Irreversible suppression of calcium entry into nerve terminals by methylmercury

Measurements of uptake of 45Ca into rat forebrain synaptosomes depolarized with high K+ and EPP amplitudes at the rat neuromuscular junction were used to assess the effects of methylmercury (MeHg) on voltage-dependent Ca++ uptake and subsequent transmitter release at model central and peripheral synapses. The objectives were to: determine whether MeHg altered uptake of 45Ca into purified synaptosomes depolarized by high K+; compare its effects with those produced by HgCl2; ascertain whether the "fast" or "slow" components of Ca++ uptake were affected preferentially by MeHg; and determine whether a functional correlate to the effects on 45Ca uptake could be observed electrophysiologically at the mammalian neuromuscular junction. HgCl2 (10-500 microM) produced a concentration-dependent decrease of total depolarization-induced 45Ca uptake. Peak inhibition occurred at 200 microM Hg++ which suppressed nerve terminal Ca++ uptake to approximately 5% of Hg-free control values, a result similar to that obtained previously by others. Similarly, MeHg also suppressed total 45Ca uptake although the maximal inhibition produced (70% at 200 microM MeHg) was less than that produced by HgCl2. The effect of MeHg was apparent both in nonpreviously depolarized synaptosomes after a 1-sec depolarization ("fast uptake") and after 10-sec incubation in synaptosomes predepolarized with 41 mM K+ in Ca-free solutions before addition of MeHg and 45Ca ("slow uptake"). A significant decrease in the slow phase of 45Ca uptake occurred with 200 and 500 microM MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of antibiotics on uptake of calcium into isolated nerve terminals

The goal of the present study was to determine whether several antibiotics which are known to block neuromuscular transmission would impair depolarization-dependent and/or -independent uptake of calcium into isolated nerve terminals prepared from forebrain synaptosomes of rats by conventional methods. Antibiotics tested for potential block of Ca++ uptake included the aminoglycosides neomycin and streptomycin, the lincosamide clindamycin, oxytetracycline and polymyxin B. Drugs were applied in concentrations ranging from 1 to 1000 microM. Uptake of 45Ca was determined during depolarization induced by an elevated K+ concentration (77.5 mM). Influxes of 45Ca during 1 and 10 sec of depolarization were used to assess Ca++ uptake via a "fast, inactivating path" and total uptake, respectively. Uptake of 45Ca during 10 sec of depolarization into synaptosomes which were previously depolarized for 10 sec in the presence of 77.5 mM K+ but in the absence of external Ca++ was used to measure uptake during a "slow, noninactivating path." Total depolarization-dependent uptake of 45Ca was depressed significantly by all antibiotics tested except oxytetracycline; however, the various agents differed with respect to their efficacy and potency as blockers of Ca influx. The fast component of uptake, which is thought to be associated with neurotransmitter release, was decreased significantly by all antibiotics. Neomycin and polymyxin were the most potent and most effective at lowering fast phase 45Ca influx; streptomycin, was intermediate in effectiveness whereas clindamycin and oxytetracycline were only effective at concentrations greater than or equal to 100 microM. Only clindamycin, streptomycin and polymyxin B caused significant reductions in the "slow" phase of 45Ca uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Modulation of the Ca++-evoked release of [3H]dopamine from striatal synaptosomes by dopamine (D2) agonists and antagonists

