated the phase II effect of extracellular ATP, and reversed the effect of a hypertonic challenge. The additive nature of the permeability changes in response to osmotic challenges and to ATP during phase II suggests that different sites are involved in each response, i.e., the resistance of the intercellular space changes with osmolarity and that of the tight junction during phase II.

Role of calcium fluxes in the action of glucagon on glucose metabolism in rat hepatocytes

The aim of the present study was to assess the role of calcium fluxes in the action of glucagon on glycogenolysis and gluconeogenesis in isolated rat hepatocytes. Calcium influx was blocked by two ways: by use of the compound tetramethrin and by reduction of extracellular calcium to 1 microM. The minimal concentration of tetramethrin that inhibited glucagon-mediated calcium entry was 7.5 x 10(-7) M. In the presence of 7.5 x 10(-7) M tetramethrin, glucagon-induced glycogenolysis was markedly attenuated when glucagon concentration was 10(-9) M or higher. In contrast, tetramethrin had no effect on glucogenolysis evoked by lower concentrations of glucagon. Similarly, tetramethrin greatly reduced gluconeogenesis induced by high concentrations of glucagon without affecting the effect of low concentrations of glucagon. The same results were obtained in the presence of 1 microM extracellular calcium. To abolish glucagon-induced elevation of cytoplasmic free calcium concentration, we heavily loaded quin2 into hepatocytes. In these cells, glycogenolysis evoked by low concentrations of glucagon was completely abolished. Glycogenolysis caused by high concentrations of glucagon was markedly inhibited. These results indicate that glucagon action on hepatic glucose metabolism is mediated by two different mechanisms, which depend on concentrations of glucagon.

Role of calcium entry and protein kinase C in the progression activity of insulin-like growth factor-I in Balb/c 3T3 cells

We previously reported that insulin-like growth factor-I (IGF-I) stimulated calcium entry (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1988) J. Biol. Chem. 263, 16561-16567) and production of 1,2-diacylglycerol in IGF-responsive "primed competent" Balb/c 3T3 cells (Kojima, I., Kitaoka, M., and Ogata, E. (1990) J. Biol. Chem. 265, 16846-16850). The present study was conducted to determine a role of protein kinase C (PKC) in the progression activity of IGF-I. To monitor the activity of PKC in intact cells, we measured phosphorylation of a synthetic KRTLRR peptide, a substrate of PKC, immediately after the permeabilization of the cells with digitonin. When 1 nM IGF-I was added to primed competent cells, KRTLRR peptide phosphorylation was augmented. IGF-I induced more than 2-fold increase in KRTLRR peptide phosphorylation that was blocked by PKC19-36, a pseudosubstrate of PKC, which blocks the activity of the kinase, and Ro31-8220, an inhibitor of PKC. The phosphorylation remained elevated for up to 6 h. To assess the role of PKC in cell cycle progression, IGF-I-induced nuclear labeling was measured in the presence of Ro31-8220. Ro31-8220 reduced the rate of entrance into S phase when added in the first quarter of the G1 phase, but did not affect cell cycle progression when added at the second quarter or later. In contrast, reduction of extracellular calcium completely blocked cell cycle progression when done in the first, second, and third quarter but had no effect in the last quarter. These results indicate that IGF-I persistently activates PKC in primed competent cells, but the activation is required only for the initiation of progression. We conclude that IGF-I promotes cell cycle progression by calcium-dependent mechanisms that are largely independent of PKC.

Calcium influx into neurons can solely account for cell contact-dependent neurite outgrowth stimulated by transfected L1.

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.

Cyclic GMP, calcium and photoreceptor sensitivity in mice heterozygous for the rod dysplasia gene designated “rd”

The rise in photoreceptor cGMP, induced by < 1.0 nM extracellular calcium, is delayed in retinas of mice heterozygous for the rod dysplasia gene (+/rd). The calcium ionophore A23187 reduces the delay, suggesting that +/rd outer segments contain more calcium than normal. In turn, this might explain the increased photosensitivity of the +/rd retina. During the response to low calcium there is no correlation in +/rd retinas between the total concentration of cGMP and the photoresponse amplitude and its time to peak. The observations imply that either free cGMP is abnormally independent of the bound pool in the +/rd photoreceptor outer segment or that factors other than cGMP and its phosphodiesterase are modulating the rising phase of the response. The time-to-peak of PIII in a +/rd retina, incubated in a standard medium and stimulated with dim light, is abnormally delayed. Reduction of extracellular calcium induces an abnormal delay as well in responses to higher light levels. In addition to this, a second delay manifests slowly in both the normal and the +/rd retina. More studies are needed to explain these observations.

