Voltage-dependent Calcium Channel Antagonist Research Articles

Analysis of intra- and intercellular calcium signaling in a mouse malignant glioma cell line.

Intra- and intercellular calcium signaling in glioma cells was examined by mechanical stimulation of a monolayer cell line of methylcholanthrene-induced mouse ependymoblastoma, 203-glioma, with a fine round-tip glass needle. A fura-2 fluorescence image of the glioma revealed a four- to eightfold increase in the cytosolic calcium ion concentration in directly stimulated signal cells. The increased calcium spread to surrounding cells at a speed of 20 microns/sec for a distance of up to 200 microns. Calcium was transmitted between adjacent cells and even in cells up to 200 microns distant from the initially stimulated cell. Microinjection of Lucifer yellow dye showed no gap junctional communication between cells. Depletion of extracellular calcium ion inhibited both cytosolic calcium elevation and propagation to neighboring cells by mechanical stimulus. An intracellular calcium blocker, TMB-8, eliminated the cytosolic calcium mobilization in a mechanically stimulated cell, but had no effect on calcium diffusion to surrounding cells. Nifedipine and verapamil, antagonists of voltage-dependent calcium channels, did not act on the mechanically induced calcium response. This suggests that some stimulating factor may trigger transmission of calcium, which may be ejected directly from single stimulated cells and mediated via a membrane receptor but not through a gap junction. The calcium signaling in a mechanically stimulated cell may be related to both an influx and a redistribution of intracellular calcium from internal stores, while calcium propagation to neighboring cells may involve calcium influx alone.

Phospholipid Metabolism in Neural Microvascular Endothelial Cells after Exposure to Lead in Vitro

Lead (Pb 2+) is known to alter the permeability of brain capillaries. A possible mechanism for this alteration may be related to the ability of Pb 2+ to substitute for Ca 2+. Products derived from phospholipid metabolism, namely eicosanoids and diacylglycerol, control endothelial permeability, are partly regulated by intracellular Ca 2+, and thus may be sensitive to Pb 2+. We asked in this study whether Pb 2+ increased arachidonic acid release or stimulated phosphatidylcholine breakdown in an in vitro model of brain capillaries, namely cultured bovine retinal endothelial (BRE) cells. Pb 2+ stimulated arachidonic acid release and phosphatidylcholine and phosphatidylinositol metabolism in the presence of ionomycin, but not by itself. More arachidonic acid was released than phosphorylcholine in BRE cells stimulated with ionomycin and Pb 2+, but the magnitudes of these responses were similar in cells exposed to ionomycin plus Ca 2+. Ionomycin plus Pb 2+ or plus Ca 2+ resulted in the activation of phospholipase A 2, since an increase in lysophosphatidylcholine and arachidonic acid was observed. Protein kinase C was not required for arachidonic acid release because release was observed in cells with a down-regulated enzyme. Ionomycin plus other metals (La 3+, Cd 2+, or Mg 2+) did not result in arachidonic acid release, but Cd 2+ or Co 2+ inhibited arachidonic acid release by more than 80% when cells were exposed to ionomycin with either Pb 2+ or Ca 2+. Thapsigargin or maitotoxin plus Ca 2+ increased arachidonic acid release that was inhibited by the receptor-dependent calcium channel antagonist SK&F 96365 but not by the voltage-dependent calcium channel antagonist nifedipine. However, thapsigargin or maitotoxin plus Pb 2+ failed to stimulate arachidonic release. Since in this in vitro model Pb 2+ stimulated phospholipid metabolism solely in the presence of an ionophore, the increase in permeability observed in Pb 2+-exposed animals is probably not due to a release of metabolites of arachidonic acid.

Calcium channels play a pivotal role in the sequence of ionic changes involved in initiation of mouse sperm acrosomal exocytosis.

