Transmembrane Influx Of Ca2 Research Articles

Proliferation of human lens epithelial cells (HLE-B3) is inhibited by blocking of voltage-gated calcium channels

Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage-gated calcium channels. T-type and L-type calcium channels are substantially present in human lens epithelial cell (hLEC), and total calcium currents are inhibited by mibefradil. Here, the hypothesis was tested if calcium influx via Ca(v) channels regulates proliferation in epithelial cells. Cell proliferation was determined by cell culture assays using the L- and T-type Ca(v) channel blockers mibefradil and verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench technique. Western blot experiments were accomplished under standard conditions using antibodies against MAPK 3. Mibefradil as well as verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of MAPK 3 was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. We conclude that the transmembrane influx of Ca(2+) through Ca(v) channels contributes to the regulation of hLEC proliferation, identifying Ca(v) channel blockers as potential therapeutic substances in ocular diseases.

Inhibition of Degranulation of RBL-2H3 Cells by Casein Kinase II Substrate Peptide

In a previous study, we demonstrated the existence of casein kinase II-like ectokinase activity on rat basophilic leukemia 2H3 (RBL-2H3) cells (Immunol. Let. 68, 369, 1999), and to determine the role of ectokinase in the degranulation of RBL-2H3 cells, in this study we investigated the effect of casein kinase II substrate peptide on the degranulation by the cells. Casein kinase II peptide (RRRDDDSDDD) dose-dependently (IC50 = 50 μM) inhibited β-hexosaminidase release by IgE-sensitized antigen-stimulated RBL-2H3 cells, whereas casein kinase II peptide analogue (RRRDDDADDD) only partially inhibited β-hexosaminidase release by antigen-stimulated RBL-2H3 cells. Preincubation of RBL-2H3 cells with 200 μM of casein kinase II peptide significantly inhibited external 130 kDa protein phosphorylation, and casein kinase II peptide inhibited the sustained increase in cytosolic calcium ion concentration in response to antigen stimulation. Our findings suggest that casein kinase II acts as an ectokinase in RBL-2H3 cells and that casein kinase II peptide acts as a competitor for phosphorylation of ectoprotein, which involves degranulation and a transmembrane influx of Ca2+ by IgE receptor cross-linking. Another casein kinase II inhibitor, GT copolymer, also inhibited the degranulation of antigen-stimulated RBL-2H3 cells. Thus, the modulator of casein kinase II activity seems to be a good tool for the inhibitor of signal transduction in RBL-2H3 cells.

Cellular activation by Ca2+ release from stores in the endoplasmic reticulum but not by increased free Ca2+ in the cytosol

Ca(2+) release from intracellular stores and/or transmembrane influx can increase the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). Such changes in [Ca(2+)](i) might transduce signals regulating transcription, motility, secretion, and so on. Surfactant secretagogues such as ATP and ionomycin stimulate the release and transmembrane influx of Ca(2+), both of which increase [Ca(2+)](i). The addition of surfactant protein A (SP-A) or depleting cellular Ca(2+) inhibited both surfactant secretion and Ca(2+) transients. Current results suggest that Ca(2+) signalling stimulates surfactant secretion by type II pneumocytes, but not via increased [Ca(2+)](i). Treatment of cells with a Ca(2+) chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM), stimulated secretion but decreased [Ca(2+)](i). Adding SP-A or depleting Ca(2+) inhibited BAPTA-AM-induced secretion. When studied directly, Ca(2+) in the endoplasmic reticulum store ([Ca(2+)](l)) decreased in response to BAPTA, ionomycin and thapsigargin, and increased in response to SP-A. Phorbol ester (PMA) induced surfactant secretion without altering [Ca(2+)](i) or [Ca(2+)](l) and was unaffected by Ca(2+) depletion. The addition of PMA to Ca(2+)-releasing secretagogues increased secretion, but combining two Ca(2+)-releasing secretagogues did not. These results suggest that (1) Ca(2+) signalling of type II cell surfactant secretion reflects changes in [Ca(2+)](l), not [Ca(2+)](i), (2) PMA elicits secretion differently from Ca(2+)-releasing secretagogues, and (3) SP-A inhibits secretion by enhancing Ca(2+) sequestration within endoplasmic reticulum stores. Whether other cell types signal via changes in [Ca(2+)](l) is unknown.