Pharmacology of a cholecystokinin receptor on 5-hydroxytryptamine neurones in the dorsal raphe of the rat brain.

1. The effect of bath application of sulphated cholecystokinin octapeptide (CCK-8) was studied on neurones in slices containing rat raphe nucleus. 2. Intracellular recordings were made from neurones in the dorsal raphe nucleus. Some of the neurones with the characteristics of 5-hydroxytryptamine (5-HT)-containing cells which were inhibited by 5-HT and excited by noradrenaline were excited by cholecystokinin. The response to cholecystokinin was dose-dependent over the range 10 to 1000 nM. 3. The response to CCK-8 persisted in the presence of tetrodotoxin. Either reduction of extracellular calcium or addition of 25 mM magnesium did not block the CCK response, suggesting it was mediated by receptors located on the membrane of the raphe neurones. 4. The agonist and antagonist specificity of the CCK response was determined. The CCKB selective agonist, pentagastrin, was inactive when applied at concentrations up to 10 microM. the CCKA receptor antagonist L-364,718 (1 to 100 nM) blocked the response to cholecystokinin. Much higher (1-10 microM) concentrations of the CCKB receptor antagonist L-365,260 were required for inhibition of the CCK response. 5. These data support the existence of a CCK receptor, located on raphe neurones in the rat, with a pharmacological profile very similar to that described for the CCKA type.

Effects of 8-Bromo-cyclic GMP on contraction and on inotropic response of ferret cardiac muscle

The effects of guanosine 3′:5′-cyclic monophosphate (cGMP) on cardiac contraction are not established. Using isolated electrically-stimulated ferret papillary muscle at 29°C, 2.0 m m calcium we investigated the effects of 8-Bromo-cGMP on (a) basal contraction for comparison with the effects of reduction in extracellular calcium or of reduction in resting muscle length; (b) contraction of preparations stimulated by isoprenaline, the dihydropyridine calcium agonist Bay K8644 or post-extrasystolic potentiation. 8-Bromo-cGMP (0.1 m m) induced a small significant reduction in isometric twitch tension (TT) (7%), isotonic shortening (PS) (6%) and in twitch duration, but had no effect on maximum unloaded shortening velocity ( V max) or rate of tension development ( + dτ dt ). Reduction in muscle length induced a similar immediate effect on contraction. Reduction of extracellular calcium (2.0 m m to 1.25 m m) reduced TT by 24% and PS by 14% as well as V max (19%) and + dτ dt (29%), but did not alter twitch duration. Bay K8644 (0.01 to 10 μ m) produced increases in TT, + dτ dt , PS and twitch duration each of which was significantly reduced in the presence of 8-Bromo-cGMP (0.1 m m). 8-Bromo-cGMP had no effect on the responses to isoprenaline 1 n m to 100 μ m—which increased TT, + dτ dt and PS but markedly reduced twitch duration—nor on post-extrasystolic potentiation which increased TT and + dτ dt but slightly reduced twitch duration. These results show that 8-Bromo-cGMP induces changes similar to the immediate effects of reduction in resting muscle length, and reduces the positive inotropic effects of Bay K8644 but not those of isoprenaline or post-extrasystolic potentiation.

Influence of Magnesium and Extracellular Calcium Reduction on Ouabain‐treated Sinoatrial Node Cells in Rabbit Heart

The influence of the extracellular Mg on the slow regenerative potential induced by Ca removal was investigated by using rabbit SA node tissue treated with ouabain. When the SA node tissue was superfused with ouabain (10(-5) M), the preparation became quiescent. Further superfusion with Mg-free and Ca-free solution produced a small regenerative potential reaching 21 +/- 3 mV (n = 5). Superfusion with high Mg, Ca-free solution containing 5.19 mM Mg inhibited the regenerative potential. Superfusion of the ouabain-treated SA node tissue with Ca-free (2 mM EGTA) solution containing normal Mg or Mg-free, Ca-free (2 mM EGTA) solution produced a slow regenerative potential reaching 45 +/- 2 mV (n = 5) or 44 +/- 3 mV (n = 5). Increasing the external Mg to 3.6 mM or 6.18 mM in the Ca-free, EGTA solution caused a significant inhibition of slow regenerative potential. The addition of diltiazem (0.08 mM) blocked both normal action potential and slow regenerative potential induced by superfusion with Mg-free, Ca-free (2 mM EGTA) solution. These results suggest that Mg inhibits the development of the slow regenerative potential resulting from inward currents through the Ca channel.

