Opening Of Na Research Articles

Lead exerts a depression of neurotransmitter release through a blockade of voltage dependent calcium channels in chromaffin cells

The present work, using chromaffin cells of bovine adrenal medullae (BCCs), aims to describe what type of ionic current alterations induced by lead (Pb2+) underlies its effects reported on synaptic transmission. We observed that the acute application of Pb2+ lead to a drastic depression of neurotransmitters release in a concentration-dependent manner when the cells were stimulated with both K+ or acetylcholine, with an IC50 of 119,57 μM and of 5,19 μM, respectively. This effect was fully recovered after washout. Pb2+ also blocked calcium channels of BCCs in a time- and concentration-dependent manner with an IC50 of 6,87 μM. This blockade was partially reversed upon washout. This compound inhibited the calcium current at all test potentials and shows a shift of the I-V curve to more negative values of about 8 mV. The sodium current was not blocked by acute application of high Pb2+ concentrations. Voltage-dependent potassium current was also shortly affected by high Pb2+. Nevertheless, the calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 24,49 μM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to Ca2+-activated K+-channels (BK) instead a direct linking to these channels. Under current-clamp conditions, BCCs exhibit a resting potential of −52.7 mV, firing spontaneous APs (1–2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of K+ channels. In spite of the effect on ionic channels exerted by Pb2+, we found that Pb2+ didn’t alter cellular excitability, no modification of the membrane potential, and no effect on action potential firing. Taken together, these results point to a neurotoxic action evoked by Pb2+ that is associated with changes in neurotransmitter release by blocking the ionic currents responsible for the calcium influx.

Mexiletine: Antiarrhythmic mechanisms, emerging clinical applications and mortality.

Mexiletine, a class Ib antiarrhythmic drug, exhibits its major antiarrhythmic effect via inhibition of the fast and late Na+ currents in myocardial tissues that are dependent on the opening of Na+ channels for their excitation. Through a comprehensive examination of mexiletine's therapeutic benefits and potential risks, we aim to provide valuable insights that reinforce its role as a vital therapeutic option for patients with ventricular arrhythmias, long QT syndrome, and other heart rhythm disorders. This review will highlight the current understandings of the antiarrhythmic effects and rationales for recent off-label use and address the mortality and proarrhythmic effects of mexiletine utilizing published basic and clinical studies over the past five decades.

Micromolar concentrations of Zn2+ depress cellular excitability through a blockade of calcium current in rat adrenal slices

The present work, using chromaffin cells in rat adrenal slices (RCCs), aims to describe what type of ionic current alterations induced by zinc underlies their effects reported on synaptic transmission. Thus, Zn2+ blocked calcium channels of RCCs in a time- and concentration-dependent manner with an IC50 of 391 μM. This blockade was partially reversed upon washout and was greater at more depolarizing holding potentials (i.e. 32 ± 5% at −110 mV, and 43 ± 6% at -50 mV, after 5 min perfusion). In ω-toxins-sensitive calcium channels (N-, P- and Q-types), Zn2+caused a lower blockade of ICa, 33.3%, than in ω-toxins-resistant ones (L-type, 55.3%; and R-type, 90%). This compound inhibited calcium current at all test potentials and shows a shift of the I-V curve to more depolarized values of about 10 mV. The sodium current was not blocked by acute application of high Zn2+concentrations. Voltage-dependent potassium current was marginally affected by high Zn2+ concentrations showing no concentration-dependence. Nevertheless, calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 453 μM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to BK channels. Under current-clamp conditions, RCCs exhibit a resting potential of -50.7 mV, firing spontaneous APs (1–2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of voltage and Ca2+-activated K+-channels (BK). We found that the blockade of these ionic currents by Zn2+ led to a drastic alteration of cellular excitability with a depolarization of the membrane potential, the slowdown and broadening of the APs and the severe reduction of the after hyperpolarization (AHP) which led to a decrease in the APs firing frequency. Taken together, these results point to a neurotoxic action evoked by zinc that is associated with changes to cellular excitability by blocking the ionic currents responsible for both the neurotransmitter release and the action potentials firing.

