Removal Of External Na Research Articles

Electrophysiological characterization of voltage-dependent calcium currents and TRPV4 currents in human pulmonary fibroblasts

We have presented indirect evidence of a key role for voltage-dependent Ca(2+) currents in TGFβ-induced synthetic function in human pulmonary fibroblast (HPF), as well as in bleomycin-induced pulmonary fibrosis in mice. Others, however, have provided indirect evidence for transient receptor potential vanilloid 4 (TRPV4) channels in both of those effects. Unfortunately, definitive electrophysiological descriptions of both currents in HPFs have been entirely lacking. In this study, we provide the first direct electrophysiological and pharmacological evidence of the currents in HPFs at rest and during overnight stimulation with TGFβ. These currents include a Ca(2+)-dependent K(+) current, a TRPV4 current, a chloride current, and an L-type voltage-dependent Ca(2+) current. Evidence for the TRPV4 current include activation of a large-conductance change by two putatively TRPV4-selective agonists (4α-phorbol-12,13-didecanoate; GSK1016790A), with a reversal potential near 0 mV, partial sensitivity to two different TRPV4-selective blockers (RN1734; HC067047), and partial reduction following removal of external Na(+) Substantial reduction of the evoked current was seen following the coapplication of RN1734, DIDS, and niflumic acid, suggesting that a chloride current is also involved. The voltage-dependent Ca(2+) current is found to be "L-type" in nature, as indicated by the voltage and time dependence of its activation, deactivation, and inactivation properties, and by its pharmacology (sensitivity to replacement with barium and inhibition by nifedipine, verapamil, or mibefradil). We also found that overnight treatment with TGFβ evoked a periodic current (inward at negative holding potentials, with reversal potential near 0 mV), which is sufficient to trigger the voltage-dependent Ca(2+) currents and, thereby, account for the rhythmic Ca(2+) oscillations, which we have described previously in these cells.

Pathological impact of hyperpolarization-activated chloride current peculiar to rat pulmonary vein cardiomyocytes

Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs+ resistant, and was not affected by removal of external Na+ or K+. The current was inhibited with Cd2+, and the reversal potential was sensitive to changes in [Cl−] on either side of the membrane in a manner consistent with a Cl− selective channel. Cl− channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl− current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl− current in rat PVs was shifted to negative potentials as [Cl−]i increased, (ii) the Cl− current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl− channel, and that the channel may play a functional role in norepinephrine-induced automaticity.

Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: Direct evidence of functional Ca2+-induced Ca2+ release

Human embryonic stem cells (hESCs) serve as a potential unlimited ex vivo source of cardiomyocytes for disease modeling, cardiotoxicity screening, drug discovery, and cell-based therapies. Despite the fundamental importance of Ca(2+)-induced Ca(2+) release in excitation-contraction coupling, the mechanistic basis of Ca(2+) handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive. To study Ca(2+) sparks as unitary events of Ca(2+) handling for mechanistic insights. To avoid ambiguities owing to the heterogeneous nature, we experimented with hESC-VCMs, purified on the basis of zeocin resistance and signature ventricular action potential after LV-MLC2v-tdTomato-T2A-Zeo transduction. Ca(2+) sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca(2+)-ATPase (thapsigargin and cyclopiazonic acid) and ryanodine receptor (RyR; ryanodine, tetracaine) but not inositol trisphosphate receptors (xestospongin C and 2-aminoethyl diphenylborinate) could be recorded. In a permeabilization model, we further showed that RyRs could be sensitized by Ca(2+). Increasing external Ca(2+) dramatically escalated the basal Ca(2+) and spark frequency. Furthermore, RyR-mediated Ca(2+) release sensitized nearby RyRs, leading to compound Ca(2+) sparks. Depolarization or L-type Ca(2+) channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca(2+)-dependent cytosolic Ca(2+) increase and reduced the sarcoplasmic reticulum content. By contrast, removal of external Na(+) or the addition of the Na(+)-Ca(2+) exchanger inhibitor (KB-R7943 and SN-6) had no effect, suggesting that the Na(+)-Ca(2+) exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca(2+) waves. Taken collectively, our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca(2+)-induced Ca(2+) release. However, the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development.