Dissociation of Cardiodepression from Cardioprotection with Calcium Antagonists

This study was performed to determine if cardiodepression can be dissociated from cardioprotection with calcium antagonists and which one (diltiazem, nifedipine, or verapamil) can maximally protect ischemic myocardium at a given level of cardiodepression. Isolated rat hearts were subjected to 0.1, 0.5, or 1.0 microM diltiazem, verapamil, or nifedipine 10 min before global ischemia. Ischemia was maintained for 25 min, at which time reperfusion was instituted for 30 min. Pre- and postischemia function, flow, and lactate dehydrogenase (LDH) release were measured. All three drugs reduced preischemic function and improved postischemic function and reflow in a dose-dependent fashion. LDH release and contracture were also mitigated with all drugs. When the efficacy of these drugs was expressed as the ratio of LDH release versus preischemic, postdrug function (ability of drug to reduce LDH release at a given level of cardiodepression), diltiazem had a significantly lower ratio as compared with verapamil or nifedipine. When similar experiments were performed with various concentrations of calcium in the perfusion buffer (2.50, 1.25, 0.75, 0.50, 0.41 mM CaCl2) administered 10 min before ischemia and reperfusion with normal (1.25 mM) buffer, preischemic function was reduced in a concentration-dependent fashion. Despite severe reductions in function at the concentration of 0.50 mM CaCl2, LDH release was not reduced. The concentration of 0.41 mM CaCl2, which depressed function to the same degree as 0.50 mM CaCl2, reduced LDH release. This reduction in LDH release, however, was not as great as that which occurred with the high dose of the calcium antagonists. Reperfusion with 0.41 mM calcium buffer, however, nearly abolished LDH release. Thus, although all three calcium antagonists reduced the severity of ischemia, diltiazem reduces it with the lowest cost in cardiac function. Reduction in extracellular calcium reduces cardiac function, but reductions in severity of ischemia, as measured by LDH release, do not parallel these changes.

The role of calcium in the pharmacology of mania

AbstractEpisodes of mania in bipolar affective disorder patients are associated with a decrease in cerebrospinal fluid calcium. A reduction in extracellular calcium in most neurones leads to a decrease in calcium‐activated potassium conductance mechanisms. Such mechanisms help govern the rate of action potential generation by neurones, and thus the rate of information processing. The dopamine hypothesis of neuroleptic action cannot explain the antimanic actions of lithium, calcium antagonists or carbamazepine. This paper proposes a common mechanism of action for such substances — namely, an action on calcium‐activated potassium conductances.

MECHANISMS AND SIGNIFICANCE OF CALCIUM ENTRY AT POSITIVE MEMBRANE POTENTIALS IN GUINEA‐PIG VENTRICULAR MUSCLE CELLS

Possible mechanisms for calcium entry at positive membrane potentials were investigated in single cells isolated from guinea-pig ventricular muscle. The cells were voltage clamped and contraction was measured by an optical technique. When prolonged (200 ms to 2 s) depolarizations at +60 mV were applied, contraction amplitude increased with pulse duration, in contrast to the contraction at 0 mV. When a 'pre-pulse' to 0 mV was applied for 200 ms to inactivate current through 'L-type' calcium channels, contraction nevertheless increased with membrane potential during a subsequent test pulse applied over the range -40 to +60 mV. Contraction during the test pulse at +60 mV was abolished when extracellular calcium was reduced to zero. This effect developed more rapidly than abolition of the contraction in response to the pre-pulse to 0 mV. Reduction of extracellular calcium from 2.5 to 1 mM reduced the contraction at +60 mV to a greater extent than that at 0 mV and caused an inward shift in the current at +60 mV. Nifedipine (5 microM) substantially reduced the contraction during the test pulse to 0 mV but had little effect on the contraction at +60 mV. Conversely, dodecylamine (20 microM) caused little or no decrease in the contraction at 0 mV but substantially reduced the contraction at +60 mV. Following a conditioning pre-pulse to 0 mV the contraction at +60 mV was not consistently reduced by exposure to 3 microM-ryanodine. The interpolation of a single 200 ms pulse to +60 mV in a train of pulses to 0 mV potentiated the following contraction to 0 mV. This potentiation decayed over the first four steps to 0 mV following an interpolated pulse and increased with the voltage of the interpolated pulse over the range -20 to +60 mV. Potentiation was abolished on exposure to 3 microM-ryanodine. These observations are consistent with entry of calcium at positive membrane potentials through voltage-dependent, non-inactivating pathways which are insensitive to nifedipine but inhibited by dodecylamine. The observations support the hypothesis that calcium entry via this mechanism may contribute, at least under some conditions, to the loading of intracellular stores of calcium during the late plateau of the action potential, and thus influence subsequent contraction. Calcium entry through Na+-Ca2+ exchange is a possibility which would allow calcium entry to increase over the range of membrane potentials at which contraction was increased. However, additional calcium entry through other nifedipine-insensitive pathways, such as calcium-activated non-selective channels, cannot be excluded.

