Dihydropyridine Agonist Research Articles

Whole cell and single-channel properties of a unique voltage-activated sustained calcium current identified in teleost retinal horizontal cells.

1. A voltage-activated, sustained calcium current in white bass retinal cone horizontal cells was characterized on the basis of electrophysiological and pharmacological criteria. Studies were performed with the use of a combination of whole cell and single-channel analysis of outside-out excised patches from isolated, cultured retinal horizontal cells. 2. We found that the white bass sustained calcium channel represents a unique type of calcium channel. On the basis of our analysis, it does not fall into any current classification scheme. The horizontal cell channel shares some biophysical and pharmacological properties with the typical high-voltage-activated L-type channel, but it also has features in common with the P-type channel. 3. The biophysical characteristics of the channel were most typical of an L-type channel. It activated above -30 mV membrane potential and only very slowly inactivated. It had a single-channel conductance of 25 pS. 4. Like the typical L-type current, the horizontal cell current was sensitive to the dihydropyridine agonist Bay K 8644. It prolonged the channel open time, which resulted in a large increase in macroscopic current flow into the cell. However, unlike the typical L current, dihydropyridine antagonists (nifedipine, nimodipine, etc.) as well as the specific L-channel inhibitor diltiazem were only moderately effective at best. 5. In a previous study, we found the current was antagonized by a factor found in funnel-web spider toxin. Here we show that the current is completely blocked by low doses of omega-agatoxin IVA. These are characteristics of the P-type calcium channel. But unlike the P current, the horizontal cell current is relatively insensitive to low or high doses of omega-conotoxin MVIIC. 6. The overall combination of calcium channel characteristics sets apart the calcium channel in bass horizontal cells from previously described channels. It appears to be a unique, tissue-specific ion channel, which we have labeled the PL channel.

Altered Pharmacologic Responsiveness of Reduced L‐Type Calcium Currents in Myocytes Surviving in the Infarcted Heart

The pharmacologic responses of macroscopic L-type calcium channel currents to the dihydropyridine agonist, Bay K 8644, and beta-adrenergic receptor stimulation by isoproterenol were studied in myocytes enzymatically dissociated from the epicardial border zone of the arrhythmic 5-day infarcted canine heart (IZs). Calcium currents were recorded at 36 degrees to 37 degrees C using the whole cell, patch clamp method and elicited by applying step depolarizations from a holding potential of -40 mV to various test potentials for 250-msec duration at 8-second intervals. A Cs+ -rich and 10 mM EGTA-containing pipette solution and a Na+ -and K+ -free external solutions were used to isolate calcium currents from other contaminating currents. During control, peak ICa,L density was found to be significantly less in IZs (4.0 +/- 1.1 pA/pF) than in myocytes dispersed from the epicardium of the normal noninfarcted heart (NZs; 6.5 +/- 1.8 pA/pF). Bay K 8644 (1 micro M) significantly increased peak ICa,L density 3.5-fold above control levels in both NZs (to 22.5 +/- 6.2 pA/pF; n = 7) and IZs (to 12.8 +/- 3.0 pA/pF; n = 5), yet peak ICa,L density in the presence of drug was significantly less in IZs than NZs. The effects of Bay K 8644 on kinetics of current decay and steady-state inactivation relations of peak ICa,L were similar in the two cell types. In contrast, the response of peak L-type current density to isoproterenol (1 micro M) was significantly diminished in IZs compared to NZs regardless of whether Ba2+ or Ca2+ ions carried the current. Thus, these results indicate an altered responsiveness to beta-adrenergic stimulation in cells that survive in the infarcted heart. Furthermore, application of forskolin (1 micro M and 10 micro M) or intracellular cAMP (200 micro M), agents known to act downstream of the beta-receptor, also produced a smaller increase in peak IBa density in IZs versus NZs, suggesting that multiple defects exist in the beta-adrenergic signaling pathway of IZs. In conclusion, these studies illustrate that reduced macroscopic calcium currents of cells in the infarcted heart exhibit an altered pharmacologic profile that has important implications in the development of drugs for the diseased heart.