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na+, Ca2+) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca2+ at the expense of the Na+ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca2+ extrusion and thus to the recovery from intracellular Ca2+ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na+ to 1 Ca2+, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na+ concentration or minor depolarisations switch it into the “reverse mode” (Ca2+ import/Na+ export). Notably, transient Na+ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na+-permeable ion channels in response to neuronal activity. Activity-related Na+ transients result in NCX reversal, which mediates Ca2+ influx from the extracellular space, thereby generating astrocyte Ca2+ signalling independent from InsP3-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca2+ overload, while dampening Na+ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na+-driven, reverse NCX-mediated astrocyte Ca2+ signals are involved neurone-glia interaction. Na+ transients, translated by the NCX into Ca2+ elevations, thereby emerge as a new signalling pathway in astrocytes.

Excitation of murine cardiac myocytes by nanosecond pulsed electric field.

Opening of voltage-gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. Fluorescent imaging of optical APs (FluoVolt) and Ca2+ -transients (Fluo-4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200-nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin-sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P < 0.001). Inhibition of l-type calcium and sodium-calcium exchanger currents reduced the SSD amplitude and increased the AP threshold ( P < 0.05). The threshold for Ca 2+ -transients (1.40 ± 0.04 kV/cm) was not significantly affected by a tetrodotoxin-verapamil cocktail, suggesting the activation of a Ca 2+ entry pathway independent from the opening of Na + or Ca 2+ voltage-gated channels. Removal of external Ca 2+ decreased the SSD amplitude ( P = 0.004) and blocked Ca 2+ -transients but not APs. The incidence of transient afterdepolarization waves was decreased by verapamil and by removal of external Ca 2+ ( P = 0.002). The study established that nsPEF stimulation caused calcium entry into cardiac myocytes (including routes other than voltage-gated calcium channels) and SSD. Tetrodotoxin-sensitive APs were mediated by SSD, whose amplitude depended on the calcium entry. Plasma membrane electroporation was the most likely primary mechanism of SSD with additional contribution from l-type calcium and sodium-calcium exchanger currents.

Axonal excitability and conduction alterations caused by levobupivacaine in rat.

In this study, effects of the long-acting amide-type local anesthetic levobupivacaine on axonal conduction and excitability parameters of the rat sciatic nerve were thoroughly examined both in vitro and in vivo. In order to deduce its effects on isolated nerve conduction, compound nerve action potential (CNAP) recordings were performed using the suction method over sciatic nerves of Wistar rats before and after administration of 0.05 % (1.7 mmol L-1) levobupivacaine. Levobupivacaine caused complete CNAP area and amplitude depression by blocking conduction in a time-dependent manner. To assess the influence of levobupivacaine on in vivo excitability properties, threshold-tracking (TT) protocols were performed at sciatic nerves of rats injected with perineural 0.05 % (1.7 mmol L-1) levobupivacaine or vehicle alone. Charge-duration TT results revealed that levobupivacaine increases the rheobase and decreases the strength-duration time constant, suggesting interference of the anesthetic with the opening of Na+ channels. Twenty and 40 % threshold electrotonus curves were found for both groups to follow the same paths, suggesting no significant effect of levobupivacaine on K+ channels for either the fastest or relatively slow conducting fibers. Current-threshold relationship results revealed no significant effect on axonal rectifying channels. However, according to the results of the recovery cycle protocol yielding the pattern of excitability changes following the impulse, potential deviation was found in the recovery characteristics of Na+ channels from the absolute refractory period. Consequently, conduction blockage caused by levobupivacaine may not be due to the passive (capacitive) properties of axon or the conductance of potassium channels but to the decrease in sodium channel conductance.

Different contributions of calcium channel subtypes to electrical excitability of chromaffin cells in rat adrenal slices.

