Voltage-clamped Rat Research Articles

Pharmacological and toxicological activity of RSD921, a novel sodium channel blocker

BackgroundRSD921, the R,R enantiomer of the kappa (k) agonist PD117,302, lacks significant activity on opioid receptors. MethodsThe pharmacological and toxicological actions were studied with reference to cardiovascular, cardiac, antiarrhythmic, toxic and local anaesthetic activity. ResultsIn rats, dogs and baboons, RSD921 dose-dependently reduced blood pressure and heart rate. In a manner consistent with sodium channel blockade it prolonged the PR and QRS intervals of the ECG. Furthermore, in rats and NHP, RSD921 increased the threshold currents for induction of extra-systoles and ventricular fibrillation (VFt), and prolonged effective refractory period (ERP). In rats, RSD921 was protective against arrhythmias induced by electrical stimulation and coronary artery occlusion. Application of RSD921 to voltage-clamped rat cardiac myocytes blocked sodium currents. RSD921 also blocked transient (ito) and sustained (IKsus) outward potassium currents, albeit with reduced potency relative to sodium current blockade. Sodium channel blockade due to RSD921 in myocytes and isolated hearts was enhanced under ischaemic conditions (low pH and high extracellular potassium concentration). When tested on the cardiac, neuronal and skeletal muscle forms of sodium channels expressed in Xenopus laevis oocytes, RSD921 produced equipotent tonic block of sodium currents, enhanced channel block at reduced pH (6.4) and marked use-dependent block of the cardiac isoform. RSD921 had limited but quantifiable effects in subacute toxicology studies in rats and dogs. Pharmacokinetic analyses were performed in baboons. Plasma concentrations producing cardiac actions in vivo after intravenous administration of RSD921 were similar to the concentrations effective in the in vitro assays utilized. ConclusionsRSD921 primarily blocks sodium currents, and possesses antiarrhythmic and local anaesthetic activity.

Systolic [Ca2+ ]i regulates diastolic levels in rat ventricular myocytes.

Key points For the heart to function as a pump, intracellular calcium concentration ([Ca2+]i) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood.The present study investigates the control of diastolic [Ca2+]i in rat ventricular myocytes.We show that diastolic [Ca2+]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+]i such that the time‐averaged [Ca2+]i remains constant.We report that diastolic [Ca2+]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole.The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i. The intracellular Ca concentration ([Ca2+]i) must be sufficently low in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+]i, in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane, are unclear. We investigated the effects on diastolic [Ca2+]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo‐3 in voltage clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+]i. This increase of [Ca2+]i was larger when SR function was impaired either by making the ryanodine receptor leaky (with caffeine or ryanodine) or by decreasing sarco/endoplasmic reticulum Ca‐ATPase activity with thapsigargin. The increase of diastolic [Ca2+]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient, such that there was no change of time‐averaged [Ca2+]i. Time‐averaged [Ca2+]i was increased by β‐adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+]i was a linear function of Ca entry per unit time. Diastolic and time‐averaged [Ca2+]i were decreased by decreasing the L‐type Ca current (with 50 μm cadmium chloride). We conclude that diastolic [Ca2+]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres that decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+]i. This identifies a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i.

Regulation of cardiac Ca 2+ channel by extracellular Na +

Hyponatremia is a predictor of poor cardiovascular outcomes during acute myocardial infarction and in the setting of preexisting heart failure [1]. There are no definitive mechanisms as to how hyponatremia suppresses cardiac function. In this report we provide evidence for direct down-regulation of Ca 2+ channel current in response to low serum Na +. In voltage-clamped rat ventricular myocytes or HEK 293 cells expressing the L-type Ca 2+ channel, a 15 mM drop in extracellular Na + suppressed the Ca 2+ current by ∼15%; with maximal suppression of ∼30% when Na + levels were reduced to 100 mM or less. The suppressive effects of low Na + on I Ca, in part, depended on the substituting monovalent species (Li +, Cs +, TEA +), but were independent of phosphorylation state of the channel and possible influx of Ca 2+ on Na +/Ca 2+ exchanger. Acidification sensitized the Ca 2+ channel current to Na + withdrawal. Collectively our data suggest that Na + and H + may interact with regulatory site(s) at the outer recesses of the Ca 2+ channel pore thereby directly modulating the electro-diffusion of the permeating divalents (Ca 2+, Ba 2+).