Release of [3H]dopamine ([3H]DA) from striatal synaptosomes is evoked most commonly by elevating potassium levels in the presence of calcium. However, it has been difficult to show that DA agonists or antagonists can modify K+-evoked release of [3H]DA. DA. In this study [3H]DA release evoked by exposure of synaptosomes (isolated and superfused previously with 0.0 mM Ca++ and 0.1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) to 1.25 mM Ca++ can be modulated by the DA (D2) agonists apomorphine, pergolide and quinpirole and antagonists l-sulpiride and domperidone. The release was evoked under low potassium (6 mM or less) concentrations and the potassium concentration in the superfusion medium was not elevated before or during Ca++ exposure. Analysis of the superfusates obtained during Ca++ exposure revealed that approximately 80% of the tritium released was [3H]DA. The ability of DA (D2) agonists to inhibit the Ca++-evoked release from synaptosomes superfused with 9 mM K+ was greatly reduced. Therefore, prolonged depolarization may block DA (D2) regulation of [3H]DA release from synaptosomes. The Ca++-evoked release of [3H]DA was reduced greatly when 1 microM tetrodotoxin was present indicating sodium channels play a role in triggering the processes involved in Ca++-evoked [3H]DA release.     

Methylmercury blocks N- and L-type Ca++ channels in nerve growth factor-differentiated pheochromocytoma (PC12) cells.

Effects of methylmercury (MeHg) on whole-cell Ba++ currents in rat pheochromocytoma (PC12) cells were examined. Based on biophysical characteristics and sensitivity to omega-conotoxin GVIA and dihydropyridine agonists and antagonists, voltage-activated Ba++ currents (IBa) in PC12 cells were mediated by N- and L-type Ca++ channels. Addition of MeHg (10 microM) to the extracellular solution caused a rapid and complete block of current carried by 20 mM Ba++. The rate of block of IBa by MeHg increased in a concentration-dependent manner between 1 and 20 microM. Increasing the frequency of stimulation from 0.1 to 0.4 Hz facilitated block of IBa by MeHg. A 2-min application of 10 microM MeHg in the absence of stimulation also reduced IBa by approximately 80%. Thus, block of IBa by MeHg is not state-dependent. Additionally, MeHg blocked IBa when the membrane holding potential was -40, -70 and -90 mV, indicating that both N- and L-type Ca++ channels are blocked by MeHg. Block of IBa by MeHg was voltage-dependent at a membrane holding potential of -40 mV, but not at holding potentials of -70 and -90 mV. Decreasing the extracellular concentration of Ba++ ([Ba++]e) from 20 mM to 10 mM increased the magnitude of block by MeHg from 45.6 to 77.3%. Increasing [Ba++]e to 30 mM caused no further antagonism of block. Block of IBa by MeHg was not reversed by washing with MeHg-free solution. The ionic permeability of PC12 cell Ca++ channels was Ca++ = Sr++ greater than Ba++. In the presence of MeHg, all three divalent cations were equally permeant.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Disruption of brain mitochondrial calcium sequestration by methylmercury.

In vitro effects of methylmercury (MeHg) on Ca2+ transport and respiratory control of mitochondria isolated from rat forebrain were examined to determine whether MeHg disrupted sequestration of Ca2+ by neuronal mitochondria. Uptake of 45Ca2+ by mitochondria and release of 45Ca2+ from preloaded mitochondria were measured in the presence and absence of ATP. Release of 45Ca2+ from preloaded mitochondria by MeHg was measured in the presence and absence of ruthenium red (RR), a putative inhibitor of the mitochondrial Ca2+ uptake uniporter. During incubation intervals ranging from 10 sec to 5 min, 10 microM MeHg reduced mitochondrial uptake of 45Ca2+ by about 50% and 100 microM MeHg completely prevented 45Ca2+ uptake. These effects of MeHg occurred in both the presence and absence of ATP. Exposure of mitochondria preloaded with 45Ca2+ to either 10 microM or 100 microM MeHg for 10 sec resulted in increased efflux of 45Ca2+ of 10% and 65%, respectively, in both the absence and presence of ATP. Loading mitochondria with 45Ca2+ in the presence of 20 microM RR reduced total uptake of 45Ca2+ and greatly attenuated MeHg-induced release of 45Ca2+ from mitochondria. RR did not inhibit the effects of MeHg on Ca2+ release by merely preventing the binding of MeHg to mitochondria because RR did not alter mitochondrial binding of methyl[203Hg]. The ratio of state 3 to state 4 respiration (respiratory control ratio) was measured as a means of assessing functional integrity of isolated mitochondria in the absence and presence of MeHg. Control ratios of from 3 to 5 were only marginally reduced by 2 microM MeHg but were greatly reduced by 10 and 20 microM MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of fast- and slow-phase depolarization-dependent synaptosomal calcium uptake by ethanol