We characterized the ionic currents underlying the cellular excitability and the Ca(2+) -channel subtypes involved in action potential (AP) firing of rat adrenal chromaffin cells (RCCs) preserved in their natural environment, the adrenal gland slices, through the perforated patch-clamp recording technique. RCCs prepared from adrenal slices exhibit a resting potential of -54 mV, firing spontaneous APs (2-3 spikes/s) generated by the opening of Na(+) and Ca(2+) -channels, and terminated by the activation of voltage and Ca(2+) -activated K(+) -channels (BK). Ca(2+) influx via L-type Ca(2+) -channels is involved in reaching threshold potential for AP firing, and is responsible for activation of BK-channels contributing to AP-repolarization and afterhyperpolarization, whereas P/Q-type Ca(2+) -channels are involved only in the repolarization phase. BK-channels carry total outward current during AP-repolarization. Blockade of L-type Ca(2+) -channels reduces BK-current ~60%, whereas blockade of N- or P/Q-type produces little effect. This study demonstrates that Ca(2+) influx through L-type Ca(2+) -channels plays a key role in modulating the threshold potential from RCCs in situ. This study demonstrates that Ca(2+) influx through L-type Ca(2+) channels plays a key role in modulating the threshold potential for action potential firing and activating BK channels contributing to repolarization and afterhyperpolarization from rat adrenal chromaffin cells in situ.

The arrhythmogenic consequences of increasing late INa in the cardiomyocyte

This review presents the roles of cardiac sodium channel NaV1.5 late current (late INa) in generation of arrhythmic activity. The assumption of the authors is that proper Na(+) channel function is necessary to the maintenance of the transmembrane electrochemical gradient of Na(+) and regulation of cardiac electrical activity. Myocyte Na(+) channels' openings during the brief action potential upstroke contribute to peak INa and initiate excitation-contraction coupling. Openings of Na(+) channels outside the upstroke contribute to late INa, a depolarizing current that persists throughout the action potential plateau. The small, physiological late INa does not appear to be critical for normal electrical or contractile function in the heart. Late INa does, however, reduce the net repolarizing current, prolongs action potential duration, and increases cellular Na(+) loading. An increase of late INa, due to acquired conditions (e.g. heart failure) or inherited Na(+) channelopathies, facilitates the formation of early and delayed afterpolarizations and triggered arrhythmias, spontaneous diastolic depolarization, and cellular Ca(2+) loading. These in turn increase the spatial and temporal dispersion of repolarization time and may lead to reentrant arrhythmias.

A Mutation in Telethonin Alters Nav1.5 Function

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na(+) channels in excitable cells results in influx of Na(+) and cellular depolarization. The function of Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na(+) channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Na(v)1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.

Sources of axonal calcium loading during in vitro ischemia of rat dorsal roots

A detailed understanding of injury mechanisms in peripheral nerve fibers will help guide successful design of therapies for peripheral neuropathies. This study was therefore undertaken to examine the ionic mechanisms of Ca2+ overload in peripheral myelinated fibers subjected to chemical inhibition of energy metabolism. Myelinated axons from rat dorsal roots were co-loaded with Ca2+-sensitive (Oregon Green BAPTA-1) and Ca2+-insensitive (Alexa Fluor 594) dextran-conjugated fluorophores and imaged using confocal laser scanning microscopy. Axoplasmic regions were clearly outlined by the Ca2+-insensitive dye, from which axonal Ca2+-dependent fluorescence changes (FCa.ax) were measured. Block of Na+-K+ ATPase (ouabain), opening of Na+ channels (veratridine), and inhibiting energy metabolism (iodoacetate + NaN3) caused a rapid rise in FCa.ax to 96% above control after 30 min. Chemical ischemia (iodoacetate + NaN3) caused a more gradual increase in FCa.ax (54%), which was almost completely dependent on bath Ca2+, indicating that most of the Ca2+ accumulation occurred via influx across the axolemma. Na+ channel block (tetrodotoxin) reduced ischemic FCa.ax rise (14%); however, inhibition of L-type Ca2+ channels (nimodipine) had no effect (60%). In contrast, Na+-Ca2+ exchange inhibition (KB-R7943) significantly reduced ischemic FCa.ax rise (18%). Together our results indicate that the bulk of Ca2+ overload in injured peripheral myelinated axons occurs via reverse Na+-Ca2+ exchange, driven by axonal Na+ accumulation through voltage-gated tetrodotoxin-sensitive Na+ channels. This mechanism may represent a viable therapeutic target for peripheral neuropathies.