Epithelial acetylcholine – a new paradigm for cholinergic regulation of intestinal fluid and electrolyte transport

Epithelial acetylcholine – a new paradigm for cholinergic regulation of intestinal fluid and electrolyte transport

Nitric oxide can acutely modulate its biosynthesis through a negative feedback mechanism on l-arginine transport in cardiac myocytes

Nitric oxide (NO) plays a central role as a cellular signaling molecule in health and disease. In the heart, NO decreases the rate of spontaneous beating and the velocity and extent of shortening and accelerates the velocity of relengthening. Since the cationic amino acid l-arginine (l-Arg) is the substrate for NO production by NO synthases (NOS), we tested whether the transporters that mediate l-Arg import in cardiac muscle cells represent an intervention point in the regulation of NO synthesis. Electrical currents activated by l-Arg with low apparent affinity in whole cell voltage-clamped rat cardiomyocytes were found to be rapidly and reversibly inhibited by NO donors. Radiotracer uptake studies performed on cardiac sarcolemmal vesicles revealed the presence of high-affinity/low-capacity and low-affinity/high-capacity components of cationic amino acid transport that were inhibited by the NO donor S-nitroso-N-acetyl-dl-penicillamine. NO inhibited uptake in a noncompetitive manner with K(i) values of 275 and 827 nM for the high- and low-affinity component, respectively. Fluorescence spectroscopy experiments showed that millimolar concentrations of l-Arg initially promoted and then inhibited the release of endogenous NO in cardiomyocytes. Likewise, l-Arg currents measured in cardiac myocytes voltage clamped in the presence of 460 nM free intracellular Ca(2+), a condition in which a Ca-CaM complex should activate endogenous NO production, showed fast activation followed by inhibition of l-Arg transport. The NOS inhibitor N-nitro-l-arginine methyl ester, but not blockers of downstream reactions, specifically removed this inhibitory component. These results demonstrate that NO acutely regulates its own biosynthesis by modulating the availability of l-Arg via cationic amino acid transporters.

Voltage Dependence of Cardiac Excitation–Contraction Coupling

Excitation-contraction coupling in cardiac myocytes occurs by Ca2+-induced Ca2+ release, where L-type Ca2+ current evokes a larger sarcoplasmic reticulum (SR) Ca2+ release. The Ca2+-induced Ca2+ release amplification factor or gain (SR Ca2+ release/I(Ca)) is usually assessed by the V(m) dependence of current and Ca2+ transients. Gain rises at negative V(m), as does single channel I(Ca) (i(Ca)), which has led to the suggestion that the increases of i(Ca) amplitude enhances gain at more negative V(m). However, I(Ca) = NP(o) x i(Ca) (where NP(o) is the number of open channels), and NP(o) and i(Ca) both depend on V(m). To assess how i(Ca) and NP(o) separately influence Ca2+-induced Ca2+ release, we measured I(Ca) and junctional SR Ca2+ release in voltage-clamped rat ventricular myocytes using "Ca2+ spikes" (confocal microscopy). To vary i(Ca) alone, we changed [Ca2+](o) rapidly at constant test V(m) (0 mV) or abruptly repolarized from +120 mV to different V(m) (at constant [Ca2+](o)). To vary NP(o) alone, we altered Ca2+ channel availability by varying holding V(m) (at constant test V(m)). Reducing either i(Ca) or NP(o) alone increased excitation-contraction coupling gain. Thus, increasing i(Ca) does not increase gain at progressively negative test V(m). Such enhanced gain depends on lower NP(o) and reduced redundant Ca2+ channel openings (per junction) and a consequently smaller denominator in the gain equation. Furthermore, modest i(Ca) (at V(m) = 0 mV) may still effectively trigger SR Ca2+ release, whereas at positive V(m) (and smaller i(Ca)), high and well-synchronized channel openings are required for efficient excitation-contraction coupling. At very positive V(m), reduced i(Ca) must explain reduced SR Ca2+ release.