Uptake of 45Ca++ by synaptosomes isolated from cerebral cortex, cerebellum, midbrain and brain stem of male, Sprague-Dawley rats was measured at 1-, 3-, 5-, 15-, 30- and 60-sec time periods. At 1 sec, the Ca++ uptake rate by cerebrocortical synaptosomes was 1.45 mumol/sec/g of protein, whereas the 60-sec rate was 0.03 mumol/sec/g of protein. In vitro addition of ethanol, 80 mM, inhibited depolarization-dependent (65 mM KCl) 45Ca++ uptake by synaptosomes but the time-response relationships varied depending upon the brain region studied. In cerebrocortical synaptosomes, ethanol significantly inhibited only the fast-phase component of 45Ca++ uptake (1 and 3 sec). Ethanol inhibited 45Ca++ uptake by midbrain synaptosomes at all measurement times studied (1, 3, 5 and 15 sec), whereas in cerebellum and brain stem ethanol inhibited 45Ca++ uptake at 3- and 5-sec time periods. Ethanol at concentrations of 25, 50, 100 and 150 mM inhibited 45Ca++ uptake by 9.0, 15.9, 24.8 and 30.7%, respectively, in cerebrocortical synaptosomes. In vitro ethanol, 80 mM, added to cerebrocortical synaptosomes isolated from rats fed a nutritionally adequate liquid ethanol diet did not significantly inhibit depolarization-dependent 45Ca++ uptake. The results of this study show that pharmacologically relevant ethanol concentrations inhibit voltage-dependent 45Ca++ uptake into synaptosomes. This inhibitory action may, at least in part, underlie some of the intoxicating effects of ethanol. In addition, chronic administration of ethanol resulted in an apparent adaptive response such that addition of ethanol no longer blocked 45Ca++ uptake. This adaptive response involving the calcium channel may represent a cellular mechanism for functional tolerance development.     

Modulation of levels of free calcium within synaptosomes by organochlorine insecticides

Effects of the organochlorine insecticides chlordecone, mirex, 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane on free intrasynaptosomal Ca2+ [( Ca++]i), synaptosomal 45Ca uptake and synaptosomal plasma and mitochondrial membrane potentials in vitro were studied. Chlordecone (10-50 microM) increased [Ca++]i from the resting level of 370 nM in a dose- and time-dependent manner to above 1.5 microM. This took place in the presence of 1 mM extrasynaptosomal Ca++ but not in nominally Ca++-free medium. Verapamil, a voltage sensitive Ca++ channel blocker, inhibited the initial increase of [Ca++]i caused by chlordecone, by 40%. Chlordecone also elevated [Ca++]i in synaptosomes in which mitochondrial Ca++ uptake had been abolished by valinomycin. Chlordecone depolarized partially the synaptosomal plasma membrane and, to a lesser extent, the potential of mitochondria within synaptosomes. However, chlordecone appeared to inhibit synaptosomal K+-stimulated and unstimulated 45Ca++ uptake by 20 to 30%. Inasmuch as chlordecone also stimulated release of 45Ca++ and the fluorescent dye fura-2 from preloaded synaptosomes, the apparent inhibition of uptake might be due to lysis of some synaptosomes by chlordecone. The effect of chlordecone on [Ca++]i decreased when the total amount of tissue in incubations was increased. [Ca++]i was only elevated marginally by mirex at the same concentration range. The results suggest that chlordecone increases free intrasynaptosomal Ca++ mainly by increasing influx of extrasynaptosomal Ca++. The principal mechanism appears to be a nonspecific leakage of Ca++ through the plasma membrane but some Ca++ may pass through voltage-sensitive Ca++ channels due to chlordecone-induced membrane depolarization.     