A Novel Role for Bcl-2 in Regulation of Cellular Calcium Extrusion

SummaryThe antiapoptotic protein Bcl-2 [1, 2] plays important roles in Ca2+ signaling [3] by influencing inositol triphosphate receptors and regulating Ca2+-induced Ca2+ release [4–6]. Here we investigated whether Bcl-2 affects Ca2+ extrusion in pancreatic acinar cells. We specifically blocked the Ca2+ pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca2+ concentration after a period of enhanced Ca2+ entry. Because external Ca2+ was removed and endoplasmic reticulum Ca2+ pumps were blocked, Ca2+ extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca2+ level much faster than control cells. The enhanced Ca2+ extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na+/Ca2+ exchange activity, because removal of external Na+ did not influence the Ca2+ extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca2+ extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca2+ extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca2+ pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca2+ extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.

Orexin-A excites pyramidal neurons in layer 2/3 of the rat prefrontal cortex

The arousal peptides, orexins, play an important role in regulating the function of the prefrontal cortex (PFC). Although orexins have been shown to increase the excitability of deep-layer neurons in the medial prefrontal cortex (mPFC), little is known about their effect on layer 2/3, the main intracortical processing layer. In this study, we investigated the effect of orexin-A on pyramidal neurons in layer 2/3 of the mPFC using whole-cell recordings in rat brain slices. We observed that orexin-A reversibly depolarized layer 2/3 pyramidal neurons through a postsynaptic action. This depolarization was concentration-dependent and mediated via orexin receptor 1. In voltage-clamp recordings, the orexin-A-induced current was reduced by the replacement of internal K+ with Cs+, removal of external Na+, or an application of flufenamic acid (an inhibitor of nonselective cation channels). A blocker of Na+/Ca2+ exchangers (SN-6) did not influence the excitatory effect of orexin-A. Moreover, the current induced by orexin-A reversed near Ek when the external solution contained low levels of Na+. When recording with Cs+-containing pipettes in normal external solution, the reversal potential of the current was approximately −25mV. These data suggest an involvement of both K+ channels and nonselective cation channels in the effect of orexin-A. The direct excitatory action of orexin-A on layer 2/3 mPFC neurons may contribute to the modulation of PFC activity, and play a role in cognitive arousal.

Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes

Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins L-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal DL-threo-β-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes.

Testicular Expression of Adora3i2 in Adora3 Knockout Mice Reveals a Role of Mouse A3Ri2 and Human A3Ri3 Adenosine Receptors in Sperm*

Adenosine is a candidate modulator of sperm motility in the female reproductive tract that increases sperm flagellar beat frequency in vitro. Past work suggested that this acceleration may involve equilibrative (ENT) and concentrative (CNT) nucleoside transporters. Here we show that Slc29a1 (ENT-1) is the predominant nucleoside transporter expressed in the mouse testis. Unexpectedly, the beat of Slc29a1-null sperm still accelerates in response to 2-chloro-2'-deoxyadenosine (Cl-dAdo). Moreover, in wild-type sperm neither blockade of CNTs by removal of external Na(+), nor inhibition of ENTs with nitrobenzylthioionosine, prevents acceleration of the sperm beat by Cl-dAdo. In contrast, pertussis toxin produces strong blockade, indicating involvement of a Gα(i/o)-coupled adenosine receptor. Although agonists selective for adenosine receptors A1R, A2aR, and A2bR are ineffective, A3R-selective agonists Cl-IB-MECA and IB-MECA do accelerate the beat. Consistent with this pharmacological profile, the predominant Adora transcripts in the testis are products of the nested Adora3i1 and Adora3i2 genes. Surprisingly, Cl-IB-MECA and Cl-dAdo still accelerate the beat of Adora3i1-null sperm indicating that the remaining Adora3i2 transcript produces an A3R that functions in sperm. When cloned Adora3i2 is heterologously expressed in tsA-201 cells, Cl-dAdo decreases forskolin-evoked accumulation of cAMP, indicating that Adora3i2 specifies a functional A3Ri2 adenosine receptor that couples through Gα(i). Database mining reveals that mouse Adora3i2 is expressed primarily in testis, almost exclusively in spermatids. Expression of the orthologous ADORA3i3 transcript also is most prominent in human testis; presumably producing an A3Ri3 receptor that is functional in sperm and that may be a target for development of male-directed contraceptives.