Spatial and Temporal Separation of Calcium Signals in Myeloid Cells Stimulated by Immune Complexes

Stimulation of large (100 μm) human myeloid cells with immune complexes resulted in Ca 2+ spiking. Both global and regional changes in the intracellular cytosolic free Ca 2+ concentration were detected in response to immune complex stimulation. The regional changes were mediated by release of Ca 2+ from stores, whereas global changes were mediated by Ca 2+ influx. They occurred independently of each other, with release of Ca 2+ from intracellular Ca 2+ stores being separated from transmembrane influx of Ca 2+. Bromophenacyl bromide, an l-plastin binding agent, inhibited store release without preventing transmembrane influx of Ca 2+. The large size of the myeloid cells used here allowed the visualisation of the spatial and temporal separation of store release from transmembrane influx of Ca 2+, providing further evidence for the existence of independent Ca 2+ store release and Ca 2+ influx mechanisms in these cells.

The relaxing effect of ketamine on isolated rabbit lower esophageal sphincter.

We used ketamine to investigate the effects and intracellular mechanisms of several anesthetics on strips of lower esophageal sphincter (LES) from rabbits. Ketamine induced dose-dependent relaxation of LES preparations. It increased the content of 3',5'-cyclic adenosine monophosphate (cAMP) dose-dependently, but decreased that of 3',5'-cyclic guanosine monophosphate (cGMP). Pretreatment with nicotinic acid, an inhibitor of adenylate cyclase, along with atropine to block neurogenic effects, antagonized ketamine-induced relaxation. Pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89), a selective antagonist for cAMP-dependent protein kinase, similarly antagonized the relaxant effect of ketamine. Cholera toxin and dibutyryl cAMP induced LES relaxation. However, dibutyryl cGMP induced little LES relaxation, and pretreatment with NG-nitro-L-arginine or methylene blue did not alter the relaxant effect. Atropine, propranolol, phentolamine, vasoactive intestinal peptide (VIP) antagonist, and tetrodotoxin did not affect the ketamine-induced relaxation. This response, however, was potentiated in the presence of indomethacin or diphenhydramine. Ketamine-induced relaxation was inhibited in the presence of verapamil. These findings suggest that ketamine induces relaxation of LES, in part, by modulating the activity of adenylate cyclase and in part by inhibiting transmembrane influx of Ca2+.

Effects of phorbol ester on intracellular Ca 2+ and membrane currents in cultured human microglia

The effects of protein kinase C (PKC) activation by phorbol ester on intracellular Ca 2+ concentration ([Ca 2+] i) and membrane currents in human microglia grown in culture were investigated. Treatment of microglia with phorbol myristate acetate (PMA) resulted in a large increase in [Ca 2+] i in cells loaded with fura-2. The increased levels of [Ca 2+] i were not altered following removal of the phorbol ester. In Ca 2+-free medium, application of PMA did not increase [Ca 2+] i. In addition, PMA application in standard Ca 2+ -solution containing lanthanum (1.8 mM) had no effect on the microglial response to PMA, suggesting that the phorbol ester actions were due to transmembrane influx of Ca 2+ but not through voltage-gated Ca 2+ channels. Whole-cell patch clamp measurements demonstrated that PMA potentiated an outward K + current and inhibited an inward rectifier K + current. This study is the first demonstration that PKC activation by phorbol ester leads to increased intracellular [Ca 2+] and changes in membrane currents in human microglia.

Depressant Effects of Prostacyclin in Human Atrial Fibers and Cardiomyocytes.

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI(2)) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI(2) (1 nM-10 &mgr;M) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K](o) (4 mM) and high [K](o) (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI(2) on Na- and Ca-dependent inward currents (I(Na) and I(Ca)) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt(max) = 101 +/- 15 Vs(-1)) in 4 mM [K](o), PGI(2) did not influence dV/dt(max) of phase 0 depolarization even at 1 &mgr;M. However, at a concentration as low as 10 nM, PGI(2) depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI(2) also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI(2) reduced I(Ca) but not I(Na). The present findings show that, in human atrial fibers and cardiomyocytes, PGI(2) induces greater depressant effects on the slow-response action potential, I(Ca) and triggered activity than on the fast-response action potential. It is suggested that PGI(2) may act through a selective reduction of transmembrane Ca influx. Copyright 1994 S. Karger AG, Basel

Stretching releases Ca2+ from intracellular storage sites in canine cerebral arteries