The sequence of ionic changes involved in initiation of acrosomal exocytosis in capacitated mouse spermatozoa was investigated. Earlier studies demonstrated that a large influx of Na+ is required for exocytosis, this Na+ apparently being associated with an increase in intracellular pH (pHi) via an Na(+)-H+ exchanger. This rise in pHi may in turn activate calcium channels and permit the influx of extracellular Ca2+ needed to trigger acrosomal exocytosis. In the present study, the dihydropyridine voltage-dependent calcium channel antagonist nifedipine was able to inhibit significantly exocytosis in sperm cells treated in various ways capable of stimulating acrosomal loss. The monovalent cation ionophore monensin can promote Na+ entry required for both capacitation and acrosomal exocytosis, as demonstrated by using chlortetracycline to monitor changes in sperm functional potential. In the presence of 10 nM nifedipine, monensin treatment accelerated capacitation but was unable to trigger exocytosis. The requirement for internalization of a high concentration of Na+ can be bypassed by the addition of 25 mM NH4Cl to raise the pHi of cells capacitated in 25 mM Na+ (insufficient Na+ to support exocytosis under usual conditions). Again, introduction of nifedipine was able to inhibit exocytosis. In a third experimental approach, amiloride-stimulated exocytosis in capacitated cells was significantly inhibited by nifedipine. In contrast to these treatments directed at specific mechanisms, the ability of the Ca2+ ionophore A23187 to promote more general entry of Ca2+ and thereby to accelerate capacitation and exocytosis was not inhibited by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca 2+ antagonists effect an antidepressant-like adaptation of the NMDA receptor complex

Chronic, but not acute treatment of mice with nimodipine and diltiazem produce significant increases in the IC 50 of glycine to inhibit [ 3H]5,7-dichlorokynurenic acid binding in cerebral cortex. Such adaptive changes in the ligand binding properties of the NMDA receptor complex are also manifested following chronic treatment with antidepressants from every principal therapeutic class. These findings indicate voltage-dependent calcium channel antagonists would be strong candidates for rigorous clinical trials in depressive disorders.

Anticonvulsant activity of δ-HCH, calcium channel blockers and calmodulin antagonists in seizures induced by lindane and other convulsant drugs

The anticonvulsant activity of δ-HCH and of a calmodulin antagonist, W-7 were investigated on convulsions induced in mice by lindane (ED 100 100 mg/kg), by GABAergic antagonists PTZ (ED 100 60 mg/kg) and PTX(ED 100 4 mg/kg), by calcium channel agonist BAY-K-8644 (ED 100 5 mg/kg), by two agonists of excitatory amino acid receptors, kainic acid (ED 100 80 mg/kg) and NMDA (ED 100 160 mg/kg and by the atypical benzodiazepine Ro 5-4864 (ED 100 40 mg/kg). The anticonvulsant activity of a voltage-dependent calcium channel antagonist, nifedipine was also investigated on convulsions induced by Ro 5-4864, BAY-K-8644, kainic acid and NMDA. δ-HCH antagonized lindane- and BAY-K-8644-induced convulsions (ED 50 231 (172–309) mg/kg and 148 (142–154) mg/kg, respectively) and at concentrations up to 300 mg/kg failed to antagonize Ro 5-4864, kainic acid and NMDA convulsions. In contrast δ-HCH potentiated PTX-induced seizures. Nifedipine antagonized BAY-K-8644- and kainic acid-induced convulsions (ED 50 6.5 (4.3–9.7) mg/kg and 30 (13–70) mg/kg but at concentrations up to 20 mg/kg failed to antagonize Ro 5-4864 and 25% of protection was observed on NMDA-induced convulsions at the highest dose (20 mg/kg). The ED 50 of W-7 to antagonize convulsions induced by lindane and BAY-K-8644 were 12 (8–19) mg/kg and 49 (29–85) mg/kg, respectively. Some anticonvulsant effect was observed against PTZ and NMDA but without any dose-dependent anticonvulsant activity. W-7 did not protect against PTX and kainic acid convulsions and 30% of protection was observed against convulsions at the highest dose of W-7 (75 mg/kg). These results suggest that voltage-dependent calcium channels are involved in the mechanism of convulsant action of lindane.