L- and N-type Ca2+ channels in adult rat carotid body chemoreceptor type I cells.

1. Whole-cell voltage-dependent Ca2+ currents recorded from chemoreceptor type I cells of the adult rat carotid body had maximum amplitudes of -94 pA in 10 mM Ca2+ and were half-inactivated at a holding potential of -38 mV. Somatostatin and dopamine inhibited whole-cell Ca2+ current in type I cells. 2. The dihydropyridine agonist (+)202-791 increased the Ca2+ current amplitude by 106% at a step potential of -18 mV. The dihydropyridine antagonist nimodipine decreased the Ca2+ current amplitude by 40% from a holding potential of -80 mV, and by 74% from a holding potential of -60 mV. The nimodipine-sensitive current had a maximum amplitude at a membrane potential of -12 mV. omega-Conotoxin GVIA (omega-CgTX GVIA) blocked the whole-cell Ca2+ current by 40%. The omega-CgTX GVIA-sensitive current had a maximum amplitude at a membrane potential of +2 mV. 3. In summary, type I cells of the adult rat carotid body have dihydropyridine-sensitive L-type and omega-conotoxin GVIA-sensitive N-type voltage-dependent Ca2+ channels. These channels may play a role in the voltage-gated entry of Ca2+ necessary for stimulus-secretion coupling in response to changes in arterial PO2, PCO2 and pH. Inhibition of the Ca2+ currents by somatostatin and dopamine may alter the chemotransduction signal in type I cells.

Evidence for voltage-sensitive, calcium-conducting channels in airway epithelial cells

In airways epithelial cultures, mechanical stimulation induces intracellular Ca2+ concentration ([Ca2+]i) changes by causing Ca2+ entry and intracellular Ca2+ release. Mechanically induced Ca2+ fluxes across the plasma membrane are blocked by Ni2+ (Boitano, S., M. J. Sanderson, and E. R. Dirksen. J. Cell. Sci. 107: 3037-3044, 1994). In this report we use fluorescence imaging microscopy with fura 2 and intracellular recording of the transmembrane potential to further characterize Ca2+ flux in the plasma membrane of these cells. Mechanically induced Ca2+ influx is blocked by nifedipine. Addition of the dihydropyridine agonist BAY K8644 (2 microM) leads to a delayed increase of [Ca2+]i that is dependent on extracellular Ca2+. Switching to high extracellular K+ concentration ([K+]o) causes depolarization of the plasma membrane and a transient increase in [Ca2+]i. The number of cells that respond to high [K+]o is significantly decreased by Ni2+ (1 mM) or nifedipine (10 microM). Mechanical stimulation causes a rapid depolarization of the stimulated cell that can be suppressed by the K+ ionophore valinomycin. Valinomycin treatment also blocks mechanically induced Ca2+ dux. These results suggest that voltage-sensitive Ca(2+)-conducting channels exist in airway epithelial cells, and these channels contribute to the [Ca2+]i changes observed after mechanical stimulation or depolarization of the plasma membrane.

Developmental changes in the inducibility of fos-like immunoreactivity in primary embryonic spinal cord cultures