Effects of activation of sodium and calcium entry on spontaneous release of acetylcholine induced by methylmercury

The effect of ionophores and channel activators for Ca and Na on the time course and magnitude of methylmercury (MeHg)-induced increase in spontaneous release of neurotransmitter was studied at the murine neuromuscular junction using intracellular microelectrode recording techniques. The goal was to test whether chemicals that increase entry of Na+ or Ca++ into nerve terminals would shorten the latent period that precedes the onset of MeHg-induced increase in MEPP frequency. Administration of MeHg (100 microM) with A23187 (25 microM), a calcium ionophore, caused a more rapid time to peak induced increase in MEPP frequency than "control" MeHg preparations. This effect also occurred in solutions to which no extracellular Ca++ was added. Use of monensin, a Na+ ionophore (25-100 microM), did not shorten the time to peak increase of MEPP frequency. The dihydropyridine Ca++ channel agonist Bay K 8644 (750 nM) produced the most marked shortening of the time to peak MEPP frequency for MeHg. This effect also occurred in solutions deficient in extracellular Ca++. Veratridine (20 microM), a sodium channel activator, decreased the time to peak MEPP frequency when used in conjunction with MeHg in both Ca++-containing and Ca++-deficient solutions. Replacement of sodium in the extracellular perfusion solution with methylamine, which does not penetrate axon sodium channels, did not prevent the MeHg-induced increase in MEPP frequency although it did prolong the time to peak increase and decreased the maximal MEPP frequency induced by MeHg compared with experiments conducted in sodium-containing solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of divalent cations on the methylmercury-induced alterations of acetylcholine release

Bath application of methylmercury (MeHg) at the murine neuromuscular junction blocks synchronous evoked release of acetylcholine (ACh) and then increases spontaneous release of ACh effects observed electrophysiologically as cessation of EPPS, and increased MEPP frequency (MEPPf), respectively. The objectives of the present study were to test whether the effect of MeHg on spontaneous release was Ca++-specific by substituting Sr++ or Ba++ for Ca++, whether the time course of MeHg-induced block of synchronous evoked release was altered by varying Ca++ concentrations or substituting Sr++ and whether the processes involved in the decay of elevated MEPPf after repetitive stimulation (asynchronous evoked release) were altered by MeHg. MEPPf was recorded continuously from the rat hemidiaphragm using conventional methods during pretreatment with 2 mM Ca++, 2 mM Sr++ or 0.5 mM Ba++ and subsequently with the cation plus 100 microM MeHg. The time to peak MEPPf in MeHg was not different under any condition; however, peak MEPPf was lower in Sr++ solutions than in Ca++ or Ba++ solutions. EPPs were recorded from the rat hemidiaphragm cut muscle preparation during pretreatment with either 2, 4 or 8 mM Ca++ or 2 or 4 mM Sr++ and subsequently with the cation plus 100 microM MeHg. The latency to block of the EPP in 4 and 8 mM Ca++ was not significantly different from the latency in 2 mM Ca++. The latency to block in 2 or 4 mM Sr++ was also not different from that in Ca++. In addition, under all conditions EPP amplitude remained virtually unchanged from pretreatment values until block occurred after 8 to 9 min exposure to MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of K+ channel-dependent as well as -independent components of pinacidil-induced vasodilation