Divergent effects of astroglial and microglial secretions on neuron growth and survival

Brain glia have a secretory capacity which can modulate neuronal function. Astrocytes release proteins which enhance neuronal survival and induce neuronal growth and differentiation. These effects can be blocked by antagonists of voltage-dependent calcium channels and may be partly mimicked by Bay K 8644, a calcium channel agonist. Two of these neurotrophic proteins appear, on the basis of their physical properties and effects on ciliary ganglion neurons, to be ciliary neurotrophic factor and basic fibroblast growth factor. Activated microglia release a heat- and protease-stable neurotoxin of low molecular weight. This neurotoxicity is blocked by NMDA receptor antagonists. Ciliary neurons exposed to the microglial neurotoxin exhibit an abnormal distribution of neurofilament immunoreactivity, which becomes concentrated in a perinuclear region, while the astroglial growth factors induce neurofilament organization into an extensive neuritic network. The astrocyte-released growth factors can counteract the effect of the microglial neurotoxin and lead to unimpaired neural differentiation in the presence of the neurotoxin.

Lindane-induced convulsions in NMRI and OF1 mice: antagonism with (+) MK-801 and voltage-dependent calcium channel blockers

The convulsant profile of lindane was investigated in OF1 and NMRI mice lines in relation to other convulsants acting at the GABA A and NMDA receptor complexes. Thus, a specific GABA-gated chloride channel blocker, PTX, a GABA A receptor antagonist, PTZ, and an excitatory amino acid receptor agonist, NMDA, were used. Antagonism of the convulsant effects of each of these drugs was investigated with (+)MK-801, a blocker of the NMDA-operated cation channel, and with nifedipine, a voltage-dependent calcium channel antagonist. While no differences in potency for PTX or PTZ to induce seizures were observed between OF1 and NMRI mice. lindane was approximately 80 and 90% more polent in its ability to induce seizures and lethality, respectively, in OF1 than NMRI mice. Brain lindane concentrations at the moment of convulsion, measured after ED 100 doses of lindane (400 and 200 mg/kg for NMRI and OF1 mice, respectively), did not differ between OF1 and NMRI mice, suggesting that the different potency of lindane between these mouse lines is a consequence of pharmacokinetic factors. Furthermore, (+)MK-801 antagonized seizures induced by either lindane, PTX or PTZ with similar potencies in both mouse lines. These results, coupled with the different pharmacokinetics of lindane in OF1 and NMRI mice, suggest that the distinct effects of lindane in these mice are not mediated by different activities at either NMDA or GABA A receptor complexes. Nonetheless, nifedipine antagonized lindane-induced seizures with a three-fold higher potency in NMRI than in OF1 mice. In contrast, nifedipine failed to antagonize PTX and PTZ convulsions in both OFI and NMRI mice. These results suggest that besides the GABA A receptor complex other mechanisms related to calcium mobilization may be involved in the convulsant action of lindane.

Properties of spontaneous inward currents recorded in smooth muscle cells isolated from the rabbit portal vein.

1. Characteristics of spontaneous transient inward currents (STICs) which produced membrane depolarization were analysed with the perforated patch technique in single smooth muscle cells isolated from the rabbit portal vein. 2. In K(+)-free solutions the amplitude of STICs was linearly related to membrane potential and the reversal potential (Er) was -3.0 +/- 0.9 mV. Replacement of external NaCl with NaI shifted Er to -40.0 +/- 1.0 mV. Substitution of external NaCl by NaSCN also moved Er to negative values but replacement of sodium with Tris and choline did not change Er. It is concluded that STICs are generated by an increase in chloride conductance. 3. STICs were abolished or reduced by the chloride channel antagonists anthracene-9-carboxylic acid (1 mM) and 4-acetamido-4'-isothiocyanato-2,2'- stilbene-disulphonic acid (2 mM). 4. STICs were blocked by noradrenaline and caffeine which deplete intracellular calcium stores. This effect was reversible and this result indicates that the primary trigger for STICs is calcium released from intracellular stores and therefore STICs are calcium-activated chloride currents (ICl(Ca)). 5. Removal of calcium from the bathing solution abolished STICs in six out of seven cells but STICs persisted in Ca(2+)-free solution in one cell. When STICs were abolished in Ca(2+)-free external solution the size of the internal calcium store, as estimated from the noradrenaline-induced ICl(Ca), was not altered. It appears that an influx of calcium is usually necessary for STICs to be observed. 6. The frequency and amplitude of STICs were not altered by the voltage-dependent calcium channel antagonist cadmium (1 mM). However, in some quiescent cells influx of calcium through voltage-dependent channels did activate STICs. 7. It was concluded that in isolated portal vein cells STICs represent a Ca(2+)-activated chloride current which leads to spontaneous depolarization of the membrane and may play an important physiological or pathophysiological role to produce smooth muscle contraction.