The immediate early gene (IEG) transcription factor c- fos coordinates changes in the pattern of long term gene expression and, therefore, it may be involved in mediating epigenetic control during neurodevelopment. We used pharmacological treatments mimicking various environmental and intracellular signals and assessed the inducibility of fos-like immunoreactivity (LIR) at various stages of neurodifferentiation in a primary embryonic spinal cord culture system by immunohistochemistry. Constitutive fos LIR exclusively found in neurons, was driven by the onset and extent of spontaneous electrical activity, as it was blockable by tetrodotoxin (TTX) at all developmental stages. Phorbol myristate 13 acetate (PMA) increased the number of fos-LIR cells equally effectively at all stages, but the predominant cellular localization of fos-LIR changed through ontogeny. The effect of veratridine, kainate and serum-derived factors in significantly inducing fos-LIR was restricted to the earliest developmental stage (4 days in vitro; DIV) investigated; whereas forskolin, the GABA A antagonist picrotoxin and NMDA failed to induce fos-LIR at this stage, but increased the number of fos-LIR neurons at later stages. Dihydropyridine agonists of the voltage-sensitive calcium channels (VSCC) raised the number of fos-LIR neurons and also prevented TTX-mediated down-regulation; whereas antagonists markedly reduced fos-LIR at all ages. Either type of NMDA antagonists (AP5 and MK801) and the GABA A agonist muscimol significantly reduced fos-LIR at all ages. These findings demonstrate that the inducibility of fos-LIR is substantially different in embryonic neurons than in adult ones and that inducibility by various first and second messengers is dependent on the developmental stage.

Extracellular heparin inhibits Ca2+ transients and contraction in mammalian cardiac myocytes.

The effect of heparin on Ca2+ transients and cell shortening was studied in isolated cardiac myocytes from rat and guinea-pig ventricles. Ca2+ signals were measured with the fluorescent indicator fura-2. Heparin reversibly decreased Ca2+ transients and cell shortening in a dose-dependent manner. Half and complete blockade were obtained with 50microg/ml and 200microg/ml heparin, respectively. The dihydropyridine agonist BAY K 8644 (50nM) antagonized the effects of heparin. However, Ca2+ release elicited by caffeine (10mM) was not affected by heparin. The actions of heparin were also studied in multicellular preparations. In papillary muscle, heparin (5mg/ml) reversibly reduced the amplitude of the plateau of the action potential and the associated peak tension. BAY K 8644 (500nM) also antagonized these effects. It is proposed that heparin interacts with dihydropyridine-sensitive Ca2+ channels to cause a decrease of Ca2+ transients and contractility in heart.

Diversity of voltage-gated calcium currents in large diameter embryonic mouse sensory neurons.

Voltage-gated Ca2+ currents were investigated in a subpopulation of dorsal root ganglion neurons (large diameter, neurofilament-positive) acutely isolated from 13-day-old mouse embryos and recorded using the whole-cell patch-clamp technique. Low- and high-voltage-activated calcium currents were recorded. These currents could be identified and separated by their distinct (i) threshold of activation, (ii) ability to run-up during the early phase of recording and (iii) decay kinetics using Ba2+ instead of Ca2+ as the charge carrier. Among high-voltage-activated currents, L-, N- and P-type Ca2+ currents were identified by their sensitivity to, respectively, the dihydropyridine agonist Bay K 8644 (5 microM) and antagonist nitrendipine (3 microM), omega-conotoxin GVIA (3 microM) and omega-agatoxin IVA (30 nM). In the combined presence of nitrendipine (3 microM), omega-conotoxin GVIA (3 microM) and omega-agatoxin IVA (30 nM), two additional high-voltage-activated components were detected. One, blocked by 500 nM omega-conotoxin MVIIC and 1 microM omega-agatoxin IVA, had properties similar to those of the Q-type Ca2+ current first reported in cerebellar granule cells. The other, defined by its resistance to saturating concentrations of all the blockers mentioned above applied in combination, resembles the R-type Ca2+ current also described in cerebellar granule cells. In conclusion, embryonic sensory neurons appear to express a large repertoire of voltage-activated Ca2+ currents with distinct pharmacological properties. This diversity suggests a great variety of pathways for Ca2+ signaling which may support different functions during development.

Voltage-dependent calcium channel β-subunits in combination with α1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by G αo in rat frontal cortex

The dihydropyridine-sensitive calcium channel agonist (−)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by G o in rat frontal cortex membranes. An anti-calcium channel β-subunit antiserum abolished the (−)-BayK 8644-stimulated hydrolysis of GTP by G o and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the β-subunit binding domain of the calcium channel complex, also attenuated (−)-BayK 8644 activation of GTPase. This study suggests that the calcium channel β-subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by G o. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal.

Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle.

Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca2+ channels. Spermine and spermidine (0.1-1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 microM), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 microM). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca2+ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca2+ current responsible for generation of action potentials.

Inhibition of the interaction of G protein G(o) with calcium channels by the calcium channel beta‐subunit in rat neurones.

1. The beta-subunit has marked effects on the biophysical and pharmacological properties of voltage-dependent calcium channels. In the present study we examined the ability of the GABAB agonist (-) -baclofen to inhibit calcium channel currents in cultured rat dorsal root ganglion neurones following depletion of beta-subunit immunoreactivity, 108-116 h after microinjection of a beta-subunit antisense oligonucleotide. 2.We observed that, although the calcium channel current was markedly reduced in amplitude following beta-subunit depletion, the residual current (comprising both N- and L-type calcium channel currents) showed an enhanced response to application of (-) -baclofen. Therefore, it is possible that there is normally competition between activated G protein G(o) and the calcium channel beta-subunit for binding to the calcium channel alpha 1-subunit; and this competition shifts in favour of the binding of activated G(o) following depletion of the beta-subunit, resulting in increased inhibition. 3. This hypothesis is supported by evidence that an antibody against the calcium channel beta-subunit completely abolishes stimulation of the GTPase activity of G(o) by the dihydropyridine agonist S-(-) -Bay K 8644 in brain membranes. This stimulation of GTPase is thought to result from an interaction of G(o) alpha-subunit (G alpha o) with its calcium channel effector which may operate as a GTPase-activating protein. 4. These data suggest that the calcium channel beta-subunit when complexed with the beta 1-subunit normally inhibits its association with activated G(o). It may function as a GTPase-activating protein to reduce the ability of activated G(o) to associate with the calcium channel, and thus limit the efficacy of agonists such as (-) -baclofen.

Boar sperm plasma membrane Ca(2+)-selective channels in planar lipid bilayers.

Entry of Ca2+ through Ca2+ channels is thought to trigger the acrosome reaction of spermatozoa during fertilization. Antagonists of the L-type Ca2+ channel are known to prevent the intracellular Ca2+ (Ca2+) increase and inhibit acrosomal exocytosis in mammalian sperm. Planar bilayer recordings were used to study Ca2- channels incorporated from partially purified boar sperm plasma membranes. With symmetrical 50 mM NaCl and 100 mM BaCl2 on the cis side, single-channel events consistent with Ba2+ flux from cis to trans were observed. These channels were activated by the dihydropyridine agonist (+/-)BAY K 8644 and blocked by the antagonist nitrendipine. Sperm Ca2- channels did not require depolarization for activation and did not inactivate. The (+/-)BAY K 8644 and (S-)BAY K 8644 enantiomers increased apparent open time in a dose-dependent [half-maximal activity constant (K0.5) = 0.9 and 0.3 microM, respectively] manner. Dihydropyridine antagonists nitrendipine (K0.5 = 0.5 microM) and (R+)BAY K 8644 (K0.5 = 2.8 microM) decreased apparent open times. The channels described in this report share some properties with brain, cardiac, and skeletal muscle t tubule Ca2+ channels and may be involved in increasing Cai2+ before the acrosome reaction.