The mechanisms of pinacidil-induced direct vasodilation were studied in vitro in RMA and RAO. In RMA, pinacidil produced dose-dependent relaxations of norepinephrine (5 microM)-induced contractions with an IC50 of 0.2 microM. This component of pinacidil relaxation appeared to be dependent on K+ conductance because pretreatment with tetraethylammonium (10 mM), Ba++ (0.5 mM), glyburide (1 microM) and 20 mM K+ all caused a rightward shift of the pinacidil dose-response curve (DRC) and a corresponding increase in the pinacidil IC50. However, additional relaxation effects of pinacidil were still evident in the presence of various K+ channel blockers. Pinacidil also showed a relaxation DRC under the condition of 80 mM K+ contraction in both RMA and RAO with IC50 values of 27 and 50 microM, respectively. Pinacidil could also produce maximal relaxation in RMA and RAO remained unaffected in 145 mM K+ (zero Na+) depolarizing solution suggesting a lack of dependence on Na(+)-Ca++ exchange mechanism for this action of pinacidil. Studies using 1 or 3 min pulse labeling with 45Ca showed an absence of an inhibitory effect of pinacidil (at 50 and 100 microM) on unidirectional 45Ca influx stimulated by high-K+. Net 45Ca uptake studies showed that pinacidil inhibited high-K+ stimulated 45Ca uptake at 100 but not at 50 microM. Ryanodine (10-100 microM) was used as a tool to investigate the role of sarcoplasmic reticulum (SR) in this action of pinacidil. Under the condition in which ryanodine (10-100 microM) treatment was found to cause the SR to be nonfunctional, pinacidil relaxation DRC remained unaltered, suggesting a lack of a stimulatory effect of pinacidil on SR Ca++ accumulation. These data thus show that the K+ channel-independent effect of pinacidil does not involve to any significant degree an effect of pinacidil on plasmalemmal voltage-sensitive Ca++ channels, SR Ca++ stores, Na(+)-Ca++ exchange or membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative action of methylmercury and divalent inorganic mercury on nerve terminal and intraterminal mitochondrial membrane potentials.

Both methylmercury (MeHg) and inorganic divalent mercury (Hg++) alter the flux of ions and small molecules across nerve terminal membranes by mechanisms that may involve membrane depolarization. We compared the effects of MeHg and Hg++ on plasma (psi p) and mitochondrial membrane potentials (psi m) in synaptosomes using the potentiometric carbocyanine dye 3,3'-diethylthiadicarbocyanine iodide [diS-C2(5)]. Both mercurials (1-20 microM) produced concentration-dependent increases in dye fluorescence after 5 min of exposure which were not altered by removal of Ca++ from the medium. To determine directly effects of mercurials on psi p, predepolarization of psi m using NaN3 and oligomycin was necessary. Under this condition, MeHg- and Hg(++)-induced increases in fluorescence were associated with depolarization of psi p. A second approach was used to assess changes in psi p. In synaptosomes, the magnitude of the increase in fluorescence resulting from depolarization of psi p with a stimulus of constant intensity is a function of the resting psi p. The fluorescence response to depolarization of synaptosomes previously exposed to either MeHg or Hg++ (1-20 microM each) was reduced in a concentration-dependent manner relative to mercury-free controls. The concentration-dependent depolarization of psi p calculated in this manner correlated (r = 0.958) with calculations of psi p using direct measurements of increases in fluorescence intensity. MeHg- and Hg(++)-induced depolarizations were not altered by lowering Na+e or by the addition of the Na+ and Ca++ channel blockers tetrodotoxin and Co++, respectively. Thus, the effects of these two neurotoxic mercurials on synaptosomal membrane potentials were similar with respect to their loci but differed in magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of methylmercury on parallel-fiber and climbing-fiber responses of rat cerebellar slices

