Tentacle regulating potentials inNoctiluca miliaris: their generation sites and ionic mechanisms

Summary Membrane potential responses that regulate movement of the food-gathering tentacle ofNoctiluca miliaris (tentacle regulating potentials, TRPs) were examined electrophysiologically under various ionic conditions. These spontaneous TRPs were modified by changing the external ionic conditions. Positive spike appeared as external Ca²⁺ concentration was lowered. The peak of the spike became more positive with increasing external Na⁺ concentration. The spike could be evoked by injecting a depolarizing current when the membrane was hyperpolarized. The positive spike is assumed to be caused by regenerative activation of depolarization-sensitive Na channels. The peak of the negative spike, reported by previous workers, became more negative with increasing external Cl^– concentration. The spike was evoked by injecting a hyperpolarizing current when the membrane was depolarized. The negative spike is assumed to be caused by regenerative activation of hyperpolarization-sensitive Cl^– channels. The waveforms and amplitudes of the TRPs recorded from the nucleus were identical to those recorded from the flotation vacuole. This suggests that the TRPs are generated on the membrane facing the external solution. Possible roles of the TRPs in the control of tentacle movement are discussed.Abbreviations ASW artificial sea water - FTP flash-triggering potential - TRP tentacle regulating potential     

Ionic mechanisms and Ca2+ dynamics underlying the glucose response of pancreatic β cells: a simulation study

To clarify the mechanisms underlying the pancreatic β-cell response to varying glucose concentrations ([G]), electrophysiological findings were integrated into a mathematical cell model. The Ca(2+) dynamics of the endoplasmic reticulum (ER) were also improved. The model was validated by demonstrating quiescent potential, burst-interburst electrical events accompanied by Ca(2+) transients, and continuous firing of action potentials over [G] ranges of 0-6, 7-18, and >19 mM, respectively. These responses to glucose were completely reversible. The action potential, input impedance, and Ca(2+) transients were in good agreement with experimental measurements. The ionic mechanisms underlying the burst-interburst rhythm were investigated by lead potential analysis, which quantified the contributions of individual current components. This analysis demonstrated that slow potential changes during the interburst period were attributable to modifications of ion channels or transporters by intracellular ions and/or metabolites to different degrees depending on [G]. The predominant role of adenosine triphosphate-sensitive K(+) current in switching on and off the repetitive firing of action potentials at 8 mM [G] was taken over at a higher [G] by Ca(2+)- or Na(+)-dependent currents, which were generated by the plasma membrane Ca(2+) pump, Na(+)/K(+) pump, Na(+)/Ca(2+) exchanger, and TRPM channel. Accumulation and release of Ca(2+) by the ER also had a strong influence on the slow electrical rhythm. We conclude that the present mathematical model is useful for quantifying the role of individual functional components in the whole cell responses based on experimental findings.     

Sarcoplasmic reticulum Ca2+ permeation explored from the lumen side in mdx muscle fibers under voltage control

Under resting conditions, external Ca(2+) is known to enter skeletal muscle cells, whereas Ca(2+) stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca(2+) entry and in the SR Ca(2+) leak is still a matter of debate, but several lines of evidence suggest that these Ca(2+) fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca(2+) dye Fura2, we first demonstrated that the rate of Ca(2+) increase in response to cyclopiazonic acid (CPA)-induced inhibition of SR Ca(2+)-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca(2+) leak. However, removal of external Ca(2+) reduced the rate of CPA-induced Ca(2+) increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca(2+) influx in mdx fibers. Fibers were then loaded with the low-affinity Ca(2+) dye Fluo5N-AM to measure intraluminal SR Ca(2+) changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca(2+) changes. Voltage dependence and magnitude of depolarization-induced SR Ca(2+) depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca(2+) reuptake activity in mdx fibers. Overall, CPA-induced SR Ca(2+) leak at -80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca(2+) in control fibers. The elevated passive SR Ca(2+) leak may contribute to alteration of Ca(2+) homeostasis in mdx muscle.     

An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.     