Pharmacological types of calcium channels and their modulation by baclofen in cerebellar granules

Voltage-dependent calcium currents were measured by whole-cell recording technique in cultured cerebellar granule neurons from 8 d old rats, in 10 mM BaCl2 and with a holding potential of -80 mV. A saturating dose (10 microM) of the dihydropyridine nimodipine reversibly inhibited the maximum current by 25% and the dose dependence showed IC50 close to 50 nM. omega-Conotoxin GVIA (cgtx, 5 microM) and omega-agatoxin IVA (agatx, 200 nM) irreversibly inhibited the current by 17% and by 47%, respectively. The effect of nimodipine was additive with that of the toxins. The GABAB agonist (+/-)baclofen, or (-)baclofen (100 microM), reduced the calcium current by 30 +/- 5%, with a IC50 4 microM. The effect was mediated by a pertussis toxin-sensitive G-protein. In cells treated with cgtx during the experiment or preincubated with the toxin for 30 min, the effect of baclofen was significantly reduced. However, the action of baclofen was not confined to cgtx-sensitive channels: application of nimodipine or agatx resulted in a 50% reduction of the baclofen effect as well. In contrast, baclofen inhibited approximately the same amount of current both before and after the increase caused by the dihydropyridine agonist BayK 8644 and did not modify the slow BayK-induced tail current. These results indicate (1) the modulation through GABAB receptors does not clearly discriminate between pharmacologically distinct calcium channels and (2) L-type calcium channels represent an heterogeneous population in these neurons.

Selective potentiation of a novel calcium channel in rat hippocampal neurones.

1. Calcium channel activity in cultured embryonic hippocampal neurones was studied with the cell-attached configuration of the patch clamp technique. Single-channel recordings revealed the presence of a novel kind of calcium channel activity characterized by marked bursts of re-openings following voltage pulses to +20 mV from a holding potential of -40 mV. 2. The re-openings were greatly prolonged by the dihydropyridine (DHP) agonist (+)-(S)-202-791, thus ruling out the possibility that they arose from T-, N- or P-type channels. Furthermore, the novel gating pattern could be readily distinguished from that of the L-type channel which showed only conventional tail currents. 3. Since the novel gating pattern was stable over many minutes, we provisionally referred to it as a novel kind of calcium channel that showed voltage-dependent potentiation (Lp channel) to distinguish it from the 'standard' L-type channel (Ls channel). 4. Lp channels could also be distinguished from Ls channels on the basis of slope conductance (24.3 vs. 26.9 pS for Lp and Ls, respectively) and mean DHP-induced long open time (2.7 vs 11 ms at +20 mV for Lp and Ls, respectively). 5. Voltage-dependent potentiation of Lp channel activity was studied using a dual-pulse protocol. When preceded by conditioning prepulses, Lp responses to test pulses were greatly increased. Ls- and N-type calcium channels showed no such enhancement of their activity. 6. Long-duration recordings revealed no clear evidence for transitions from Ls to Lp gating (or vice versa), suggesting that Ls and Lp activities arose from different kinds of calcium channels or that Lp gating is an unusually long-lived mode of Ls channel gating.

Omega 3 polyunsaturated fatty acid modulates dihydropyridine effects on L-type Ca2+ channels, cytosolic Ca2+, and contraction in adult rat cardiac myocytes.

The effect of docosahexaenoic acid (DHA; C22:6) on dihydropyridine (DHP) interaction with L-type Ca2+ channel current (ICa), cytosolic Ca2+ (Cai), and cell contraction in isolated adult rat cardiac myocytes was studied. The DHP L-type Ca(2+)-channel blocker nitrendipine (10 nM) reduced peak ICa (measured by whole-cell voltage clamp from -45 to 0 mV) and reduced the amplitude of the Ca2+ transient (measured as the transient in indo-1 fluorescence, 410/490 nm) and the twitch amplitude (measured via photodiode array) during steady-state electrical stimulation (0.5 Hz). The DHP L-type Ca2+ channel agonist BAY K 8644 (10 nM) significantly increased ICa, the amplitude of the Cai transient, and contraction. When cells were exposed to DHA (5 microM) simultaneously with either BAY K 8644 or nitrendipine, the drug effects were abolished. Arachidonic acid (C20:4) at 5 microM did not block the inhibitory effects of nitrendipine nor did it prevent the potentiating effects of BAY K 8644. DHA modulation of DHP action could be reversed by cell perfusion with fatty acid-free bovine serum albumin at 1 mg/ml. Neither DHA nor arachidonic acid alone (5 microM) had any apparent effect on the parameters measured. DHA (5 microM) had no influence over beta-adrenergic receptor stimulation (isoproterenol, 0.01-1 microM)-induced increases in ICa, Cai, or contraction. The findings that DHA inhibits the effect of DHP agonists and antagonists on Ca(2+)-channel current but has no effect alone or on beta-adrenergic-induced increases in ICa suggests that DHA specifically binds to Ca2+ channels at or near DHP binding sites and interferes with ICa modulation.