The environmental neurotoxicant methylmercury (MeHg) causes profound disruption of cerebellar function. Previous studies have shown that acute exposure to MeHg impairs synaptic transmission in both the peripheral and central nervous systems. However, the effects of MeHg on cerebellar synaptic function have never been examined. In the present study, effects of acute exposure to MeHg on synaptic transmission between parallel fibers or climbing fibers and Purkinje cells were compared in 300- to 350-microm cerebellar slices by using extracellular and intracellular microelectrode-recording techniques. Field potentials of parallel-fiber volleys (PFVs) and the associated postsynaptic responses (PSRs) were recorded in the molecular layer by stimulating the parallel fibers in transverse cerebellar slices. The climbing-fiber responses were also recorded in the molecular layer by stimulating white matter in sagittal cerebellar slices. At 20, 100, and 500 microM, MeHg reduced the amplitude of both PFVs and the associated PSRs to complete block, however, it blocked PSRs more rapidly than PFVs. MeHg also decreased the amplitudes of climbing-fiber responses to complete block. For all responses, an initial increase in amplitude preceded MeHg-induced suppression. Intracellular recordings of excitatory postsynaptic potentials of Purkinje cells were compared before and after MeHg. At 100 microM and 20 microM, MeHg blocked the Na+-dependent, fast somatic spikes and Ca++-dependent, slow dendritic spike bursts. MeHg also hyperpolarized and then depolarized Purkinje cell membranes, suppressed current conduction from parallel fibers or climbing fibers to dendrites of Purkinje cells, and blocked synaptically activated local responses. MeHg switched the pattern of repetitive firing of Purkinje cells generated spontaneously or by depolarizing current injection at Purkinje cell soma from predominantly Na+-dependent, fast somatic spikes to predominantly Ca++-dependent, low amplitude, slow dendritic spike bursts. Thus, acute exposure to MeHg causes a complex pattern of effects on cerebellar synaptic transmission, with apparent actions on both neuronal excitability and chemical synaptic transmission.     

Regulation of the number of functional voltage-sensitive Ca++ channels on PC12 cells by chronic changes in membrane potential

The properties of the various types of voltage-sensitive Ca++ channels (VSCC) are becoming increasingly well characterized, but the mechanisms which control the number and types of channels expressed by cells are virtually unknown. To study the regulation of VSCC in neuronal cells we have used PC12 pheochromocytoma cells. Binding of [3H]nitrendipine was used to determine the number of dihydropyridine-sensitive channels, and the uptake of 45Ca++ was used to determine the functional state of VSCC on the cell surface. Prolonged depolarization by elevation of extracellular K+ caused concomitant time and concentration-dependent decreases in both [3H]nitrendipine binding and depolarization-dependent uptake of 45Ca++. Changes in binding and ion flux plateaued at about a 50% decrease with 3 days of depolarization and an extracellular K+ concentration of 50 mM. Return of the cells to normal K+ caused the recovery of both [3H]nitrendipine binding and 45Ca++ uptake within 24 hr. Measurements of the intracellular free Ca++ concentration determined that it remained elevated for several hours with K+ depolarization, but returned to normal within 15 hr. Growth of the cells with a concentration of ionomycin, which caused a similar increase in intracellular free Ca++, also caused a loss of [3H]nitrendipine binding sites. Thus, it appears that the number of functional VSCC can be regulated by changes in intracellular Ca++ such as those associated with prolonged depolarization. However, because Ca++ channel number remained depressed while intracellular free Ca++ returned to normal, other mechanisms controlling channel number also must be involved.     

Membrane-mediated effects of the steroid 17-alpha-estradiol on adrenal catecholamine release

The effects of 17-alpha-estradiol on the secretion of catecholamines from the perfused bovine and cat adrenal gland and bovine chromaffin cells in culture elicited by dimethylphenylpiperazinium (DMPP), methacholine and high potassium were studied. In perfused cat adrenal glands, secretion of catecholamines evoked by pulses of DMPP (1 microM for 30 sec) was decreased by 17-alpha-estradiol at concentrations of 1 and 10 microM by 50 and 80%, respectively. However, secretion evoked by pulses of methacholine (3 microM for 30 sec) was not affected by 1 microM of 17-alpha-estradiol and was affected to a variable extent by 10 microM 17-alpha-estradiol. Catecholamine secretion evoked by higher concentrations of methacholine (100 microM for 60 sec) was reduced by 50% by 10 microM 17-alpha-estradiol. 17-alpha-Estradiol decreased secretion evoked by pulses of 120 mM K+ for 10 sec to a similar extent in the perfused bovine and cat adrenal gland. The 45Ca++ uptake into bovine chromaffin cells in culture stimulated by DMPP (100 microM for 10 sec) or high K+ (59 mM for 10 sec) was almost inhibited completely by 100 microM 17-alpha-estradiol. The rapid action precludes a classical genomic mechanism and suggests effects at the cell membrane.     