Differential effects of ceramide species on exocytosis in rat PC12 cells

Increases in several ceramide species have been shown by non-targeted lipid profiling (lipidomics) of the rat hippocampus after kainate lesions (Guan et al. FASEB J 20:1152-1161, 2006). This study was carried out to examine possible effects of ceramide species on exocytosis. Significant increase in membrane capacitance in voltage-clamped rat pheochromocytoma (PC12) cells, an indication of exocytosis, was detected immediately after external application of C2, C6, and C18 ceramide. In contrast, no increase in capacitance was found after addition of C16 and C20 ceramide, or DMSO vehicle. The effect of ceramide on exocytosis was dependent on the integrity of lipid rafts. Treatment of cells with the cholesterol binding agent/disruptor of lipid rafts, methyl beta cyclodextrin, prior to addition of C18 ceramide suppressed the increase in capacitance induced by this lipid species. The ability of C2, C6 and C18 ceramide to trigger exocytosis was confirmed using total internal reflection fluorescence microscopy (TIRFM) experiments. External application of these species caused an exponential decrease in the number of subplasmalemmal neuropeptide Y (NPY)-enhanced green fluorescence protein (EGFP) labeled vesicles, indicating exocytosis. Interestingly, C18 is also the ceramide species that showed the greatest increase in the rat hippocampus after kainate excitotoxicity. It is postulated that C18 ceramide might facilitate exocytosis of glutamate from damaged neurons, thus propagating neuronal injury.

L‐Arginine currents in rat cardiac ventricular myocytes

L-Arginine (L-Arg) is a basic amino acid that plays a central role in the biosynthesis of nitric oxide, creatine, agmantine, polyamines, proline and glutamate. Most tissues, including myocardium, must import L-Arg from the circulation to ensure adequate intracellular levels of this amino acid. This study reports novel L-Arg-activated inward currents in whole-cell voltage-clamped rat ventricular cardiomyocytes. Ion-substitution experiments identified extracellular L-Arg as the charge-carrying cationic species responsible for these currents, which, thus, represent L-Arg import into cardiac myocytes. This result was independently confirmed by an increase in myocyte nitric oxide production upon extracellular application of L-Arg. The inward movement of Arg molecules was found to be passive and independent of Na(2+), K(2+), Ca(2+) and Mg(2+). The process displayed saturation and membrane potential (V(m))-dependent kinetics, with a K(0.5) for l-Arg that increased from 5 mm at hyperpolarizing V(m) to 20 mm at +40 mV. L-Lysine and L-ornithine but not D-Arg produced currents with characteristics similar to that activated by L-Arg indicating that the transport process is stereospecific for cationic L-amino acids. L-Arg current was fully blocked after brief incubation with 0.2 mm N-ethylmaleimide. These features suggest that the activity of the low-affinity, high-capacity CAT-2A member of the y(2+) family of transporters is responsible for L-Arg currents in acutely isolated cardiomyocytes. Regardless of the mechanism, we hypothesize that a low-affinity arginine transport process in heart, by ensuring substrate availability for sustained NO production, might play a cardio-protective role during catabolic states known to increase Arg plasma levels severalfold.

Increasing Ryanodine Receptor Open Probability Alone Does Not Produce Arrhythmogenic Calcium Waves

Diastolic waves of Ca 2+ release have been shown to activate delayed afterdepolarizations as well as some cardiac arrhythmias. The aim of this study was to investigate whether increasing ryanodine receptor open probability alone or in the presence of β-adrenergic stimulation produces diastolic Ca release from the sarcoplasmic reticulum (SR). When voltage-clamped rat ventricular myocytes were exposed to caffeine (0.5 to 1.0 mmol), diastolic Ca 2+ release was seen to accompany the first few stimuli but was never observed in the steady state. We attribute the initial phase of diastolic Ca 2+ release to a decrease in the threshold SR Ca 2+ content required to activate Ca 2+ waves and its subsequent disappearance to a decrease of SR content below this threshold. Application of isoproterenol (1 μmol/L) increased the amplitude of the systolic Ca 2+ transient and also the SR Ca 2+ content but did not usually produce diastolic Ca 2+ release. Subsequent addition of caffeine, however, resulted in diastolic Ca 2+ release. We estimated the time course of recovery of SR Ca 2+ content following recovery from emptying with a high (10 mmol/L) concentration of caffeine. Diastolic Ca 2+ release recommenced only when SR content had increased back to its final level. We conclude that increasing ryanodine receptor open probability alone does not produce arrhythmogenic diastolic Ca 2+ release because of the accompanying decrease of SR Ca 2+ content. β-Adrenergic stimulation increases SR content and thereby allows the increased ryanodine receptor open probability to produce diastolic Ca 2+ release. The implications of these results for arrhythmias associated with abnormal ryanodine receptors are discussed.