Ca2+ stabilizes the membrane potential of moth olfactory receptor neurons at rest and is essential for their fast repolarization

The role of Ca(2+) in insect olfactory transduction was studied in the moth Spodoptera littoralis. Single sensillum recordings were made to investigate in vivo the role of sensillar Ca(2+) on the electrophysiological properties of sex pheromone responsive olfactory receptor neurons (ORNs). Lowering the sensillar Ca(2+) concentration to 2 x 10(-8) M increased ORN spontaneous firing activity and induced long bursts of action potentials (APs) superimposed on spontaneous negative deflections of the transepithelial potential. We inferred that Ca(2+) stabilizes the membrane potential of ORNs, keeping the spontaneous firing activity at a low and regular level. Neither the amplitude and kinetics of the rising phase of sensillar potentials (SPs) recorded in response to pheromone stimuli nor the AP generation during stimulation depended on the extracellular Ca(2+) concentration. Thus, extracellular Ca(2+) is not absolutely necessary for ORN response. Partial inhibition of responses with a calmodulin antagonist, W-7, also indicates that intracellular Ca(2+) contributes to the ORN response and suggests that Ca(2+) release from internal stores is involved. In 2 x 10(-8) M Ca(2+), the repolarization of the SP was delayed when compared with higher Ca(2+) concentrations. Therefore, in contrast to depolarization, ORN repolarization depends on extracellular Ca(2+). Ca(2+)-gated K(+) channels identified from cultured ORNs with whole-cell recordings are good candidates to mediate ORN repolarization.     

Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca2+ release in rat skeletal myotubes

Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca(2+) levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca(2+) signals evoked by PbTx-3 were inhibited by blocking either IP(3) receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP(3) mass levels, dependent on extra-cellular Na(+). A similar increase in IP(3) mass was induced by high K(+) depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP(3) mass induced by either PbTx-3 or K(+) was also detected in Ca(2+)-free medium. These results establish that the effect of the toxin on both intracellular Ca(2+) and IP(3) levels occurs via a membrane potential sensor instead of directly by Na(+) flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP(3)-dependent calcium signal involved in regulation of gene expression.     

The ionic basis of cardiac activity in the bivalve mollusc Perna perna

The ionic basis of cardiac activity and aspects of excitation-contraction (E-C) coupling were investigated in the isolated heart of the bivalve mollusc Perna perna, using the sucrose-gap technique. The role of the principal ions was established employing artificial seawater, in which specific ion concentrations were modified, and ion channel blockers. The mean membrane resting potential (MP) and the action potential (AP) were -33+/-0.7 mV (n=89) and 13+/-0.3 mV (n=71), respectively. The MP potential was primarily dependent on K(+) ions. Three types of cardiac APs were identified: fast, slow and spike-plateau potentials. Cardiac activity was maintained in Na(+)- or Ca(2+)-free salines but ceased when either Cd(2+) or EDTA was added to these salines. Other Ca(2+) channel blockers reduced the amplitude and increased duration of the cardiac APs. Tetrodotoxin (TTX) and procaine did not alter the AP. The data showed that the depolarizing phase of the AP was dependent on Ca(2+) influx while the plateau phase, when present, resulted from Na(+) influx that was modulated by Ca(2+). The mechanical responses were more sensitive to changes in extracellular Ca(2+) concentration than were the electrical responses.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Oscillatory zones and their role in normal and abnormal sheep purkinje fiber automaticity

The mechanisms by which low [K(+)](o) induces spontaneous activity was studied in sheep Purkinje fibers. Purkinje strands were superfused in vitro and membrane potentials were recorded by means of a microelectrode technique. The results show that low [K(+)](o) increases the slope and amplitude of early diastolic depolarization, sharpens the transition between early and late diastolic depolarizations, induces an after-potential and large pre-potentials through a negative shift of an oscillatory zone. Pre-potentials occur progressively sooner during diastole and merge with the after-potential to induce uninterrupted spontaneous discharge. During recovery, when the rate slows, after- and pre-potentials separate once more, the slower discharge decreasing the after-potentials but not the pre-potentials. Low [K(+)](o) has little effect on the plateau, but markedly slows phase 3 repolarization and may altogether prevent it. At depolarized levels, voltage oscillations, slow responses, sinusoidal fluctuations or quiescence may be present depending on voltage. During the recovery, a train of either sub-threshold oscillations or spontaneous action potentials appear towards the end of phase 3 repolarization. The cessation of the action potentials unmasks large sub-threshold oscillations, that occur in the oscillatory zone. Drive, high [Ca(2+)](o) and norepinephrine increase slope and amplitude of early diastolic depolarization as low [K(+)](o) does. In low [K(+)](o), Cs(+) prevents spontaneous discharge at polarized levels, but not the decrease in resting potential nor the onset of slow responses at depolarized levels. Cs(+) blocks the train of oscillations and of action potentials occurring during recovery. We conclude that low [K(+)](o) steepens early diastolic depolarization and increases its amplitude through an after-potential that results from an increased Ca(2+) load; allows the attainment of the threshold through Cs(+)-sensitive voltage oscillations which develop when the oscillatory zone is entered either by diastolic depolarization or by phase 3 repolarization; and causes voltage oscillations also at depolarized levels, but through a Cs(+)-insensitive different mechanism.     