Dihydropyridine ligands influence the evoked release of oxytocin and vasopressin dependent on stimulation conditions

The effects of dihydropyridine ligands on the electrically evoked release of neurohypophysical hormones from isolated, rat neurointermediate lobes were investigated as a function of all combinations of two pulse widths (0.2 and 2 ms) and three stimulation frequencies (6.5, 13 and 30 Hz). The dihydropyridine agonist ( S)-(+)-202-791 potentiated concentration dependently the release of both oxytocin and vasopressin at a pulse width of 2 ms and a frequency of 6.5 Hz. This effect of ( S)-(+)-202-791 was abolished by the antagonist (−)-nitrendipine and stereospecifically by ( R)-(−)-202-791 (only vasopressin). The antagonist ( R_-(−)-202-791 alone inhibited the release of oxytocin at 13 Hz and 2 ms. The results presented show that the effects of the dihydropyridine ligands are dependent on the stimulation conditions, and thus demonstrate that the entry of Ca 2+ through the dihydropyridine sensitive L-type Ca 2+ channel is associated with electrically evoked release of neurohypophysical hormones under certain conditions.

Regulation of the frequency-dependent facilitation of L-type Ca2+ currents in rat ventricular myocytes.

1. An increase in the rate of stimulation induces an augmentation of L-type Ca2+ currents (ICa) and concomitant slowing of current decay in rat ventricular cells. This facilitation is quasi immediate (1-3 s), graded with the rate of stimulation, and occurs only from negative holding potentials. We investigated this effect using trains of stimulation at 1 Hz and the whole-cell patch-clamp technique (18-22 degrees C). 2. The decay of ICa is normally bi-exponential and comprises fast and slow current components (ICa,fc and ICa,sc, respectively). Facilitation of ICa was observed only when ICa,fc was predominant. 3. Facilitation developed during the run-up of ICa with the interconversion of ICa,sc into ICa,fc, and vanished during the run-down of ICa with the loss of ICa,fc.Ni2+ (300 microM) and nifedipine (1 microM) suppressed facilitation owing to the preferential inhibition of ICa,fc. 4. Facilitation of ICa was not altered (when present) or favoured (when absent) by the cAMP-dependent phosphorylation of Ca2+ channels promoted by isoprenaline or by intracellular application of cAMP or of the catalytic subunit of protein kinase A (C-sub). A similar effect was observed when the dihydropyridine agonist Bay K 8644 was applied. In both cases, facilitation was linked to a preferential increase of ICa,fc. 5. Following intracellular application of inhibitors of protein kinase A in combination with a non-hydrolysable ATP analogue, ICa consisted predominantly of ICa,sc and no facilitation was observed. The calmodulin antagonist naphthalenesulphonamide had no effect on facilitation. 6. When Bay K 8644 was applied in combination with isoprenaline, cAMP or C-sub, the decay of ICa was slowed with the predominant development of ICa,sc, and facilitation of ICa was nearly abolished. Facilitation also depended on extracellular Ca2+, and was suppressed when Ba2+ replaced Ca2+ as the permeating ion. 7. When no EGTA was included in the patch pipette, facilitation was not further enhanced but a use-dependent decrease of ICa frequently occurred. When BAPTA was used in place of EGTA, the rate of inactivation of ICa was reduced and facilitation was abolished. 8. In conclusion, the facilitation of ICa that reflects a voltage-driven interconversion of ICa,fc into ICa,sc is also regulated by Ca2+ and by cAMP-dependent phosphorylation. The presence of the gating pattern typified by ICa,fc is required. Ca2+ may exert its effect near the inner pore of the Ca2+ channel protein and control the distribution between the closed states of the two gating pathways.