Similarities of SP-, NKA- and NKB-induced currents in rat dorsal root ganglion neurons

Tachykinin (TK; including substance P (SP), neurokinin A (NKA) and neurokinin B (NKB))-induced currents ( I TKs) were studied in freshly isolated rat dorsal root ganglion (DRG) neurons using whole-cell patch clamp recording and repatch techniques. All the three I TKs manifested features of fast activating kinetics, such as short latency and fast τ on and τ off, and very slow desensitization. The concentration–response relationships for TKs show: (1) compared with the concentration–response curve for NKA, the curve for NKB shifted upwards, while that for SP shifted downwards; (2) the EC 50 values for NKB-, NKA- and SP-activated currents were very close to each other. The I– V curves for the three TKs were basically linear and arrayed in the order of NKB>NKA>SP; the reversal potentials for the three I TKs were all around +15 mV. Replacement of NaCl in the external solution by equimolar N-methyl- d-glucamine (NMDG) attenuated both NKA- and NKB-activated currents markedly, as it was the case with SP-activated current caused by the opening of Na + preferring non-selective cation channels observed in our previous work. All the three TKs proved to inhibit coexistent GABA A receptor-mediated current ( I GABA); this effect was removed by intracellular dialysis of GDP-β-S or H-7. However, these drugs did not block the SP-, NKA- and NKB-activated currents at all, which indicated that I TKs were G-protein independent. In short, the responses of rat DRG neurons to SP, NKA and NKB were similar in essence, although the amplitudes of currents induced by the same concentration of the three TKs were different. Taking the results of this study and our previous studies together, we hypothesized that SP, NKA and NKB may induce inward currents through undiscovered channels that are associated with tachykinins receptors (NK1R, NK2R, NK3R, which have already been cloned), but independent of G-protein coupling and remains to be further investigated.

Three kinds of current in response to substance P in bullfrog DRG neurons

In response to SP applied externally, neurons freshly isolated from bullfrog dorsal root ganglion (DRG) showed three kinds of current ( I SP), i.e. slow, fast and moderately activating I SPs. All the three kinds of I SP were inward currents, and were completely blocked by either peptide antagonist of SP receptor spantide or non-peptide antagonist of SP receptor WIN51708. The slow activating I SP showed slow kinetic features. Replacement of NaCl in external solution by NMDG had no effect on this kind of I SP, while Ba 2+ abolished it almost completely, thus the ionic mechanism underlying slow activating I SP was deduced to be the closure of K + channels. The fast activating I SP in bullfrog DRG neurons, just as in rat DRG neurons, was proved to be caused by the opening of Na + preferring non-selective cation channel, for it was abolished almost completely by replacement of NaCl in external solution with equimolar NMDG. The moderately activating I SP was similar to the fast activating I SP in current configuration, however, its kinetic characteristics lay between those of fast and slow activating I SPs. Either NMDG or Ba 2+ suppressed this kind of I SP partially. Therefore the moderately activating I SP might be mediated by non-selective cation channel. We used repatch technique to explore the intracellular mechanism underlying the three kinds of I SP and found that the three kinds of I SP were caused by the activity of either G-protein coupled channel (slow activating I SP) or directly opened channel (fast activating I SP) or both (moderately activating I SP ).