Opposite regulation of KCNQ5 and TRPC6 channels contributes to vasopressin-stimulated calcium spiking responses in A7r5 vascular smooth muscle cells

Physiologically relevant concentrations of [Arg 8]-vasopressin (AVP) induce repetitive action potential firing and Ca 2+ spiking responses in the A7r5 rat aortic smooth muscle cell line. These responses may be triggered by suppression of KCNQ potassium currents and/or activation of non-selective cation currents. Here we examine the relative contributions of KCNQ5 channels and TRPC6 non-selective cation channels to AVP-stimulated Ca 2+ spiking using patch clamp electrophysiology and fura-2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 channel expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown resulted in more positive resting membrane potentials and induced spontaneous action potential firing and Ca 2+ spiking. However physiological concentrations of AVP induced additional depolarization and increased Ca 2+ spike frequency in KCNQ5 knockdown cells. AVP activated a non-selective cation current that was reduced by TRPC shRNA treatment or removal of external Na +. Neither resting membrane potential nor the AVP-induced depolarization was altered by knockdown of TRPC6 channel expression. However, both TRPC6 shRNA and removal of external Na + delayed the onset of Ca 2+ spiking induced by 25 pM AVP. These results suggest that suppression of KCNQ5 currents alone is sufficient to excite A7r5 cells, but AVP-induced activation of TRPC6 contributes to the stimulation of Ca 2+ spiking.

Amiloride inhibits hydrogen peroxide-induced Ca 2+ responses in human CNS pericytes

The aims of the present study were to investigate the mechanisms of Ca 2+ signaling caused by hydrogen peroxide in CNS pericytes. In cultured human brain microvascular pericytes, cytosolic Ca 2+ concentration was measured by means of fura-2 fluorescence. Reverse transcription and polymerase chain reaction was performed to examine the expression of mRNA. Knockdown of Na +/H + exchanger (NHE) was done by transfecting the cells with specific double-strand siRNAs for NHE. Externally applied hydrogen peroxide dose-dependently (100 μM–10 mM) increased cytosolic Ca 2+ in human CNS pericytes. Cytosolic Ca 2+ remained high after wash-out of hydrogen peroxide. However, the addition of dithiothreitol rapidly reversed cytosolic Ca 2+ to the resting level. The hydrogen peroxide-induced Ca 2+ increase was not inhibited by nicardipine, Gd 3+, La 3+, or omission of external Ca 2+. Neither thapsigargin nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone attenuated the hydrogen peroxide-induced Ca 2+ rise. Amiloride and its derivatives, benzamil and hexamethylene amiloride reversed the hydrogen peroxide-induced Ca 2+ increase. Human CNS pericytes expressed acid sensing ion channel (ASIC) 1a, Na +/Ca 2+ exchanger (NCX) 1, Na +/H + exchanger (NHE) 1, and NHE7. However, the removal of external Na +, treatment with KB-R 7943 and mibefradil, or knockdown of NHE1 and NHE7 did not affect the hydrogen peroxide-induced Ca 2+ increase. Hydrogen peroxide releases Ca 2+ from intracellular Ca 2+ pool via an amiloride-sensitive protein, which is controlled by oxidation of thiol group in human CNS pericytes.

Contribution of Kv7.5 potassium channels inhibition and TRPC6 non‐selective channels activation in AVP induced calcium oscillations in A7r5 smooth muscle cells

Activation of TRPC6 non‐selective cation channels and inhibition of KCNQ5 voltage‐gated K+ channels have both been implicated in Ca2+ signaling in response to [Arg8]‐vasopressin (AVP) in vascular smooth muscle cells. In A7r5 cells, AVP (25 pM) induces repetitive Ca2+‐dependent action potential firing and Ca2+ spiking. The contributions of KCNQ5 channels and TRPC6 channels to AVP‐stimulated Ca2+ spiking were examined using fura‐2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown was sufficient to induce spontaneous Ca2+ spiking, but the frequency was less than that induced by 25 pM AVP (2.5 ± 0.6 spikes/min (n=3) and 5.1 ± 0.4 spikes/min (n=10), respectively). AVP‐activated non‐selective cation currents (ICAT) were significantly reduced by knockdown of TRPC6 channel expression or removal of external Na+. Both of these treatments also significantly delayed the onset of Ca2+ spiking induced by 25 pM AVP (a 2.9 ± 1.0 min delay with TRPC6 knockdown (n=6) and an 11.3 ± 2.8 min delay in Na+‐free medium (n=4)). Spike frequency was also significantly reduced by the latter treatment (by 3.3 ± 0.7 spikes/min, n=4). These results suggest that although suppression of KCNQ5 currents alone is sufficient to excite A7r5 vascular smooth muscle cells, AVP‐induced activation of TRPC6 non‐selective cation channels contributes to the stimulation of Ca2+ spiking.