Responses of the solitary olfactory receptor cell of the guinea pig.

Olfactory receptor cells were isolated from the nasal mucosa of the guinea pig, using enzymatic digestion and mechanical dissociation. Under a phase-contrast microscope, the cells can be readily distinguished from other tissues, because of morphological characteristics. The intracellular free calcium ion concentrations ([Ca2+]i) of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 microM ionomycin, calcium-ionophore, there was an irreversible increase in [Ca2+]i in all the cells. During prolonged stimulation by 10 mM n-Amyl acetate under standard conditions, 16 of the 40 isolated olfactory cells responded with [Ca2+]i. There was no evidence of any increase in [Ca2+]i in the olfactory receptor cells in nominally Ca2+ free solution and stimulated by amyl acetate. When 3 mM CaCl2 was added, the [Ca2+]i increased remarkably. When cells were stimulated with amyl acetate in a CaCl2-depleted extracellular fluid supplemented with 10 microM verapamil (an L-type voltage-dependent calcium channel antagonist), no change occurred in [Ca2+]i. The addition of CaCl2 to this fluid did not elevate [Ca2+]i, in any cell. This would suggest that the [Ca2+]i increase in the presence of amyl acetate may mainly depend on the influx of calcium from the extracellular space. Our finding that the influx of extracellular calcium into cells was suppressed by verapamil suggests that the calcium channel, which is opened by amyl acetate, is a voltage-dependent L-type Ca2+ channel.

Time Course and Calcium Dependence of Sustained Bronchoconstriction Induced by Deep Inhalation in Asthma

We studied six asthmatic patients who showed a progressive decrease in FEV1 when successive forced expiratory maneuvers were performed at 1-min intervals. We determined the time course of changes in specific airway conductance following a single deep inhalation (DI) and the ratio of maximum expiratory flow at 40% of FVC from maximal and partial flow-volume curves (MEF40M/P) during a series of forced expiratory maneuvers. Specific airway conductance measured 3 s after DI was increased by 11 +/- 6 (SE)%, which was not significantly different from an increase of 23 +/- 8% observed in six healthy control subjects. Later (i.e., 10 to 40 s after DI) specific airway conductance was significantly less than the pre-DI value in asthmatic but not in healthy subjects. Mean FEV1 decreased significantly by 28% from the first to the eighth forced expiratory maneuver performed during a period of 15 min, whereas MEF40M/P was not significantly changed and remained always significantly greater than 1. The voltage-dependent calcium channel antagonist nifedipine significantly prevented the reduction of FEV1 without affecting MEF40M/P. We conclude that, in some asthmatic individuals, DI may induce a transient bronchodilatation followed by a calcium-dependent sustained bronchoconstriction. We suggest that the initial bronchodilatation is due to the mechanical interdependence between airways and lung parenchyma, whereas the sustained bronchoconstriction is due to contraction of the airway smooth muscle.

Effects of NMDA and calcium channel antagonists on regional cerebral blood flow

N- Methyl- d-aspartate (NMDA) antagonists and voltage-dependent calcium channel antagonists were tested to determine potential effects on regional cerebral blood flow in the normal rabbit brain. Ketamine had no effects on cortical or hippocampal blood flow, but was found to significantly decrease blood flow in the inferior colliculus. MK-801 decreased blood flow in almost all regions of the brain tested. On the other hand, nimodipine significantly increased flow in the cortex, hippocampus, and tegmentum. Dextromethorphan and dextrorphan, which have been shown to act at the NMDA receptor as well as the dihydropyridine calcium channel, decreased blood flow in the inferior colliculus, but showed no effects in the cortex or hippocampus. These results suggest that the neuroprotective NMDA antagonists do not increase blood flow primarily in the normal brain.

HIV-related neurotoxicity.