RESPONSES OF THE SHORE CRAB, CARCINUS MAENAS (DECAPODA, BRACHYURA), TO AN ACUTE DOSE OF CYPERMETHRIN

The shore, or green, crab Carcinus maenas (Linnaeus, 1758), is an endemic species of northern Europe. It has become an important species internationally due to its introduction to foreign waters where it has become so successful that it is now considered one of the most serious marine pests in the U.S.A. and Australia (Geller et al., 1997). Carcinus is used in pollution studies, not only because of the widespread and topical nature of the animal, but also because it is easy to maintain in the laboratory, and at high stocking densities. Cypermethrin is a synthetic pyrethroid used as an insecticide and also to treat salmon against the infestation of sea lice (i.e., parasitic Copepoda). It was authorized within Scotland in 1997 for use on salmon farms. Cypermethrin acts on nervous systems by prolonging the opening of Na+ channels leading to trains of spikes in neurons (WHO, 1989). Cypermethrin is especially toxic to Crustacea (cf. Clark et al., 1989), in particular marine decapods (WHO, 1989). This study was designed to investigate the effect of 5 /xg/1 of cypermethrin on the shore crab (the same concentration used to kill sea lice). These findings are reported here in order to share some new ideas for the use of Carcinus as a biomonitoring species. Two 125 litre plastic, static seawater tanks were set up with aeration, each containing 12 Carcinus (70-85 mm carapace width) at 12?C and a light : dark cycle of 12: 12 hours. The crabs were acclimated for 4 days prior to the experiment and were not fed during acclimation or the experiment. The treatment dose of 5 /xg cypermethrin/1 was created in the treatment tank using ethanol as a carrier. The control tank was treated with an equal dose of pure ethanol (0.5 ml). The water in each tank was then mixed to equilibrate the treatments within the tanks. Crabs were observed at various intervals after the start of the experiment to remove mortalities and look for any visually recognizable effects of the cypermethrin. At 96 hours the experiment was stopped and the remaining crabs were externally and

Energetics of functional activation in neural tissues.

Glucose utilization (ICMRglc) increases linearly with spike frequency in neuropil but not perikarya of functionally activated neural tissues. Electrical stimulation, increased extracellular [K+] ([K+]o), or opening of Na+ channels with veratridine stimulates ICMRglc in neural tissues; these increases are blocked by ouabain, an inhibitor of Na+,K+-ATPase. Stimulating Na+,K+-ATPase activity to restore ionic gradients degraded by enhanced spike activity appears to trigger these increases in ICMRglc. Cultured neurons behave similarly. Astrocytic processes that envelop synapses in neuropil probably contribute to the increased ICMRglc. ICMRglc in cultured astroglia is unaffected by elevated [K+]o but is stimulated by increased intracellular [Na+] ([Na+]i), and this stimulation is blocked by ouabain or tetrodotoxin. L-Glutamate also stimulates ICMRglc in astroglia. This effect is unaffected by inhibitors of NMDA or non-NMDA receptors, blocked by ouabain, and absent in Na+-free medium; it appears to be mediated by increased [Na+]i due to combined uptake of Na+ with glutamate via Na+/glutamate co-transporters.

Contribution of Astroglia to Functionally Activated Energy Metabolism

Studies of local glucose utilization in neural tissues in vivo with the autoradiographic [14C]deoxyglucose method have demonstrated that energy metabolism increases almost linearly with the degree of functional activation, i.e. spike frequency, in the terminal projection zones of activated pathways. The increased metabolism is found in neuropil and is minimal or undetectable in neuronal cell bodies. Electrical stimulation, increased extracellular [K+] ([K+]o), or opening of Na+ channels with veratridine stimulates metabolism in neutral tissues, and this increase is blocked by ouabain, a specific inhibitor of Na+,K(+)-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to mediate the function-related increase in energy metabolism. The metabolic activation is, therefore, not directly related to the functional activity itself but to processes operating to recover from that activity. The limited spatial resolution of the [14C]DG method precludes identification of cellular elements in neuropil participating in the metabolic activation, e.g. axonal terminals, dendrites, or astrocytic processes enveloping the synapses. We have, therefore, attempted to stimulate in vitro conditions to be expected from functional activation and increased spike activity in vivo, e.g. increased extracellular [K+], intracellular [Na+], or extracellular neurotransmitter levels, and examined their effects on glucose metabolism in neurons and astroglia in culture. Increased [K+]o stimulated [14C]DG phosphorylation in neuronal and mixed neuronal-astroglial cultures, but not in astroglial cultures assayed in bicarbonate buffer; it did occasionally stimulate metabolism in astroglia when assayed in HEPES or phosphate buffers, but these effects were variable and inconsistent. Veratridine (75 microM) stimulated [14C]DG phosphorylation in neurons and astroglia; these stimulations were blocked by 1 mM ouabain or 10 microM tetrodotoxin (TTX), which blocks voltage-dependent Na+ channels. The Na+ ionophore monensin (10 microM) doubled the rate of metabolism, a stimulation that was only partially blocked by ouabain and unaffected by TTX. L-Glutamate (500 microM) stimulated [14C]DG phosphorylation in astroglia, but this stimulation was probably secondary to Na+ uptake into the cells via a sodium/glutamate co-transporter because it was not blocked by inhibitors of NMDA or non-NMDA receptors but was absent in Na(+)-free medium. These results indicate that astroglia contribute to the increased energy metabolism in neuropil during functional activation by mechanisms that promote Na+ entry into the cells.