Mechanical stretch applied to canine cerebral artery produced myogenic contraction. The contraction of the artery in response to quick stretch was dependent on not only the transmembrane influx of Ca2+ through 1,4-dihydropyridine-sensitive Ca2+ channels but also the release of Ca2+ from intracellular storage sites: the stretch-produced contractile component that was resistant to 0.1 microM nicardipine, a Ca(2+)-channel antagonist, was inhibited by about 50% after treatment with ryanodine, and was almost completely suppressed by 0.1 mM 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, a putative phospholipase C inhibitor, or by lowering the temperature from 35 to 20 degrees C. The results suggest that in addition to transmembrane influx of Ca2+ through L-type Ca2+ channels, the release of Ca2+ from both ryanodine-sensitive and -insensitive intracellular storage sites, which increases intracellular Ca2+, accounts for the stretch-induced contraction of canine basilar artery. It seems also possible that inositol 1,4,5-trisphosphate is a common mediator for the release of Ca2+ from both types of intracellular storage sites.

Cross-linking of IgG receptors inhibits membrane immunoglobulin-stimulated calcium influx in B lymphocytes.

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor-induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg-mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2-loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.

Thapsigargin discharges intracellular calcium stores and induces transmembrane currents in human endothelial cells.

We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca(2+)-adenosine 5'-triphosphatase (Ca(2+)-ATPase), on membrane currents and on the intracellular Ca2+ concentration ([Ca2+]i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patch-clamp technique in the whole-cell mode and [Ca2+]i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 mumol/l induced a slow increase in [Ca2+]i to a peak value of 400 +/- 110 nmol/l above a resting level of 120 +/- 35 nmol/l, and then slowly declined to a new steady-state level of 315 +/- 90 nmol/l (n = 33). The thapsigargin-induced increase in [Ca2+]i depended on the extracellular Ca2+ concentration ([Ca2+]o: it declined after removal of extracellular Ca2+, but increased again when [Ca2+]o was augmented, indicating that the response depends on a transmembrane influx of Ca2+ ions. The peak amplitude of the histamine-induced Ca2+ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca2+]i induced by thapsigargin than during the rising phase of the changes in [Ca2+]i. The decline of the Ca2+ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant tau equal to 24.5 +/- 5 s (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ and Ca(2+)-activated Cl- currents in rabbit oesophageal smooth muscle.

1. An inward current carried by Ca2+ was recorded from single smooth muscle cells of rabbit oesophageal muscularis mucosae using a whole-cell gigaseal technique with physiological (2 mM) external calcium concentration ([Ca2+]o) in the presence of intracellular Cs+ ([Cs+]i 130 mM). Only one type of Ca2+ current could be identified. The threshold for its activation was approximately -30 mV and maximum inward current (approximately 300 pA) was recorded at 0 mV. 2. This inward current was blocked by Co2+ (4 mM), Cd2+ (0.5 mM) and nifedipine (1 microM) and was enhanced by Bay K 8644 (5 microM). We therefore classify it as a L-type Ca2+ current and denote it ICa. 3. Steady-state inactivation data were well-fitted by a Boltzmann distribution, indicating that inactivation of the Ca2+ current is strongly modulated by membrane potential. However, the inactivation of ICa slowed significantly and became less complete when BaCl2 replaced CaCl2 in the Tyrode solution suggesting that the inactivation of ICa may also be dependent on [Ca2+]i. The steady-state activation and inactivation curves for ICa overlap between -40 and 0 mV indicating that there may be a Ca2+ window current in this range of potentials. 4. When EGTA was omitted from the pipette-filling solution, depolarizations positive to -10 mV resulted in a transient as opposed to a maintained inward Ca2+ current which was followed by a relatively large outward current. Under these conditions, slowly decaying inward tail currents were also recorded upon repolarization to the holding potential, -60 mV. However, when EGTA was omitted from the pipette, marked 'run-down' of the Ca2+ current occurred within 10 min after starting the whole-cell recording. 5. This run-down of ICa was reduced significantly when the nystatin perforated patch technique was used. Under these conditions stable ICa records could be obtained for over 1 h. Outward currents and slow decaying inward tail currents similar to those recorded with no EGTA in the pipette were also obtained consistently using the nystatin recording technique. 6. In nystatin perforated patch recordings, CoCl2 (2 mM) completely abolished the Ca2+ current, the outward currents and the slow inward tails. These findings suggest that the outward currents and slow inward tails are activated by a transmembrane influx of Ca2+. 7. Ion replacement and pharmacological tests provided evidence that both the outward currents and the slow inward tails are due to Ca(2+)-activated Cl- current (ICl(Ca)).(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of ethanol on the chemotactic peptide-induced second messenger generation and superoxide production in polymorphonuclear leukocytes.