Potential contribution of a voltage-activated proton conductance to acid extrusion from rat hippocampal neurons

We examined the potential contribution of a voltage-gated proton conductance ( g H +) to acid extrusion from cultured postnatal rat hippocampal neurons. In neurons loaded with Ca 2+- and/or pH-sensitive fluorophores, transient exposures to 25–139.5 mM external K + (K + o) or 20 μM veratridine in the presence of 2 mM Ca 2+ o (extracellular pH (pH o) constant at 7.35) caused reversible increases and decreases in intracellular free calcium concentration ([Ca 2+] i) and intracellular pH (pH i), respectively. In contrast, under external Ca 2+-free conditions, the same stimuli failed to affect [Ca 2+] i but caused an increase in pH i, the magnitude of which was related to the [K +] o applied and the change in membrane potential. Consistent with the properties of g H +s in other cell types, the magnitude of the rise in pH i observed in the absence of external Ca 2+ was not affected by the removal of external Na + but was sensitive to external Zn 2+ and temperature and was dependent on the measured transmembrane pH gradient (ΔpH memb). Increasing ΔpH memb by pretreatment with carbonylcyanide- p-trifluoromethoxyphenylhydrazone augmented both the high-[K +] o-evoked rise in pH i and the Zn 2+-sensitive component of the rise in pH i, suggestive of increased acid extrusion via a g H +. The inhibitory effect of Zn 2+ at a given ΔpH memb was further enhanced by increasing pH o from 7.35–7.8, consistent with a pH o-dependent inhibition of the putative g H + by Zn 2+. Under conditions designed to isolate H + currents, a voltage-dependent outward current was recorded from whole-cell patch-clamped neurons. Although the outward current appeared to show some selectivity for protons, it was not sensitive to Zn 2+ or temperature and the H +-selective component could not be separated from a larger conductance of unknown selectivity. Nonetheless, taken together, the results suggest that a Zn 2+-sensitive proton conductive pathway is present in rat hippocampal neurons and contributes to H + efflux under depolarizing conditions.

Latrunculin A depolarizes starfish oocytes

Depolymerization of the actin cytoskeleton may liberate Ca 2+ from InsP 3-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca 2+ influx in others. However, no information is available on the modulation of membrane potential ( V m) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V m in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V m which reached the threshold of activation of voltage-gated Ca 2+ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca 2+-free sea water, while it was abolished upon removal of external Na +. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca 2+ from InsP 3Rs. The Ca 2+ signal in turn activates a Ca 2+-dependent Na + entry, which causes the positive shift in V m and stimulates the VGCC.

Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells

Fluid secretion across the blood-brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO(3)(-), which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pH(i)). To analyse the transporters that may be involved in the maintenance of pH(i) and hence secretion of HCO(3)(-), we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO(2)/HCO(3)(-). pH(i) in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO(3)(-) than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO(3)(-) were similar. In the nominal absence of HCO(3)(-) the rate of acid loading was increased equally by removal of external Na(+) or by inhibition of Na(+)/H(+) exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO(3)(-) the increase in the rate of acid loading when Na(+) was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na(+) and HCO(3)(-). Removal of Cl(-) in the presence of HCO(3)(-) produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na(+) following removal of Cl(-) increased the rate of acid gain. In the presence of HCO(3)(-) and initial presence of Na(+) and Cl(-), DIDS inhibited the changes in pH(i) produced by removal of either Na(+) or Cl(-). These are the expected results if these cells possess an AE-like Cl(-)/HCO(3)(-) exchanger, a 'channel-like' permeability allowing slow influx of acid (or efflux of HCO(3)(-)), a NBC-like Cl(-)-independent Na(+)-HCO(3)(-) cotransporter, and a NHE-like Na(+)/H(+) exchanger. The in vitro rates of HCO(3)(-) loading via the Na(+)-HCO(3)(-) cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO(3)(-) into the brain.