Central colonic antipropulsive effect of two natural tachykinins, PG-SPI and PG-KII, in the rat

Tachykinin (TK; including substance P (SP), neurokinin A (NKA) and neurokinin B (NKB))-induced currents (ITKs) were studied in freshly isolated rat dorsal root ganglion (DRG) neurons using whole-cell patch clamp recording and repatch techniques. All the three ITKs manifested features of fast activating kinetics, such as short latency and fast τon and τoff, and very slow desensitization. The concentration–response relationships for TKs show: (1) compared with the concentration–response curve for NKA, the curve for NKB shifted upwards, while that for SP shifted downwards; (2) the EC50 values for NKB-, NKA- and SP-activated currents were very close to each other. The I–V curves for the three TKs were basically linear and arrayed in the order of NKB>NKA>SP; the reversal potentials for the three ITKs were all around +15 mV. Replacement of NaCl in the external solution by equimolar N-methyl-d-glucamine (NMDG) attenuated both NKA- and NKB-activated currents markedly, as it was the case with SP-activated current caused by the opening of Na+ preferring non-selective cation channels observed in our previous work. All the three TKs proved to inhibit coexistent GABAA receptor-mediated current (IGABA); this effect was removed by intracellular dialysis of GDP-β-S or H-7. However, these drugs did not block the SP-, NKA- and NKB-activated currents at all, which indicated that ITKs were G-protein independent. In short, the responses of rat DRG neurons to SP, NKA and NKB were similar in essence, although the amplitudes of currents induced by the same concentration of the three TKs were different. Taking the results of this study and our previous studies together, we hypothesized that SP, NKA and NKB may induce inward currents through undiscovered channels that are associated with tachykinins receptors (NK1R, NK2R, NK3R, which have already been cloned), but independent of G-protein coupling and remains to be further investigated.

Role of voltage-gated Na+ and Ca2+ channels in gonadotropin-releasing hormone-induced membrane potential changes in identified rat gonadotropes.

We have previously reported that GnRH induces rhythmic hyperpolarizations in male rat (35- to 45-day-old) gonadotropes by periodically opening apamin-sensitive Ca(2+)-activated K+ channels. Using the whole cell recording technique, we now show that these gonadotropes, identified with the reverse hemolytic plaque assay, express tetrodotoxin-sensitive Na+ channels and omega-conotoxin-insensitive, high voltage-activated Ca2+ channels that are partially sensitive to dihydropyridines. We found no low voltage-activated Ca2+ channels in these cells. At the normal resting potential, about 93% of the Na+ channels and 50% of the Ca2+ channels are inactivated. The GnRH-induced hyperpolarizations transiently remove the resting inactivation of Na+ and Ca2+ channels, enabling them to initiate action potentials at the termination of each hyperpolarization. Opening of Na+ channels accounts for the high rate of rise and the positive peak of the action potential. In addition, a significant fraction of Ca2+ channels should be activated during the action potentials, allowing a voltage-gated entry of extracellular Ca2+ that can enhance the frequency and amplitude of GnRH-induced intracellular Ca2+ oscillations. Therefore, we envision the following role for action potentials in GnRH-stimulated Ca2+ responses: action potentials will open voltage-gated Ca2+ channels that allow entry of extracellular Ca2+, which can help to replenish the intracellular Ca2+ store and act as a coactivator in the stimulation of intracellular Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive store.

Function-related changes in energy metabolism in the nervous system: localization and mechanisms.