The generation of oxygen radicals by polymorphonuclear leukocytes (PMNL) plays a pivotal role for host defense. Since ethanol reduced FMLP- but not PMA-induced superoxide ion (O2-) formation by PMNL, the effects of ethanol on second messenger systems in PMNL were studied. FMLP induced a biphasic rise in cytosolic calcium concentrations, [Ca2+]i. Ethanol treatment abolished the second phase (believed to reflect Ca2+ influx), an effect also observed in PMNL treated with La3+ or suspended in Ca(2+)-free buffer. The FMLP-induced inositol trisphosphate generation was unaffected by ethanol, whereas diacylglycerol formation was, as expected, markedly reduced. Propranolol, an inhibitor of diacylglycerol formation from phosphatidic acid, caused a prolonged transmembrane influx of Ca2+ and partially reversed the inhibitory effect of ethanol on FMLP-induced O2- production. Thus, the ability of ethanol to inhibit FMLP-induced O2- generation in neutrophils seems to be due to both impaired influx of Ca2+ across the plasma membrane and reduced phospholipase D-mediated generation of phosphatidic acid.

K-current mediation of prolactin-induced proliferation of malignant (Nb2) lymphocytes.

The effects of different concentrations of various K-current blockers on prolactin-induced proliferation and membrane K-currents in malignant lymphocytes (Nb2 cells) were investigated. Membrane currents were measured with the whole cell patch-clamp technique, and lymphocyte density was quantified by both spectrophotometric and conventional methods. K-current blockers tested (quinidine, 4-aminopyridine, barium, and tetraethylammonium) exhibited similar rank order potency for K-current block and inhibition of prolactin-induced proliferation of malignant lymphocytes. Because Nb2 cells proliferate independently of a transmembrane Ca-influx, these results suggest that K-currents per se rather than K-current modulation of Ca-influx is an essential event for lymphocyte proliferation.

Responses to barbiturates of isolated dog cerebral and mesenteric arteries contracted with KCl and prostaglandin F2a

In helical strips of dog cerebral and mesenteric arteries previously contracted with KCl or prostaglandin F2 alpha (PGF2 alpha), the addition of pentobarbital (10(-5) to 10(-3) mol.l-1) caused dose-related relaxation, whereas thiamylal and thiopental in a low concentration (10(-5) to 10(-4) mol.l-1) caused further contraction and in a high concentration (10(-3) mol.l-1) profound relaxation. Thiobarbiturate-induced contractions were greater in mesenteric than in cerebral arteries previously contracted with PGF2 alpha. In cerebral and mesenteric arteries exposed to Ca(++)-free media for 60 min and treated with KCl or PGF2 alpha, reintroduction of Ca++ produced a transient contraction, a transient relaxation and a persistent contraction. The Ca(++)-induced persistent contraction was attenuated by pretreatment with pentobarbital (10(-4) to 10(-3) mol.l-1) and thiamylal (10(-3) mol.l-1); the attenuation was greater in arteries treated with KCl than with PGF2 alpha. The Ca(++)-induced contractions of mesenteric artery treated with PGF2 alpha were potentiated by 10(-4) mol.l-1 thiamylal. It is concluded that pentobarbital possesses only a vasodilator effect, whereas thiamylal and thiopental have both constrictor and dilator effects on vascular smooth muscle. The vasodilator effect of barbiturates is associated in part with inhibition of transmembrane influx of Ca++; the inhibition is more predominant on the influx evoked by KCl-induced depolarization than by a stimulation of PGF2 alpha receptors. Thiamylal in low concentrations appears to enhance Ca++ influx through a receptor-operated Ca++ channel for PGF2 alpha.