Icilin Activates the δ-Subunit of the Human Epithelial Na+ Channel

The amiloride-sensitive epithelial Na(+) channel (ENaC) regulates Na(+) homeostasis in cells and across epithelia. Four homologous ENaC subunits (alpha, beta, gamma, and delta) have been isolated in mammals. The chemical activators acting on ENaC, however, are largely unknown. More recently, we have found that capsazepine activates human ENaCdelta (hENaCdelta), which is mainly expressed in the brain. In addition, here we show that icilin, which is a tetrahydropyrimidine-2-one derivative unrelated structurally to capsazepine, markedly enhanced the activity of hENaCdeltabetagamma heteromultimer expressed in Xenopus laevis oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were increased by the application of icilin in a concentration-dependent manner with an EC(50) value of 33 microM. The icilin-elicited current was mostly abolished by the addition of 100 microM amiloride or by the removal of external Na(+). Homomeric hENaCdelta was also significantly activated by icilin, whereas hENaCalpha activity was not affected by icilin, and icilin caused a slight inhibition of the hENaCalphabetagamma current. Furthermore, icilin acted together with protons or capsazepine on hENaCdeltabetagamma. These findings identify icilin as a novel chemical activator of ENaCdelta, providing us with a lead compound for drug development in the degenerin/ENaC superfamily.

Cardiac Na+–Ca2+ exchanger current induced by tyrphostin tyrosine kinase inhibitors

Tyrosine kinase (TK) inhibitors genistein and tyrphostin A23 (A23) inhibited Ca(2+) currents in guinea-pig ventricular myocytes investigated under standard whole-cell conditions (K(+)-free Tyrode's superfusate; EGTA-buffered (pCa-10.5) Cs(+) dialysate). However, the inhibitors (100 microM) also induced membrane currents that reversed between -40 and 0 mV, and the objective of the present study was to characterize these currents. Genistein-induced current behaved like Cl(-) current, and was unaffected by either the addition of divalent cations (0.5 mM Cd(2+); 3 mM Ni(2+)) that block the Na(+)-Ca(2+) exchanger (NCX), or the removal of external Na(+) and Ca(2+). A23-induced current was independent of Cl(-) driving force, and strongly suppressed by addition of Cd(2+) and Ni(2+), and by removal of either external Na(+) or Ca(2+). These and other results suggested that A23 activated an NCX current driven by submembrane Na(+) and Ca(2+) concentrations higher than those in the bulk cytoplasm. Improved control of intracellular Na(+) and Ca(2+) concentrations was obtained by suppressing cation influx (10 microM verapamil) and raising dialysate Na(+) to 7 mM and dialysate pCa to 7. Under these conditions, stimulation by A23 was described by the Hill equation with EC(50) 68 +/- 4 microM and coefficient 1.1, tyrphostin A25 was as effective as A23, and TK-inactive tyrphostin A1 was ineffective. Phosphotyrosyl phosphatase inhibitor orthovanadate (1 mM) antagonized the action of 100 microM A23. The results suggest that activation of cardiac NCX by A23 is due to inhibition of genistein-insensitive TK.

Orexins cause depolarization via nonselective cationic and K+ channels in isolated locus coeruleus neurons

The locus coeruleus (LC) contains noradrenergic neurons that are innervated by orexin (ORX)-like immunoreactive axons and express both orexin receptor-1 and -2. We studied effects of ORX-A and -B (ORX-A/B) on dissociated LC neurons by using whole-cell patch clamp techniques. In current-clamp mode, LC neurons were depolarized by application of ORX-A (10(-7) M) [53% of neurons tested; 9.0+/-0.2 mV (n=5)], or ORX-B (10(-7) M) [38% of neurons tested; 4.0+/-0.1 mV (n=5)]. Firing frequencies of action potentials increased during application [1.1+/-0.2 Hz (n=5) in ORX-A; 0.8+/-0.2 Hz (n=5) in ORX-B] and returned to the control level [0.2+/-0.1 Hz (n=5)] after removal. The ORX-A/B-induced depolarization was well maintained in the presence of TTX (3x10(-7) M), CNQX (10(-6) M) and AP5 (10(-5) M). In voltage-clamp mode, removal of external Na+ suppressed both ORX-A/B-induced currents and shifted their reversal potentials from approximately -45 mV to -60 mV. In addition, ORX-A/B inhibited sustained K+ currents. These results suggest that ORX-A/B increase the firing frequency of LC neurons through the depolarization probably produced by both augmentation of the nonselective cationic conductance and inhibition of the sustained K+ conductance.