The central nervous system manifestations of AIDS were originally thought to consist solely of white matter lesions, but recent evidence has shown that a substantial degree of neuronal loss can also occur. This review presents evidence for HIV-related toxic factors that may account at least in part for this newly-recognized neuronal injury. One potential neurotoxin is the HIV-1 envelope glycoprotein gp-120 or a fragment of this molecule. This coat protein is shed by the virus and potentially released from HIV-infected immune cells. In tissue culture experiments on rodent neurons, gp120 produces an early rise in intracellular calcium concentration and, subsequently, delayed-onset neurotoxicity. In addition, HIV-infected macrophages or microglia release as yet undefined toxic factor(s) that kill rodent, chick, and human neurons in vitro. It is as yet unknown if one of these macrophage toxic factors might represent a gp120 fragment, or alternatively, if gp120, in the absence of HIV-1 infection, might be capable of activating macrophages to release these toxic factor(s). In at least some neuronal cell types, gp120-induced neurotoxicity can be prevented by antagonists of L-type voltage-dependent calcium channels or by antagonists of N-methyl-D-aspartate (NMDA, a subtype of glutamate receptor). Degradation of endogenous glutamate also protects neurons from gp120-related neuronal injury, suggesting that gp120 and glutamate are both necessary for neuronal cell death as synergistic effectors. Antagonists acting at the other types of glutamate receptors (non-NMDA antagonists) are ineffective in affording protection from gp120.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of action of endothelin in human lung

1 The peptides endothelin-1 (ET-1) and endothelin-2 (ET-2) elicited potent and sustained contractions of human isolated bronchus and pulmonary artery. 2 ET-1 is one of the most potent contractile agonists investigated in these tissues with an EC50 value of 18.3 nM (95% confidence interval: 12.9, 25.9 nM: n = 26) in bronchus and 3.2 nM (95% confidence interval: 0.4, 23.9 nM; n = 5) in the arterial preparation. 3 ET-1 is 2.5 times more potent than ET-2 in both the airway and vascular tissues, and both forms of the peptide have geometric mean EC50 values 5 times greater than in the isolated bronchial tissue than in the pulmonary artery. 4 Neither pretreatment with the voltage-dependent calcium (VDC) channel antagonist verapamil (10 microM) nor with indomethacin (25 microM) significantly altered the response curve to ET-1 in human isolated bronchus. Removal of calcium from the Krebs-Henseleit solution did not affect ET-1-induced responses. 5 Specific binding on the smooth muscle of human airway and pulmonary arterial tissue to both ET-1 and ET-2 was detected in autoradiographic studies. There appeared to be no difference between the peptides in the location nor the density of binding sites. 6 We conclude that contraction of human bronchial tissue by ET-1 is not dependent upon influence of extracellular calcium nor release of prostaglandins or thromboxane A2. It is likely that the action of ET-1 in this tissue is due to binding of this peptide to specific receptors located on the smooth muscle.

Effects of aminoglycoside antibiotics on the synaptic activity of rat hippocampal pyramidal cells

Activity-dependent increases in the responsiveness of spinal neurons to their normal afferent input, termed central sensitization, have been suggested to play a key role in abnormal pain sensation. We investigated the role of distinct voltage-dependent calcium channel (VDCC) subtypes in the long-term potentiation (LTP) of C-fiber-evoked field potentials (FPs) recorded in the spinal dorsal horn of rats, that is, a synaptic model to describe central sensitization. When spinally applied, we observed that omega-conotoxin GVIA (ω-CgTx), an N-type VDCC antagonist, produced a dose-dependent and prolonged inhibition of basal C-fiber-evoked FPs in naïve animals. ω-CgTx did not perturb the induction of LTP by high-frequency stimulation (HFS) of the sciatic nerve; however, potentiation was maintained at a lower level. Following the establishment of spinal LTP in naïve animals, the inhibitory effect of ω-CgTx on C-fiber-evoked FPs was significantly increased. Furthermore, in animals with chronic pain produced via peripheral nerve injury, where spinal LTP was barely induced by HFS, basal C-fiber-evoked FPs were strongly inhibited by ω-CgTx. As a result, ω-CgTx exerted a similar inhibitory profile on C-fiber-evoked FPs following the establishment of spinal LTP and chronic pain. In contrast, spinally administered omega-agatoxin IVA (ω-Aga-IVA), a P/Q-type VDCC antagonist, showed little effect on C-fiber-evoked FPs either before or after the establishment of LTP, but strongly suppressed LTP induction. These results demonstrate the requirement of N- and P/Q-type VDCCs in the maintenance and induction of LTP in the spinal dorsal horn, respectively, and their distinct contribution to nociceptive synaptic transmission and its plasticity.