Interactions of adenosine and magnesium on rat hippocampal slices

The potency of adenosine in reducing orthodromically evoked population potentials elicited in area CA1 of rat hippocampal slices was greatly reduced by removal of magnesium from the bathing medium. Removal of magnesium increased cell excitability, but this did not account for the loss of potency as the addition of (±)-2-amino-5-phosphonovaleric acid (2-APV), or a reduction in extracellular calcium failed to restore adenosine inhibition. However, addition of cobalt (500 μM) did restore inhibition. The results may indicate a requirement for magnesium, or a similar divalent cation, at the A 1-inhibitory adenosine receptor. The observation of excitation by adenosine in zero magnesium/low calcium may suggest an A 2-excitatory receptor interaction in the absence of adenosine inhibition.

The effect of halothane on the free intracellular calcium concentration of isolated rat heart cells.

The free intracellular calcium concentration of suspensions of isolated rat heart cells was monitored during sequential exposures to halothane and caffeine to evaluate cellular mechanisms of the negative inotropic effect of halothane. The calcium-sensitive, fluorescent dye quin2 was used as the indicator of free intracellular calcium. The acute addition of halothane in concentrations greater than or equal to 0.062 mM (0.19 vol%) to suspensions of quiescent rat heart cells at 37 degrees C caused a transient (approximately 1.5 min) increase in free intracellular calcium concentration. The intracellular calcium concentration after the decay of this transient was not detectably different from that prior to the addition of halothane. Neither the reduction of extracellular calcium from 1 mM to 100 nM, nor the prior addition of verapamil (5 microM) decreased this halothane-induced calcium transient. The transient was completely blocked by the prior addition of 10 mM caffeine, which depletes the sarcoplasmic reticulum of calcium. Also, the prior addition of halothane caused a reduction in the calcium transient due to caffeine. The depression of the caffeine-induced calcium transient by halothane was independent of the time interval (up to 4 min) between the additions of halothane and caffeine. These results indicate that halothane causes a net loss of calcium from the sarcoplasmic reticulum of quiescent rat heart cells. Thus, halothane has a direct effect at the sarcoplasmic reticulum, probably an enhancement of calcium release, which may explain its depression of myocardial contractility.

Pharmacological and physiological properties of the after‐hyperpolarization current of bullfrog ganglion neurones.

1. The slowly decaying, calcium-dependent after-hyperpolarization (a.h.p.) that follows action potentials in bullfrog ganglion B cells has previously been shown to be generated by a potassium current called IAHP. We have recorded IAHP using a switched, single-electrode hybrid clamp where current-clamp mode was changed to voltage-clamp mode immediately after repolarization of a spike or the last spike of a train. 2. Reduction of extracellular calcium reduced the decay time of IAHP following a single spike. At all levels of extracellular calcium tested (0.5-4 mM), the decay time of IAHP was longer following a train of action potentials than following a single action potential. Thus, the time course of IAHP evoked by action potentials is a function of the calcium load induced by the action potentials. Conversely, agents that reduce the amount of IAHP activated without affecting its rate of decay, probably do not affect calcium influx. 3. Muscarine (2 or 10 microM) inhibits IAHP following an action potential by at most 30% and has no effect on decay rate of IAHP. These results suggest that muscarine has little or no effect on either calcium influx or sequestration. Decay of the a.h.p. is accelerated by muscarine but this effect is due to an increased leak conductance. 4. Charybdotoxin (CTX) between 4 and 20 nM, prolongs action potential duration in a manner consistent with blockade of the voltage- and calcium-dependent potassium current (Ic) involved in spike repolarization in these cells. This action is consistent with its reported action on analogous channels in other systems. However, CTX also reduces IAHP. Thus, in bullfrog ganglion neurones, two distinct calcium-dependent potassium currents exhibit a comparable sensitivity to CTX. This cannot be due to a decreased influx of calcium because the decay rate of IAHP following an action potential is unchanged. The action of CTX was observed with both crude and purified preparations of CTX. 5. Apamin (25 nM) and (+)-tubocurarine (concentration giving 50% of maximal inhibition = 20 microM) block IAHP without affecting action potential duration. The action of (+)-tubocurarine is more readily reversible than apamin. Approximately 20% of IAHP is resistant to blockade by either apamin or (+)-tubocurarine. 6. Muscarine was used to block the M-current (IM) selectively and (+)-tubocurarine was used to inhibit IAHP selectively. Both currents were shown to contribute to spike frequency adaptation. Inhibition of both IM and IAHP has a synergistic action to increase repetitive firing.