Control of interval‐force relation in canine ventricular myocardium studied with ryanodine

1. The mechanism of post-extrasystolic, rest and frequency potentiation was studied in canine isolated ventricular muscle. 2. Ryanodine, which impairs Ca availability from the sarcoplasmic reticulum (SR), reduced the amplitude of the extrasystole less than that of the steady state contraction. Ryanodine also inhibited post-extrasystolic potentiation and converted rest-potentiation into rest depression. Rest-potentiation was blocked preferentially by ryanodine compared to post-extrasystolic potentiation. An increase in the contribution of extracellular Ca to the extrasystolic contraction could not entirely account for the post-extrasystolic potentiation. 3. Prolonged rest, by itself, also caused depression of the first post-rest contraction. During rest-potentiation, SR Ca seemed to play a greater role in contraction than transmembrane Ca influx. However, the ability of the 'release pool' of Ca in the SR to be reprimed after a contraction was reduced. This was seen as a decrease in post-extrasystolic potentiation elicited immediately after rest. 4. A decrease in stimulus interval was associated with a transient decrease in contraction amplitude followed by an increase. An abrupt increase in stimulus interval had the opposite effect. Ryanodine blocked the initial transient changes and accelerated the delayed changes. These results suggest that the transient changes in contraction after sudden changes in drive interval are dependent on the SR. 5. Transmembrane Ca entry and the rate of recovery of the Ca release process (repriming) in the SR after a contraction seem to be interval-dependent. The data also indicate that different mechanisms are involved in post-extrasystolic and rest-potentiation. 6. The results are consistent with a model which proposes 'recirculation' of activator Ca within the SR after a contraction or of the presence of an appreciable amount of inactivation of the SR Ca release process during normal stimulation. An increased pool of releasable Ca due to longer recirculation time or a time-dependent decay in the level of inactivation of Ca release from the SR may give rise to rest-potentiation.

Suppressive effects of somatostatin in dog Purkinje fibres.

1. The effects of somatostatin (SS, 1 nM-3 microM) on the electrical and mechanical activities of isolated Purkinje fibres of the dog were studied. 2. In most Purkinje fibres driven electrically in normal [K]o Tyrode solution, SS decreased the force of contraction slightly and had very little effect on the fast response action potential. However, in sensitive fibres SS induced a moderate reduction of action potential duration and contractile force in normal [K]o and depressed the slow response action potentials in high [K]o. 3. In spontaneously beating Purkinje fibres, SS decreased the regular rhythms slightly but abolished bursts of fast rhythms at a concentration as low as 1 nM. 4. When the fibres were depolarized in the presence of 0.2 mM barium or in Na-free solution, SS suppressed the Ca-dependent slow response action potentials. 5. These findings suggest that SS may suppress abnormal automatic activity of dog Purkinje fibres through a reduction of transmembrane Ca influx or a modulation of intracellular calcium.

Comparison of the skeletal muscle relaxant properties of Portulaca oleracea extracts with dantrolene sodium and methoxyverapamil

The effects of aqueous (AEE), dialysable (DIF) and methanol (MEE) extracts of Portulaca oleracea stems and leaves were compared with those of dantrolene sodium and methoxyverapamil (D-600) with respect to inhibition of twitch tension on the rat phrenic nerve-hemidiaphragm and with respect to contracture induced by nicotinic agonists on the frog rectus abdominis preparations. The extracts, dantrolene and D-600 inhibited twitch tension due to indirect electrical stimulation via the phrenic nerve (NS) on hemidiaphragm muscle, whereas the extracts and dantrolene inhibited, in addition, twitch amplitude due to direct muscle stimulation (MS). The extracts, dantrolene and D-600 also attenuated K + and caffeine-induced contractures with the extracts and D-600 also reducing the time taken for the K +induced contracture to fall to basal tension. In addition, the tetanic tension due to NS and MS was attenuated with only the extracts and dantrolene reducing the twitch/tetanus ratio (MS). There was a non-significant but consistent tendency for mutual potentiation between the extracts and dantrolene with respect to their inhibitory effect on twitch amplitude (MS) resulting in a shift to the left of the concentration-response curves to the extracts or dantrolene. This was not evident with the extracts and D-600 or dantrolene and D-600. Simultaneous addition of the extracts and dantrolene resulted in an increase in the rate of twitch tension inhibition and a decrease in the time to maximum relaxation of twitch amplitude (MS). The extracts and D-600 proved more effective in attenuating nicotinic agónist (acetylcholine, carbachol and nicotine)-induced contractures on the rectus abdominis muscle than dantrolene. From these observations, it appears that the Portulaca oleracea extracts mimic, in part, the effect of D-600 and dantrolene on the rat hemidiaphragm and frog rectus abdominis muscles; therefore, the muscle relaxant properties of the extracts may be due, in part, to inhibition of trans-membrane Ca influx, interference with the Ca-induced Ca release process and/or inhibition of the release of intracellular Ca from stores in the sarcoplasmic reticulum.