Induction of c-fos mRNA and protein in neurons and glia after traumatic brain injury: Pharmacological characterization

Focal brain injury in mice induced c-fos mRNA and protein in neurons throughout the damaged neocortex, including the piriform and the entorhinal cortices, as well as in nonneural brain cells (e.g., glia, pia, ependyma). The pattern of c-fos induction after injury suggested that injury led to spreading depression which then led to c-fos induction in neurons. Human neurons in the temporal cortex and hippocampus also showed c-fos protein induction after neurosurgical trauma. The c-fos mRNA and protein induction in mouse neurons was prevented by the noncompetitive NMDA antagonist ketamine but only partially inhibited by the voltage-dependent calcium channel antagonist nifedipine and the calmodulin antagonist trifluoperazine. The c-fos protein induction in nonneural brain cells after injury was not affected by these drugs. Thus, induction of c-fos in neocortical neurons after focal brain injury is partly NMDA receptor mediated.

ω-Conotoxin GVIA and nifedipine inhibit the depolarizing action of the fungal metabolite, destruxin B on muscle from the tobacco budworm (Heliothis virescens)

G. A. Bradfisch and S. L. Harmer. ω-Conotoxin GVIA and nifedipine inhibit the depolarizing action of the fungal metabolite, destruxin B on muscle from the tobacco budworm ( Heliothis virescens). Toxicon 28, 1249–1254, 1990.—Recent studies on a group of fungal metabolites, collectively called the destruxins, have suggested that these compounds activate calcium influx in insect skeletal muscle. In this study, we have investigated the sensitivity of destruxin B to the voltage-dependent calcium channel antagonists; ω-conotoxin GVIA, nifedipine, diltiazem and methoxyverapamil on skeletal muscle from the lepidopteran insect pest, tobacco budworm ( Heliothis virescens). At a concentration of 1.7 μM, destruxin B caused a rapid decrease in the transmembrane resting potential. The effect of destruxin B on insect muscle was blocked by micromolar concentrations of ω-conotoxin GVIA and nifedipine but not by methoxyverapamil or diltiazem. The inhibitory activity of ω-conotoxin GVIA on invertebrate muscle tissue was surprising since this compound was previously thought to be selective to vertebrate nervous tissue. The sensitivity of the destruxin-stimulated depolarization to the two antagonists suggested that destruxin B activated a voltage-dependent calcium channel. Neuromuscular transmission was monitored in the presence of ω-conotoxin GVIA and nifedipine to investigate the physiological role of the destruxin-activated channel. Neither antagonist altered the waveform of graded action potentials produced by synaptic activation. The lack of effect of ω-conotoxin GVIA and a high dose of nifedipine could be explained by the existence of two populations of pharmacologically distinct voltage-dependent calcium channels on the muscle membrane. One population which is involved with the production of graded action potentials is insensitive to ω-conotoxin GVIA and nifedipine. The other population is activated by destruxin B and inhibited by ω-conotoxin GVIA and nifedipine.

Calcium dependence of osmolality-, potassium-, and angiotensin II-induced aldosterone secretion.