Simultaneous Release of Glutamate and Acetylcholine from Single Magnocellular “Cholinergic” Basal Forebrain Neurons

Basal forebrain (BF) neurons provide the principal cholinergic drive to the hippocampus and cortex. Their degeneration is associated with the cognitive defects of Alzheimer's disease. Immunohistochemical studies suggest that some of these neurons contain glutamate, so might also release it. To test this, we made microisland cultures of single BF neurons from 12- to 14-d-old rats. Over 1-8 weeks in culture, neuronal processes made autaptic connections onto the neuron. In 34 of 36 cells tested, a somatically generated action potential was followed by a short-latency EPSC that was blocked by 1 mM kynurenic acid, showing that they released glutamate. To test whether the same neuron also released acetylcholine, we placed a voltage-clamped rat myoball expressing nicotinic receptors in contact with a neurite. In six of six neurons tested, the glutamatergic EPSC was accompanied by a nicotinic (hexamethonium-sensitive) myoball current. Stimulation of the M2-muscarinic presynaptic receptors (characterized using tripitramine and pirenzepine) produced a parallel inhibition of autaptic glutamatergic and myoball nicotinic responses; metabotropic glutamate receptor stimulation produced similar but less consistent and weaker effects. Atropine enhanced the glutamatergic EPSCs during repetitive stimulation by 25 +/- 6%; the anti-cholinesterase neostigmine reduced the train EPSCs by 37 +/- 6%. Hence, synaptically released acetylcholine exerted a negative-feedback inhibition of coreleased glutamate. We conclude that most cholinergic basal forebrain neurons are capable of releasing glutamate as a cotransmitter and that the release of both transmitters is subject to simultaneous feedback inhibition by synaptically released acetylcholine. This has implications for BF neuron function and for the use of cholinesterase inhibitors in Alzheimer's disease.

Diversity of atrial local Ca2+ signalling: evidence from 2‐D confocal imaging in Ca2+‐buffered rat atrial myocytes

Atrial myocytes, lacking t-tubules, have two functionally separate groups of ryanodine receptors (RyRs): those at the periphery colocalized with dihydropyridine receptors (DHPRs), and those at the cell interior not associated with DHPRs. We have previously shown that the Ca(2+) current (I(Ca))-gated central Ca(2+) release has a fast component that is followed by a slower and delayed rising phase. The mechanisms that regulate the central Ca(2+) releases remain poorly understood. The fast central release component is highly resistant to dialysed Ca(2+) buffers, while the slower, delayed component is completely suppressed by such exogenous buffers. Here we used dialysis of Ca(2+) buffers (EGTA) into voltage-clamped rat atrial myocytes to isolate the fast component of central Ca(2+) release and examine its properties using rapid (240 Hz) two-dimensional confocal Ca(2+) imaging. We found two populations of rat atrial myocytes with respect to the ratio of central to peripheral Ca(2+) release (R(c/p)). In one population ('group 1', approximately 60% of cells), R(c/p) converged on 0.2, while in another population ('group 2', approximately 40%), R(c/p) had a Gaussian distribution with a mean value of 0.625. The fast central release component of group 2 cells appeared to result from in-focus Ca(2+) sparks on activation of I(Ca). In group 1 cells intracellular membranes associated with t-tubular structures were never seen using short exposures to membrane dyes. In most of the group 2 cells, a faint intracellular membrane staining was observed. Quantification of caffeine-releasable Ca(2+) pools consistently showed larger central Ca(2+) stores in group 2 and larger peripheral stores in group 1 cells. The R(c/p) was larger at more positive and negative voltages in group 1 cells. In contrast, in group 2 cells, the R(c/p) was constant at all voltages. In group 1 cells the gain of peripheral Ca(2+) release sites (Delta[Ca(2+)]/I(Ca)) was larger at -30 than at +20 mV, but significantly dampened at the central sites. On the other hand, the gains of peripheral and central Ca(2+) releases in group 2 cells showed no voltage dependence. Surprisingly, the voltage dependence of the fast central release component was bell-shaped and similar to that of I(Ca) in both cell groups. Removal of extracellular Ca(2+) or application of Ni(2+) (5 mM) suppressed equally I(Ca) and Ca(2+) release from the central release sites at +60 mV. Depolarization to +100 mV, where I(Ca) is absent and the Na(+)-Ca(2+) exchanger (NCX) acts in reverse mode, did not trigger the fast central Ca(2+) releases in either group, but brief reduction of [Na(+)](o) to levels equivalent to [Na(+)](i) facilitated fast peripheral and central Ca(2+) releases in group 2 myocytes, but not in group 1 myocytes. In group 2 cells, long-lasting (> 1 min) exposures to caffeine (10 mM) or ryanodine (20 microM) significantly suppressed I(Ca)-triggered central and peripheral Ca(2+) releases. Our data suggest significant diversity of local Ca(2+) signalling in rat atrial myocytes. In one group, I(Ca)-triggered peripheral Ca(2+) release propagates into the interior triggering central Ca(2+) release with significant delay. In a second group of myocytes I(Ca) triggers a significant number of central sites as rapidly and effectively as the peripheral sites, thereby producing more synchronized Ca(2+) releases throughout the myocytes. The possible presence of vestigial t-tubules and larger Ca(2+) content of central sarcoplasmic reticulum (SR) in group 2 cells may be responsible for the rapid and strong activation of central release of Ca(2+) in this subset of atrial myocytes.