Slow excitatory transmission in rat spinal dorsal horn and the effects of capsaicin

High intensity repetitive stimulation of a dorsal root elicited slow depolarization in more than half of the dorsal horn neurons examined in the rat spinal cord slice preparation. There was a significantly smaller group of neurons showing slow hyperpolarization as well. Slow depolarization was not observed when synaptic activity was blocked by perfusing the slice with a TTX- or a low-Ca2+ high-Mg2+ solution. This result is consistent with a presynaptic origin of the slow response. Capsaicin treatment of neonatal rats significantly reduced the incidence of slow depolarization, suggesting that the slow depolarization was generated by small diameter afferent fibres, probably unmyelinated afferents. DR-evoked slow depolarization and SP-induced depolarization were similar in several important aspects: a) Both responses caused depolarization and increased the excitability of dorsal horn neurons; b) They were frequently associated with similar membrane conductance changes; c) The size of both responses varied in parallel when the membrane potential was shifted over a wide range; d) Both responses were markedly reduced or abolished by an analogue of SP having antagonist properties, and by polyclonal and monoclonal antibodies to SP; e) The depression of the DR-elicited slow depolarization during and after the SP-induced depolarization suggested that SP and the natural transmitter for the DR-elicited slow depolarization were bound to the same receptors. The results suggest that SP or, SP-like peptide, is an agonist that mimics in some aspects the action on the natural transmitter for the slow depolarizing potential.     

CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm

As sperm prepare for fertilization, surface Ca(2+) channels must open to initiate required, Ca(2+)-mediated events. However, the molecular identity and functional properties of sperm Ca(2+) channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca(2+)] evoked by approximately 10-s applications of an alkaline high [K(+)] medium directly reports activity of voltage-gated Ca(2+) channels. Little response occurs if external Ca(2+) is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 micrometer Ni(2+) and more completely by 100-300 micrometer Cd(2+). They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 micrometer mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 micrometer) inhibits approximately 50%. GVIA also partially blocks transient, low voltage activated Ca(2+) currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni(2+) and Cd(2+) and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca(2+) channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca(V)2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca(2+) channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca(V)1, Ca(V)2.1, and Ca(V)3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 micrometer mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni(2+) suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca(2+) entry indicates that mature sperm possess a larger palette of voltage-gated Ca(2+) channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.     

Transient release of acetylcholine from Torpedo synaptosomes in response to prolonged depolarization

The release of ACh (acetylcholine) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay. A maintained depolarization by high KCl in the presence of Ca2+ triggered only a transient ACh release. It was shown that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response. The termination of release was probably caused by inactivation of voltage-dependent Ca2+ entry and rapid removal of intraterminal Ca2+ by a (Na+)0 dependent mechanism. It was found that exposure of the synaptosomes for a short period to low Ca2+-high K+ solutions greatly reduced the responses to Ca2+ reintroduction, as compared to the control release obtained when high K+ was applied in the presence of normal Ca2+. The response to Ca2+ reintroduction was measured following various times of preincubation with high K+ and low Ca2+; thus, an estimate of the time course of the inactivation of Ca2+ permeability during a depolarization could be made. A two component exponential kinetic was observed, with a rapid (tau = 3.6 s) and a slow phase (tau = 77 s). This inactivation was more pronounced when a higher KCl concentration was used to induce a greater depolarization. The presence of EGTA during the preincubation with high KCl greatly increased the response provoked by Ca2+ reintroduction, whereas increases in Ca2+ during the preincubation period caused proportional reduction in the subsequent response to Ca2+ reintroduction, indicating that the Ca2+ influx itself was involved in the inactivation process.     