Previous neuroanatomical and electrophysiological methods to localize functional activity in the nervous system focus on perikarya as the sites of the activity. Metabolic mapping of local functional activity in the nervous system provides a new dimension, the activity in the neuropil. Studies of local glucose utilization have shown that energy metabolism is increased by functional activation, but the effects are mainly in the terminal projection zones of the activated pathway. Electrical stimulation of a pathway raises glucose utilization in the projection zones of the pathway in almost direct proportion to the spike frequency. For example, electrical stimulation of the sciatic nerve produces frequency-dependent metabolic activation in the dorsal horn of the lumbar cord, where the axonal terminals of the afferent pathway reside, but no apparent metabolic effects in the cell bodies of the pathway in the dorsal root ganglia. Functional activation of the hypothalamo-hypophysial pathway by salt-loading increases glucose utilization in the pituitary neural lobe, where the terminal axons of the pathway reside, but not in the paraventricular and supraoptic nuclei, the sites of the cell bodies of origin of the pathway. Reflex activation by hypotension of pathways to these nuclei from brain stem structures involved in baroreceptor reflexes, however, increases glucose utilization in these nuclei. Depolarization induced by electrical stimulation, extracellular K+, or opening of Na+ channels with veratridine, stimulates glucose utilization in neural tissues, and this increase is blocked by ouabain, a specific inhibitor of Na+, K(+)-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to trigger the functional activation of energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Sites and Mechanisms of Function-Related Changes in Energy Metabolism in the Nervous System

Traditional neuroanatomical and electrophysiological methods to localize functional activities in the nervous system focus on perikarya as the sites of activity. Metabolic mapping of local functional activity in the nervous system with the deoxyglucose method has directed interest toward the activity in neuropil. Studies of local glucose utilization (lCMRglc) indicate that energy metabolism is increased by functional activation mainly in terminal projection zones of activated pathways. Electrical stimulation of a pathway raises lCMRglc in the projection zones of the pathway in almost direct proportion to the spike frequency. For example, stimulation of the sciatic nerve produces frequency-dependent metabolic activation in the dorsal horn of the lumbar cord, where the axonal terminals of the afferent pathway reside, with no apparent metabolic effects in the cell bodies of the pathway in the dorsal root ganglia. Functional activation of the hypothalamo-hypophysial pathway by salt-loading increases lCMRglc in the neurohypophysis, the site of the terminal axons of the pathway, but not in the paraventricular and supraoptic nuclei, where the cell bodies of origin of the pathway reside. Activation by hypotension of pathways to these nuclei from brain stem structures involved in baroceptor reflexes does, however, increase lCMRglc in these nuclei. Depolarization induced by electrical stimulation, increased extracellular K+, or opening of Na+ channels with veratridine stimulate lCMRglc in neural tissues, and this increase is blocked by ouabain, a specific inhibitor of Na+,K(+)-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to trigger the function-related increase in energy metabolism. The metabolic activation is the consequence not of the functional activity itself but of processes operating to recover from that activity.

Stimulation of cortisol production in isolated bovine zona fasciculata cells by phorbol ester: role of ion fluxes.

The role of protein kinase C activation in the control of cortisol synthesis was studied using the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Bovine zona fasciculata cells were incubated with various concentrations of TPA in the presence and absence of EGTA, verapamil or nitrendipine to see whether cortisol stimulation was dependent on extra-cellular calcium ions. When free extracellular concentration of Ca2+ was reduced to approximately 10 mumol/l the cortisol response at all concentrations of TPA was reduced by approximately 25% indicating that protein kinase C activation is only partially dependent on extracellular calcium ions. This is confirmed by the effects of the voltage-dependent calcium channel blocker verapamil, which partially inhibited the cortisol response to a maximally effective concentration of TPA (1 mumol/l). However, a second channel blocker, nitrendipine, proved to be ten times more potent than verapamil and totally inhibited the TPA response. The partial effects of EGTA and verapamil and the contrast between verapamil and nitrendipine do not exclude the possibility that intracellular calcium ions are important in protein kinase C activation and may indicate that nitrendipine has better access to an additional site of inhibitory action than verapamil. It is significant that the ionophore A23187, which facilitates Ca2+ entry independently of voltage-sensitive channels, failed to overcome the inhibitory effects of nitrendipine in TPA-stimulated cells. In some other tissues, the effects of protein kinase C activation are mediated by the opening of Na+/H+ exchange ports. The involvement of this port in the cortisol response has been tested by incubating TPA- and ACTH-treated cells with amiloride, an inhibitor of Na+/H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)