Inhibition of [3H]catecholamine release and Ca2+ currents by prostaglandin E2 in rabbit carotid body chemoreceptor cells.

Basal release of [3H]catecholamine ([3H]CA) from rabbit carotid bodies (CBs), previously incubated in the presence of [3H]tyrosine, was not significantly modified by prostaglandin E2 (PGE2). On the contrary, PGE2 (3-300 nM) produced a dose-dependent inhibition of the low PO2-evoked release of [3H]CA. The inhibition was greatest (55%) at a low intensity of hypoxic stimulation (incubating solution PO2 approximately 66 mmHg) and decreased with increasing intensities of hypoxia. Chronic denervation of the CB did not modify the response to PGE2. The release of [3H]CA induced by incubating the CBs in a hypercapnic-acidic solution (PCO2 approximately 132 mmHg; pH = 6.60) and by dinitrophenol (100 microM) was not significantly modified by 300 nM PGE2. PGE2 (300 nM) inhibited the release of [3H]CA elicited by incubating the CBs in a high K+ (35 mM)-containing solution. The release response elicited by high K+ (25 mM) was strongly augmented by a dihydropyridine agonist of Ca2+ channels, Bay K 8644, at a concentration of 1 microM. The Bay K 8644 effect was partly inhibited by PGE2 (300 nM). Using whole-cell recordings in freshly dispersed or short-term cultured chemoreceptor cells from adult rabbits it was found that Ca2+ currents (ICa) were reversibly inhibited by bath application of PGE2. A good parallelism exits between the dose-response curves for PGE2 inhibition of ICa in isolated chemoreceptor cells and high extracellular [K+]- or hypoxia-evoked release of [3H]CA from the whole CB.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.

Endogenous ganglioside GM1 modulates L-type calcium channel activity in N18 neuroblastoma cells

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

Acutely isolated neurons of the rat globus pallidus exhibit four types of high-voltage-activated Ca2+ current

1. Large, projection-like neurons from the adult (> 3 wk post-natal) rat globus pallidus (GP) were acutely isolated and subjected to whole-cell voltage-clamp (n = 37). Ca2+ currents were isolated pharmacologically in cells with whole-cell capacitances of 15-34 pF. 2. With 5 mM Ba2+ as a charge carrier, whole-cell currents began to activate near -40 mV and peaked near 0 mV. Based on activation threshold and inactivation kinetics, currents appeared to be of the high-voltage-activated type. 3. Cd2+ blocked whole-cell currents with an IC50 near 2 microM. Currents activated at negative potentials were not relatively resistant to Cd2+, supporting the inference that low-voltage-activated currents were not prominent in these neurons. 4. The dihydropyridine, L-channel antagonist, nifedipine (5 microM), reduced peak current by 21 +/- 4% (SD) (n = 10). The dihydropyridine agonist, BayK 8644 (1-2 microM) enhanced peak current and slowed current deactivation (n = 4). 5. The N-channel antagonist, omega-conotoxin GVIA (omega-CgTx, 2 microM) blocked 25 +/- 7% of the peak whole-cell current (n = 10). The blocks produced by omega-CgTx and nifedipine were additive, blocking an average of 46 +/- 8% of the current (n = 10). 6. The current resistant to the selective N- and L-channel antagonists was partially blocked by the P-channel antagonist omega-agatoxin IVA (omega-AgTx, 100 nM). omega-AgTx blocked about one-half of the current not attributable to N- and L-type channels (22 +/- 5% of the total current, n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)