Xestospongin B, a competitive inhibitor of IP3-mediated Ca2+ signalling in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells

Xestospongin B, a macrocyclic bis-1-oxaquinolizidine alkaloid extracted from the marine sponge Xestospongia exigua, was highly purified and tested for its ability to block inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. In a concentration-dependent manner xestospongin B displaced [(3)H]IP(3) from both rat cerebellar membranes and rat skeletal myotube homogenates with an EC(50) of 44.6 +/- 1.1 microM and 27.4 +/- 1.1 microM, respectively. Xestospongin B, depending on the dose, suppressed bradykinin-induced Ca(2+) signals in neuroblastoma (NG108-15) cells, and also selectively blocked the slow intracellular Ca(2+) signal induced by membrane depolarization with high external K(+) (47 mM) in rat skeletal myotubes. This slow Ca(2+) signal is unrelated to muscle contraction, and involves IP(3) receptors. In highly purified isolated nuclei from rat skeletal myotubes, Xestospongin B reduced, or suppressed IP(3)-induced Ca(2+) oscillations with an EC(50) = 18.9 +/- 1.35 microM. In rat myotubes exposed to a Ca(2+)-free medium, Xestospongin B neither depleted sarcoplasmic reticulum Ca(2+) stores, nor modified thapsigargin action and did not affect capacitative Ca(2+) entry after thapsigargin-induced depletion of Ca(2+) stores. Ca(2+)-ATPase activity measured in skeletal myotube homogenates remained unaffected by Xestospongin B. It is concluded that xestospongin B is an effective cell-permeant, competitive inhibitor of IP(3) receptors in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells.     

Beyond Bowditch: the convergence of cardiac chronotropy and inotropy

The ability of the heart to acutely beat faster and stronger is central to the vertebrate survival instinct. Released neurotransmitters, norepinephrine and epinephrine, bind to beta-adrenergic receptors (beta-AR) on pacemaker cells comprising the sinoatrial node, and to beta-AR on ventricular myocytes to modulate cellular mechanisms that govern the frequency and amplitude, respectively, of the duty cycles of these cells. While a role for sarcoplasmic reticulum Ca(2+) cycling via SERCA2 and ryanodine receptors (RyR) has long been appreciated with respect to cardiac inotropy, recent evidence also implicates Ca(2+) cycling with respect to chronotropy. In spontaneously beating primary sinoatrial nodal pacemaker cells, RyR Ca(2+) releases occurring during diastolic depolarization activate the Na(+)-Ca(2+) exchanger (NCX) to produce an inward current that enhances their diastolic depolarization rate, and thus increases their beating rate. beta-AR stimulation synchronizes RyR activation and Ca(2+) release to effect an increased beating rate in pacemaker cells and contraction amplitude in myocytes: in pacemaker cells, the beta-AR stimulation synchronization of RyR activation occurs during the diastolic depolarization, and augments the NCX inward current; in ventricular myocytes, beta-AR stimulation synchronizes the openings of unitary L-type Ca(2+) channel activation following the action potential, and also synchronizes RyR Ca(2+) releases following depolarization, and in the absence of depolarization, both leading to the generation of a global cytosolic Ca(i) transient of increased amplitude and accelerated kinetics. Thus, beta-AR stimulation induced synchronization of RyR activation (recruitment of additional RyRs to fire) and of the ensuing Ca(2+) release cause the heart to beat both stronger and faster, and is thus, a common mechanism that links both the maximum achievable cardiac inotropy and chronotropy.     

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.     

Electrophysiological response of rat atrial myocytes to acidosis

The effect of acidosis on the electrical activity of isolated rat atrial myocytes was investigated using the patch-clamp technique. Reducing the pH of the bathing solution from 7.4 to 6.5 shortened the action potential. Acidosis had no significant effect on transient outward or inward rectifier currents but increased steady-state outward current. This increase was still present, although reduced, when intracellular Ca(2+) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); BAPTA also inhibited acidosis-induced shortening of the action potential. Ni(2+) (5 mM) had no significant effect on the acidosis-induced shortening of the action potential. Acidosis also increased inward current at -80 mV and depolarized the resting membrane potential. Acidosis activated an inwardly rectifying Cl(-) current that was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which also inhibited the acidosis-induced depolarization of the resting membrane potential. It is concluded that an acidosis-induced increase in steady-state outward K(+) current underlies the shortening of the action potential and that an acidosis-induced increase in inwardly rectifying Cl(-) current underlies the depolarization of the resting membrane potential during acidosis.     