Control of dual isoforms of Ca2+ release channels in muscle

Here we compare excitation-contraction coupling in single muscle cells of frogs and rats. Because amphibians have isoform 3 (or 3) of the ryanodine receptor/Ca2+ release channel, in addition to 1 (alpha), which is also present in the mammal, any extra feature present in the frog may in principle be attributed to isoform 3. Ca2+ release under voltage clamp depolarization has a peak and a steady phase in both taxonomic classes, but the peak is more marked in the frog, where the ratio of amplitudes of the two phases is voltage-dependent. This dependence is a hallmark of CICR. Confocal imaging identified Ca2+ sparks in the frog, but not in the voltage-clamped rat cells. Because Ca2+ sparks involve CICR both observations indicate that the contribution of CICR is minor or null in the mammal. The "couplon" model well accounts for observations in the frog, but assumes a structure that we now know to be valid only for the rat. A revised model is proposed, whereby the isoform 3 channels, located parajunctionally, are activated by CICR and contribute its characteristic global and local features. Several issues regarding the roles of different channels remain open to further study.

Spatiotemporal characteristics of junctional and nonjunctional focal Ca2+ release in rat atrial myocytes.

Atrial myocytes have two functionally separate Ca2+ release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the Ca2+ channels of surface membrane and those in central SR not associated with Ca2+ channels. Recently, we have reported on the gating of these two different Ca2+ release sites by Ca2+ current. In the present study, we report on the spatiotemporal properties of focal Ca2+ releases (sparks) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid 2-dimensional (2-D) confocal Ca2+ imaging. Peripheral and central sparks were similar in size and release time (approximately 300 000 Ca2+ ions for congruent with 12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd2+ and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had approximately 5-fold higher frequency, approximately 2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2 to 5 unitary focal releases) aligned longitudinally and occurred more frequently at the center. The diversity of peripheral and central sparks with respect to shape, frequency, and speed of spatial development and decay is consistent with regional ultrastructural heterogeneity of SR. The retarded dissipation of central atrial sparks, and high prevalence of compound sparks in cell center may be critical in facilitating the propagation of Ca2+ waves in atrial myocytes lacking t-tubular system and provide the atrial myocytes with functional Ca2+ signaling diversity. The full text of this article is available at http://www.circresaha.org.

Effects of FPL 64176 on Ca transients in voltage-clamped rat ventricular myocytes.