Different calcium-dependent mechanisms may be involved in mediating stimulus-induced aldosterone secretion. Using isolated perfused canine adrenal glands, we determined the effect of reductions in extracellular [Ca2+] and of infusion of a voltage-dependent calcium channel antagonist, nifedipine, on aldosterone secretion induced by decreases in osmolality, by increases in [K+], or by infusion of angiotensin II (ANG II). Aldosterone secretion was stimulated to a similar level by reducing osmolality, by increasing [K+], or by infusing ANG II. After 50 min of stimulation, lowering [Ca2+] from 1.25 to 0.10 mM caused a marked and similar inhibition of osmolality- and [K+]-induced aldosterone secretion that was significantly greater than inhibition of ANG II-induced aldosterone secretion. Similarly, nifedipine at 3.3 X 10(-8) M caused marked and similar inhibition of osmolality- and [K+]-induced aldosterone secretion that was significantly greater than the inhibition of ANG II-induced aldosterone secretion. These data demonstrate that calcium-dependent processes are involved in osmolality-, [K+]-, and ANG II-induced aldosterone secretion. However, the calcium-dependent process(es) evoked by reductions in osmolality or increases in [K+] are considerably different from that evoked by ANG II. Osmolality and potassium appear to induce aldosterone secretion primarily by activating voltage-dependent calcium channels.

Calcium-Dependent Control of Corticotropin Release in Rat Anterior Pituitary Cell Cultures

The role of calcium in the stimulation of ACTH secretion by CRF and other regulators was studied in rat anterior pituitary cells. Incubation of cultured pituitary cells in normal calcium with CRF, vasopressin, angiotensin II, or norepinephrine increased the rate of ACTH release for up to 45 min and then became constant for up to 3 h. In the absence of extracellular calcium, the initial rate of stimulated secretion was unaffected, but after 45 min the secretion rate decreased by 40% for CRF and to a greater extent for the other stimuli. Addition of calcium after 90 min in calcium-free medium restored the CRF-stimulated ACTH release rate to the control value. The absence of extracellular calcium had no effect on CRF-stimulated cAMP accumulation, but intracellular calcium depletion by preincubation of the cells with EGTA completely inhibited CRF-stimulated cAMP production and ACTH release. The voltage-dependent calcium channel antagonist nitrendipine and the calcium channel agonist BK 8644 had little effect on the CRF-stimulated ACTH release rate, while they, respectively, inhibited and enhanced the stimulation by vasopressin and high potassium. In calcium-depleted cells incubated with the calcium ionophore A23187, CRF stimulation of cAMP production and ACTH release were dependent upon extracellular calcium concentrations from 0.1-100 microM. These findings have defined two phases in the stimulation of ACTH release by CRF and cAMP-independent stimuli in cultured pituitary cells: an early phase with a rapid increase in the ACTH release rate which is independent of extracellular calcium, and a late phase of constant secretion rate, with partial extracellular calcium dependence for the stimulation by CRF and complete calcium dependence for the stimulation by non-cAMP-mediated stimuli.

Ionic mechanisms implicated in the stimulation of cerebellar cyclic GMP levels by N-methyl-D-aspartate.

N-Methyl-D-aspartate (NMDA) increases cyclic GMP levels in immature rat cerebellar slices incubated in magnesium-containing Krebs buffer in vitro. This effect is blocked by 2-amino-5-phosphonovalerate and by D-alpha-aminoadipate, but not by glutamic acid diethyl ester or gamma-D-glutamylaminomethylsulfonic acid, indicating specific involvement of the NMDA receptor. The response produced by NMDA is abolished by removal of calcium from the medium, proportional to the concentration of extracellular calcium, and blocked by a number of inorganic (Ni2+, Co2+, Cd2+, La3+, Mn2+) calcium antagonists. The responses to NMDA are not blocked by barium or strontium and persist when these ions are substituted for calcium in the incubation medium. The effects of NMDA are blocked by, but are not particularly sensitive to, the organic voltage-dependent calcium channel antagonists. Nifedipine (10 microM) produces partial inhibition of the effects of NMDA, which are also antagonized by high (greater than 200 microM) concentrations of diltiazem and verapamil. The effects of NMDA are tetrodotoxin insensitive but are abolished by omission of sodium from the medium and inhibited by a tetrodotoxin-insensitive sodium channel blocker, Zn2+. The results suggest that calcium channel opening is a consequence of NMDA receptor activation in this model. However, the sodium dependence of the response argues against the use of receptor-operated calcium channels, whereas the weak activity of the organic voltage-sensitive calcium channel antagonists argues either against the use of voltage-dependent calcium channels, or that those implicated in the effects of NMDA are insensitive to these agents.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of maitotoxin on 45Ca2+ flux and hormone release in GH3 rat pituitary cells.

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.