Effects of membrane depolarization and divalent cations on anaphylactic histamine secretion

The effects of membrane depolarization and divalent cations on histamine release have been studied in sensitized mast cells. Membrane potential of these cells has been measured with intracellular microelectrodes. Our results show that mast cells have a large resting potential (-61 +/- 12 mV) however they do not generate active membrane electrical responses when are depolarized by passing current through the recording microelectrode. High external K+ does not increase histamine release. Histamine secretion is supported by alkali-earth divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) but strongly inhibited by transition metals. Ca2+ concentrations above 1 mM inhibit histamine release, however, this effect is not mimicked by Sr2+ and Ba2+.     

Mitochondrial iPLA2 activity modulates the release of cytochrome c from mitochondria and influences the permeability transition

The mitochondrial Ca(2+)-independent phospholipase A(2) is activated during energy-dependent Ca(2+) accumulation under conditions where there is a sustained depression of the membrane potential. This activation is not dependent on induction of the mitochondrial permeability transition. Bromoenol lactone, which inhibits the phospholipase, is effective as an inhibitor of the transition, and this action can be overcome by low levels of exogenous free fatty acids. Apparently, activation of the Ca(2+)-independent phospholipase is a factor in the mechanisms by which depolarization and Ca(2+) accumulation promote opening of the permeability transition pore. Sustained activity of the Ca(2+)-independent phospholipase A(2) promotes rupture of the outer mitochondrial membrane and spontaneous release of cytochrome c on a time scale similar to that of apoptosis occurring in cells. However, more swelling of the matrix space must occur to provoke release of a given cytochrome c fraction when the enzyme is active, compared with when it is inhibited. Through its effects on the permeability transition and release of intermembrane space proteins, the mitochondrial Ca(2+)-independent phospholipase A(2) may be an important factor governing cell death caused by necrosis or apoptosis.     

豚鼠主动脉前庭自发性慢反应电位去极离子流的初步分析

为研究主动脉前庭自发慢反应电位的去极离充性质,利用豚鼠的离体以及心脏,常规玻璃微电极细胞内记录方法和离子通道组断剂,观测最大舒张电位（ＭＤＰ）、０相除极幅度（ＡＰＡ）、０相最大除极速度（Ｖｍａｘ）、４个自动除极速度（ＶＤＤ）、复极５０％（ＡＰＤ５０）和９０％（ＡＰＤ９０）的时间以及自发放电频率（ＲＰＦ）。结果发现：⑴０．５μｍｏｌ／Ｌ尼索地平（Ｎｉｓ）可使该慢电位的ＡＰＡ、Ｖｍａｘ、ＶＤＤ明显减小     

Ca2+ entry-dependent inactivation of L-type Ca current: a novel formulation for cardiac action potential models

Cardiac L-type Ca current (I(Ca,L)) is controlled not only by voltage but also by Ca(2+)-dependent mechanisms. Precise implementation of I(Ca,L) in cardiac action potential models therefore requires thorough understanding of intracellular Ca(2+) dynamics, which is not yet available. Here, we present a novel formulation of I(Ca,L) for action potential models that does not explicitly require the knowledge of local intracellular Ca(2+) concentration ([Ca(2+)](i)). In this model, whereas I(Ca,L) is obtained as the product of voltage-dependent gating parameters (d and f), Ca(2+)-dependent inactivation parameters (f(Ca): f(Ca-entry) and f(Ca-SR)), and Goldman-Hodgkin-Katz current equation as in previous studies, f(Ca) is not a instantaneous function of [Ca(2+)](i) but is determined by two terms: onset of inactivation proportional to the influx of Ca(2+) and time-dependent recovery (dissociation). We evaluated the new I(Ca,L) subsystem in the framework of the standard cardiac action potential model. The new formulation produced a similar temporal profile of I(Ca,L) as the standard, but with different gating mechanisms. Ca(2+)-dependent inactivation gradually proceeded throughout the plateau phase, replacing the voltage-dependent inactivation parameter in the LRd model. In typical computations, f declined to approximately 0.7 and f(Ca-entry) to approximately 0.1, whereas deactivation caused fading of I(Ca,L) during final repolarization. These results support experimental findings that Ca(2+) entering through I(Ca,L) is essential for inactivation. After responses to standard voltage-clamp protocols were examined, the new model was applied to analyze the behavior of I(Ca,L) when action potential was prolonged by several maneuvers. Our study provides a basis for theoretical analysis of I(Ca,L) during action potentials, including the cases encountered in long QT syndromes.