Regulation of Ca++ influx into striatal neurons by kainic acid

We investigated the mechanisms by which kainic acid (KA) produces increases in [Ca++]i in single striatal neurons in vitro using fura-2-based microfluorimetry. When neurons were depolarized by perfusion with high K+ or veratridine containing solutions, [Ca++]i rose rapidly to a peak and then declined to a lower sustained plateau that persisted as long as the depolarizing stimulus. The peak high K+-induced rise in [Ca++]i occurred at [K+]o greater than 50 mM and the plateau was largest at 30 mM K+. [K+]o that was greater than 70 mM caused the magnitude of the plateau to decrease. Responses to high K+ stimulation were completely dependent on [Ca++]o and presumably represented Ca++ influx. Nitrendipine partially blocked the peak of the high K+-induced response and completely blocked the sustained plateau Ca++ influx. The nitrendipine-resistant portion of the high K+ response could be completely blocked by predepolarization of the cell in Ca++-free solution. KA also produced large increases in [Ca++]i that were abolished on removal of external Ca++. Predepolarization/nitrendipine greatly reduced the effect of lower [KA] (100 microM). However, KA-induced increases in [Ca++]i became increasingly resistant to block of voltage-sensitive Ca++ channels as [KA] rose above 100 microM, indicating a second route of Ca++ entry that may be the KA receptor-gated ionophore. About one-half the responses to KA (100 microM) also displayed a large oscillation. [Ca++]i rose to a peak, fell and then rose again before finally declining to a plateau level. This oscillation was abolished when all external Na+ was replaced by Li+ and may result from alterations in the buffering of [Ca++]i as a result of KA-induced Na+ influx.     

Action of bepridil, a new calcium channel blocker on oxidative phosphorylation, oligomycin-sensitive adenosine triphosphatase activity, swelling, Ca++ uptake and Na+-induced Ca++ release processes of rabbit heart mitochondria in vitro

Effects of bepridil [1-[3-isobutoxy-2]benzylphenyl-amino)propyl pyrrolidine) on oxidative phosphorylation, oligomycin-sensitive adenosine triphosphatase, swelling, Ca++ uptake and Na+-induced Ca++ release processes of mitochondria isolated from rabbit heart were investigated. Bepridil, in concentrations greater than 5 microM, produced uncoupling of oxidative phosphorylation and stimulated oligomycin-sensitive adenosine triphosphatase activity. At low concentrations it prevented inorganic phosphate-induced swelling and associated depression of oxidative phosphorylation. Its effectiveness in preventing swelling and depression of oxidative phosphorylation was found to be dependent on inorganic phosphate concentration. A concentration of 1 microM of bepridil was effective in producing 50% less depression of phosphorylating respiration in the presence of 10 mM inorganic phosphate. Concentrations of bepridil above 25 microM inhibited the rate of Ca++ uptake. A 50% inhibition of Ca++ uptake was observed at 93 microM bepridil. The rate of Na+-induced Ca++ release was also inhibited by bepridil. A 50% inhibition of the rate of Na+-induced Ca++ release occurred at 11 microM of bepridil. When the Na+-dependent Ca++ release process was about 80% inhibited by 25 microM bepridil, the uptake process still remained at the same level as the untreated control. Results suggest that in addition to reported effects on sarcolemma and sarcoplasmic reticulum, mitochondria are also affected by bepridil.     

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

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

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

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