1. The L-type Ca channel agonist FPL 64176 increased the amplitude of both Ca currents and Ca transients elicited from isolated voltage clamped rat ventricular myocytes far more than it increased the rate of rise of the Ca transients. Consequently, the gain function relating the amplitude of peak Ca current to Ca transient rate of rise was greatly reduced at all potentials. 2. Furthermore, an increase in this gain function normally observed at negative potentials is abolished by FPL 64716. 3. Despite slowing the rate of decline of Ca transients, FPL 64176, at the concentration of 1 microM used throughout, had no direct effect on sarcoplasmic reticulum (SR) Ca uptake or release using isolated cardiac membranes. 4. Arguments based on results presented here and elsewhere suggest that decreased gain was not due to increased ryanodine receptor adaptation or inactivation, to decreased L-type single channel current, to decreased SR Ca content, or to decreased synchronization of release. Decreased gain instead appears to reflect a form of decrease in coupling efficiency due either to differential effects of long openings on whole cell currents as opposed to the Ca release the long openings trigger or to some compensatory mechanism activated by the increased Ca trigger or resting free [Ca(2+)](i). 5. Abolition by FPL 64176 of the increased gain normally seen at negative potentials rendered it impossible to confirm or refute the claim that a single Ca ion suffices to activate ryanodine receptors.

Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current.

The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca(2+) content and systolic Ca(2+) are controlled when Ca(2+) entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Increasing external Ca(2+) concentration ([Ca(2+)](o)) from 1 to 2 mmol/L increased the amplitude of the systolic Ca(2+) transient with no effect on SR Ca(2+) content. This constancy of SR content is shown to result because the larger Ca(2+) transient activates a larger Ca(2+) efflux from the cell that balances the increased influx. Decreasing [Ca(2+)](o) to 0.2 mmol/L decreased systolic Ca(2+) but produced a small increase of SR Ca(2+) content. This increase of SR Ca(2+) content is due to a decreased release of Ca(2+) from the SR resulting in decreased loss of Ca(2+) from the cell. An increase of [Ca(2+)](o) has two effects: (1) increasing the fraction of SR Ca(2+) content, which is released on depolarization and (2) increasing Ca(2+) entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca(2+) transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca(2+)](o) than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca(2+) entry and greater fractional release of Ca(2+) is an important factor in regulating excitation-contraction coupling.

ATP and beta-adrenergic stimulation enhance voltage-gated K current inactivation in brown adipocytes.

Sympathetic activation of brown fat thermogenesis stimulates adrenergic and purinergic receptors. We examined the effects of extracellular ATP and beta-adrenergic agonists on voltage-activated K currents (IKv) in voltage-clamped rat brown adipocytes. ATP or the beta-adrenergic agonist isoproterenol increased the development of IKv inactivation during depolarizing voltage steps in perforated patch-clamped cells. The effects on inactivation developed slowly in the presence of agonist and continued to increase for long times following agonist washout. 8-bromo-cAMP or forskolin had similar effects on IKv inactivation. Development of IKv inactivation during depolarizations was consistently enhanced by ATP or beta-adrenergic stimulation in perforated-patch voltage-clamped cells but was not altered by these agents in whole cell recordings, suggesting that cytosolic factors are necessary for inactivation modulation. In either recording configuration, ATP or isoproterenol shifted the activation voltage dependence of IKv to more negative potentials, indicating the activation effect is mediated by a different pathway. Since both P2 purinergic and beta-adrenergic signaling pathways generate fatty acids, we tested whether fatty acids could reproduce these modulations of IKv. Linoleic or arachidonic acid applied in whole cell recordings had effects similar to those of ATP or isoproterenol in perforated-patch experiments. These results are consistent with the possibility that beta-adrenergic and P2 receptor stimulation modulate IKv through generation of fatty acids.

The effect of acidosis on systolic Ca2+ and sarcoplasmic reticulum calcium content in isolated rat ventricular myocytes.

We have investigated the mechanisms responsible for the changes of systolic Ca2+ that occur in voltage-clamped rat ventricular myocytes during acidosis produced by application of the weak acid butyrate (30 mM). Intracellular pH regulation was inhibited with dimethylamiloride (bicarbonate-free solution). The application of butyrate produced an intracellular acidification of 0.33 pH units. This was accompanied by a decrease in systolic Ca2+ to about 50% of control. However, within 2 min, systolic Ca2+ returned to control levels. The decrease in systolic Ca2+ was accompanied by a decrease in the Na+-Ca2+ exchange current observed on repolarisation so that the calculated Ca2+ efflux on Na+-Ca2+ exchange was less than the entry on the L-type Ca2+ current. The magnitude of the Na+-Ca2+ exchange current recovered along with systolic Ca2+ until it equalled the Ca2+ entry on the L-type Ca2+ current. From the measurement of Ca2+ fluxes, it was calculated that, during acidosis, the cell gains 121.6+/-16.2 micromol l(-1) of Ca2+. This is equal to the measured increase of sarcoplasmic reticulum (SR) calcium content obtained by applying caffeine (20 mM) and integrating the resulting Na+-Ca2+ exchange current. We conclude that the recovery of the amplitude of the systolic Ca2+ transient is due to decreased SR calcium release, resulting in reduced Ca2+ efflux from the cell leading to increased SR calcium content.