Involvement of anion channel(s) in the modulation of the transient outward K(+) channel in rat ventricular myocytes.

The cardiac Ca(2+)-independent transient outward K(+) current (I(to)), a major repolarizing ionic current, is markedly affected by Cl(-) substitution and anion channel blockers. We reexplored the mechanism of the action of anions on I(to) by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs(+) substitution for intracellular K(+). Replacement of most of the extracellular Cl(-) with less permeant anions, aspartate (Asp(-)) and glutamate (Glu(-)), markedly suppressed the current. Removal of external Na(+) or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on I(to). In contrast, the permeant Cl(-) substitute Br(-) did not markedly affect the current, whereas F(-) substitution for Cl(-) induced a slight inhibition. The I(to) elicited during Br(-) substitution for Cl(-) was also sensitive to blockade by 4-AP. The ability of Cl(-) substitutes to induce rightward shifts of the steady-state inactivation curve of I(to) was in the following sequence: NO(3)(-) > Cl(-) approximately Br(-) > gluconate(-) > Glu(-) > Asp(-). Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of I(to) similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp(-) substitution for Cl(-) was without significant effect on the intensity. These results suggest that the I(to) channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated.

Forced rotation of Na +-driven flagellar motor in a coupling ion-free environment

Rotational characteristics of Na +-driven flagellar motor in the presence and absence of coupling ion were analyzed by electrorotation method. The motor rotated spontaneously in the presence of Na +, and the rotation accelerated or decelerated following the direction of the applied external torque. The spontaneous motor rotation was inhibited by removal of external Na +, however, the motor could be forcibly rotated by relatively small external torque applied by the electrorotation apparatus. The observed characteristic of the motor was completely different from that of ATP-driven motor systems, which form rigor bond when their energy source, ATP, is absent. The internal resistance of the flagellar motor increased significantly when the coupling ion could not access the inside of the motor, suggesting that the interaction between the rotor and the stator is changed by the binding of the coupling ion to the internal sites of the motor.

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes.

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.

K+-dependent Na+/Ca2+ exchange is a major Ca2+ clearance mechanism in axon terminals of rat neurohypophysis.

Two different families of Na+/Ca2+ exchangers, K+-independent NCX and K+-dependent NCKX, are known. Exploiting the outward K+ gradient, NCKX is able to extrude Ca2+ more efficiently than NCX, even when the Na+ gradient is reduced. The NCKX, which was originally thought to be limited to the retinal photoreceptor, was shown recently to be widely distributed in the brain. We investigated the contribution of Na+/Ca2+ exchange to Ca2+ clearance mechanisms in neurohypophysial (NHP) axon terminals, using patch-clamp and microfluorometry techniques. In the presence of internal K+, Ca2+ decay was significantly slowed by the removal of external Na+, indicative of the role of Na+/Ca2+ exchange. As internal [K+] was decreased, Ca2+ decay rate and its dependence on Na+ were greatly attenuated. In the absence of internal K+, Ca2+ decay rate was little affected by Na+ removal. Quantitative analysis using Ca2+ decay rate constant indicated that >60% of Ca2+ extrusion is mediated by Na+/Ca2+ exchange when peak [Ca2+] level is higher than 500 nm, and approximately 90% of Na+/Ca2+ exchange activity is K+ dependent. In situ hybridization confirmed the expression of NCKX2 transcripts in the supraoptic nucleus in which soma of NHP axon terminals are located. To our knowledge, this is the first report to show the significant role of K+-dependent Na+/Ca2+ exchange in neuronal cells other than photoreceptors. Considering that axon terminals are subject to an invasion by high-frequency Na+ spikes, which may lower Na+ gradients, the presence of NCKX may have a functional significance in intracellular Ca2+ regulation.