Ionic dependence of glutamate neurotoxicity

The cellular mechanisms by which excess exposure to the excitatory neurotransmitter glutamate can produce neuronal injury are unknown. More than a decade ago it was hypothesized that glutamate neurotoxicity (GNT) is a direct consequence of excessive neuronal excitation ("excitotoxicity" hypothesis); more recently, it has been hypothesized that a Ca influx triggered by glutamate exposure might mediate GNT (Ca hypothesis). A basic test to discriminate between these hypotheses would be to determine the dependence of GNT on the extracellular ionic environment. The excitotoxicity hypothesis predicts that GNT should depend critically on the presence of extracellular Na, since that ion appears to mediate glutamate neuroexcitation in the CNS; the Ca hypothesis predicts that GNT should depend critically on the presence of extracellular Ca. The focus of the present experiments was to determine the effects of several alterations in the extracellular ionic environment upon the serial morphologic changes that occur after mouse neocortical neurons in cell culture receive toxic exposure to glutamate. The results suggest that GNT in cortical neurons can be separated into 2 components distinguishable on the basis of differences in time course and ionic dependence. The first component, marked by neuronal swelling, occurs early, is dependent on extracellular Na and Cl, can be mimicked by high K, and is thus possibly "excitotoxic." The second component, marked by gradual neuronal disintegration, occurs late, is dependent on extracellular Ca, can be mimicked by A23187, and is thus possibly mediated by a transmembrane influx of Ca. While either component alone is ultimately capable of producing irreversible neuronal injury, the Ca-dependent mechanism predominates at lower exposures to glutamate. Glutamate exposure likely leads to a Ca influx both through glutamate-activated cation channels and through voltage-dependent Ca channels activated by membrane depolarization. Addition of 20 mM Mg, however, did not substantially block GNT; this finding, together with the observation that GNT is largely preserved in sodium-free solution, supports the notion that the activation of voltage-dependent Ca channels may not be required for lethal Ca entry. The possibility that N-methyl-D-aspartate receptors may play a dominant role in mediating glutamate-induced lethal Ca influx is discussed.

Reduction in transmembrane Ca influx induced by the negative inotropic action of nicardipine in frog ventricular muscle.

The effects of nicardipine, a new 1,4-dihydropyridine derivative, on electrical and mechanical properties of frog ventricular muscle were examined. Nicardipine (3 X 10(-7) M) reduced the twitch tension, and this reduction was frequency dependent, and considerable, in case of high frequencies. The resting potential was not affected by nicardipine (3 X 10(-7) M), but the plateau height of the action potential was decreased and the duration of the action potential was shortened. The suppression of this plateau height was frequency dependent. The nicardipine-induced suppression of tension and action potential could be almost completely antagonized by raising the concentration of [Ca]o or by applying isoprenaline (8 X 10(-7) M). These results suggest that the negative inotropic action of nicardipine is induced mainly by a reduction in the transmembrane Ca influx.

Electromechanical effects of caffeine in isolated human atrial fibres

Effects of caffeine on the action potential and contractile force of human atrial fibres obtained at cardiac surgery were studied with standard microelectrode technique. In 4 mmol . litre-1 [K]o, the only significant action produced by 0.3 to 3 mmol . litre-1 caffeine on the electro-mechanical activity of relatively normal atrial fibres was a slight shortening of the action potential duration at 50% repolarisation. When the fibres were depolarised in 27 mmol . litre-1 [K]o or in atrial fibres showing slow responses in 4 mmol . litre-1 [K]o, however, caffeine could increase the upstroke of slow response and the force. In 18% of atrial fibres showing slow responses in 4 mmol . litre-1 [K]o, caffeine induced spontaneous discharges and potentiated afterdepolarisations. The positive inotropic and the arrhythmogenic effects of caffeine could be diminished by pretreating the fibres with propranolol or Ca antagonists (diltiazem and verapamil). In fibres beating spontaneously in normal [K]o, caffeine accelerated spontaneous rhythms initially and then depressed them. Propranolol potentiated the later depression but did not block the initial acceleration. The results suggest that caffeine increases the transmembrane Ca influx and enhances the release of Ca from the intracellular stores in human atrial fibres. As a consequence, caffeine could induce arrhythmias in atria from certain individuals.