Inositol 1,4,5-trisphosphate induces bursts of calcium release inside Limulus ventral photoreceptors

Injection of inositol 1,4,5-trisphosphate (InsO 3) into dark-adapted Limulus ventral photoreceptors produces a series of discrete bursts of membrane depolarization. Prior injection of aequorin, a luminescent calcium indicator, reveals that the bursts of depolarization are accompanied by individual bursts of intracellular calcium elevation with a similar time course. Reduction of extracellular calcium increased rather than decreased the InsP 3-induced rise in calcium. These results suggest that small numbers of InsP 3 molecules can trigger discrete and rapid releases of large amounts of calcium from intracellular stores. In some cells, InsP 3 injection induces a delayed and prolonged elevation of intracellular calcium in addition to the brief bursts.

Depolarisation-evoked release of acetylcholine can mediate phosphoinositide hydrolysis in slices of rat cerebral cortex

Depolarisation of [ 3]H]inositol prelabelled slices of cerebral cortex of the rat, with elevated extracellular K + or the alkaloid veratrine, induced a marked accumulation of [ 3H]inositol monophosphate in the presence of 5mM Li +. The effects of these stimuli were concentration-related with maximal responses obtained at 30 mM K + and 30 μM veratrine. Larger concentrations produced submaximal responses but also markedly suppressed the incorporation of [ 3H]inositol into phospholipid. The response to K + or veratrine were not sensitive to atropine, prazosin, mepyramine, ketanserin or the peptidase inhibitor bacitracin. However, in the presence of the cholinesterase inhibitor physostigmine, the responses to these stimuli were greatly enhanced and this could be blocked by atropine. Both veratrine and K + markedly stimulate release of endogenous acetylcholine from the slices. Release appears to be linear with time over the 45 min period of continuous stimulation. Reduction of extracellular calcium severely suppressed both the release of acetylcholine and the atropine-sensitive component of the phosphoinositide response to K +. The results suggest that endogenous acetylcholine can stimulate phosphoinositide metabolism by interacting with muscarinic receptors. The atropine-insensitive component, at least in part, represents entry of Ca 2+ through voltage-sensitive channels and perhaps a direct effect on phosphoinositide metabolism.

The protective effect of transient extracellular calcium reduction against reperfusion-induced ventricular fibrilation in the isolated rat heart

The protective effect of transient extracellular calcium reduction against reperfusion-induced ventricular fibrilation in the isolated rat heart

A calcium-activated sodium conductance contributes to the fertilization potential in the egg of the nemertean worm Cerebratulus lacteus

The fertilization potential of the egg of the nemertean worm Cerebratulus lacteus consisted of a rapid shift from a resting potential of about −65 mV to a peak of about +44 mV; the peak was followed by a positive plateau at about +24 mV, lasting an average of 80 min. Reduction of extracellular calcium reduced the peak of the fertilization potential, indicating that the peak resulted from a calcium conductance, while reduction of extracellular sodium reduced the plateau potential, indicating that the plateau resulted from a sodium conductance. Microinjection of ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) or 1,2-bis( o-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)/CaBAPTA buffers, having a free calcium concentration of less than or equal to about 0.1 μ M lowered the fertilization potential plateau. Injection of a BAPTA/CaBAPTA mixture with a free calcium concentration of about 1 μM resulted in a prolonged positive potential at the level of the fertilization potential plateau. These observations indicated that the fertilization potential of the Cerebratulus egg depended on a calcium-activated sodium conductance. The plateau potential was reduced little, if any, when calcium-free seawater was perfused through the bath during the fertilization potential; nor was it reduced in seawater containing cadmium. These observations suggested the possibility that intracellular calcium stores could be important in producing the fertilization potential.

Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N6 O2'-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3.5 mmol/l), significantly (P less than 0.01) increased aldosterone output by at least 1.5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P less than 0.01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 +/- 14 vs 105 +/- 19 pmol/10(6) cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion. Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P less than 0.01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine. These observations suggest that: the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.