Purification and amino acid sequence of a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria nigriventer which inhibits NMDA-evoked currents in rat hippocampal neurones

A new insecticidal toxin Tx4(5-5) was isolated from the fraction PhTx4 of the venom of the spider Phoneutria nigriventer by reverse phase high performance liquid chromatography (HPLC) and anion exchange HPLC. The complete amino acid sequence determined by automated Edman degradation showed that Tx4(5-5) is a single chain polypeptide composed of 47 amino acid residues, including 10 cysteines, with a calculated molecular mass of 5175 Da. Tx4(5-5) shows 64% of sequence identity with Tx4(6-1), another insecticidal toxin from the same venom. Tx4(5-5) was highly toxic to house fly ( Musca domestica), cockroach ( Periplaneta americana) and cricket ( Acheta domesticus ), producing neurotoxic effects (knock-down, trembling with unco-ordinated movements) at doses as low as 50 ng/g (house fly), 250 ng/g (cockroach) and 150 ng/g (cricket). In contrast, intracerebroventricular injections (30 μg) into mice induced no behavioural effects. Preliminary electrophysiological studies carried out on whole-cell voltage-clamped rat hippocampal neurones indicated that Tx4(5-5) (at 1 μ M) reversibly inhibited the N-methyl- D-aspartate-subtype of ionotropic glutamate receptor, while having little or no effect on kainate-, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid- or γ-aminobutyric acid-activated currents.

One calcium ion may suffice to open the tetrameric cardiac ryanodine receptor in rat ventricular myocytes.

1. The release of Ca2+ from sarcoplasmic reticulum in response to Ca2+ entering through L-type Ca2+ channels was studied in isolated voltage clamped rat ventricular myocytes at room temperature using the fluorescent Ca2+ indicators fluo-3 and Oregon Green 488 Bapta 5N. 2. Depolarizations to positive potentials elicited fluo-3 Ca2+ transients with rates of rise that were linearly related to the magnitude of the peak measured Ca2+ current in the presence of Cs+-containing pipette solutions. 3. Further experiments utilizing prepulses to preactivate a constant number of channels also revealed a linear relationship between the Ca2+ transient rate of rise and the magnitude of entering Ca2+ current at positive potentials. Under these conditions as well, the maximal rates of rise of global myoplasmic Ca2+ transients were due primarily to Ca2+ release from the sarcoplasmic reticulum as revealed by effects of ryanodine and caffeine on the Ca2+ transients. Using such prepulses, linearity between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was found under a variety of pulse protocols. 4. Using one such pulse protocol, linearity between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was also found when Ca2+ currents assessed at one potential were reduced in magnitude during the onset of block by application of Co2+. Using the same pulse protocol, linearity between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was also found when use of Cs+ was avoided by blocking K+ currents with extracellular TEA and 4-aminopyridine. Linearity in the relationship between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was also found when Ca2+ transients were measured using the low affinity Ca2+ indicator Oregon Green 488 Bapta 5N in place of fluo-3. 5. These results appear to indicate that the cardiac ryanodine receptor is capable of being activated by only one calcium ion. Alternative interpretations of the data are discussed.

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells.

Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca2+ current (ICa) and exocytosis. The dependency of exocytosis on ICa evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of ICa, implying the existence of a Ca2+-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca2+ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca2+ concentration. Pertussis toxin blocked the inhibitory effects of ATP on ICa and exocytosis. Although RB-2, a P2Y antagonist, blocked the inhibitory effect of ATP on ICa, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited ICa via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of ICa by activation of a pertussis-toxin-sensitive G-protein linked to P2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis.