Nitric oxide synthase inhibitors enhance mechanosensitive Ca(2+) influx in cultured dorsal root ganglion neurons

Nitric oxide (NO) can have opposite effects on peripheral sensory neuron sensitivity depending on the concentration and source of NO, and the experimental setting. The aim of this study was to determine the role of endogenous NO production in the regulation of mechanosensitive Ca(2+) influx of dorsal root ganglion (DRG) neurons. Adult mouse DRG neurons were grown in primary culture for 2-5 days, loaded with Fura-2, and tested for mechanically mediated changes in [Ca(2+)](i) by fluorescent ratio imaging. In the presence of the NOS inhibitors L-NAME, TRIM, or 7-NI, but not the inactive analogue D-NAME, peak [Ca(2+)](i) transients to mechanical stimulation were increased more than 2-fold. Neither La(3+) (25 microM), an inhibitor of voltage activated Ca(2+) channels, or tetrodotoxin (TTX, 1 microM), a selective inhibitor of voltage-gated Na(+) channels, had an effect on mechanically activated [Ca(2+)](i) transients under control conditions. However, in the presence of L-NAME, both La(3+) and TTX partially blocked the [Ca(2+)](i) response. Addition of Gd(3+), a blocker of mechanosensitive cation channels and L-type Ca(2+) channels, at a concentration (100 microM) that markedly inhibited the mechanical response under control conditions, only partially inhibited the response in the presence of L-NAME. The combination of either La(3+) or TTX with Gd(3+) caused near complete inhibition of mechanically stimulated [Ca(2+)](i) transients in the presence of L-NAME. We conclude that focal mechanical stimulation of DRG neurons causes Ca(2+) influx occurs primarily through mechanosensitive cation channels under control conditions. In the presence of NOS inhibitors, additional Ca(2+) influx occurs through voltage-sensitive Ca(2+) channels. These results suggest that endogenously produced NO in cultured DRG neurons decreases mechanosensitivity by inhibiting voltage-gated Na(+) and Ca(2+) channels.     

Na(+)/Ca(2+) exchange in human myotubes: intracellular calcium rises in response to external sodium depletion are enhanced in DMD

This study aims to investigate the sodium/calcium exchanger expression in human co-cultured skeletal muscle cells and to compare the effects of Na(+)/Ca(2+) exchange activity in normal and dystrophic (Duchenne's muscular dystrophy) human co-cultured myotubes. For this purpose, variations of intracellular calcium concentration ([Ca(2+)](int)) were monitored, as the variations of the fluorescence ratio of indo-1 probe, in response to external sodium depletion. External sodium withdrawal induced [Ca(2+)](int) rises within several seconds in both normal and Duchenne's muscular dystrophy myotubes. These Na(+)-free-induced [Ca(2+)](int) elevations were attributed to the reverse mode of the Na(+)/Ca(2+) exchange mechanism since the phenomenon was dependent on extracellular calcium concentration ([Ca(2+)](ext)), and since it was sensitive to external Ni(2+) ions. Amplitudes of Na(+)-free-induced [Ca(2+)](int) rises were significantly greater in Duchenne's muscular dystrophy cells than in normal ones. Such a difference disappeared when the sarcoplasmic reticulum was pharmacologically blocked, suggesting that the reverse mode of the Na(+)/Ca(2+) exchange mechanism was able to generate enhanced calcium-induced calcium-release in Duchenne's muscular dystrophy myotubes. Immunostaining images of Na(+)/Ca(2+) exchanger (NCX) isoforms, obtained by confocal microscopy, revealed the presence of NCX1 and NCX3 at the sarcolemmal level of both normal and Duchenne's muscular dystrophy myotubes. No differences were observed in the location of NCX isoforms expression between normal and Duchenne's muscular dystrophy co-cultured myotubes.     

Cobalt-dependent potentiation of net inward current density in Helix aspersa neurons.

A low concentration of transition metal ions Co2+ and Ni2+ increases the inward current density in neurons from the land snail Helix aspersa. The currents were measured using a single electrode voltage-clamp/internal perfusion method under conditions in which the external Na+ was replaced by Tris+, the predominant external current carrying cation was Ca2+, and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was added externally to block K+ current. In the presence of Co2+ (3 mM) or Ni2+ (0.5 mM) inward Ca2+ currents were stimulated normally by voltage-dependent activation of Ca2+ channels. There was a 5-10% decrease in the rate of rise of the inward current. The principal effect of Co2+ and Ni2+ in increasing the current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage-dependent or Ca(2+)-dependent outward current and/or an inhibition of Ca2+ channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+ efflux through the voltage-activated or Ca(2+)-activated nonspecific cation channels. Co2+ is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normal Helix Ringer's solution showed that the Ca(2+)-dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co(2+)-dependent increase in inward (depolarizing) current seen in voltage-clamp experiments.     

Functional expression of constitutive nitric oxide synthases regulated by voltage-gated Na+ and Ca2+ channels in cultured human astrocytes

We report the functional characterization of constitutive nitric oxide synthase(s) (NOS) such as neuronal and endothelial NOS in cultured human astrocytes. Exposure of cultured human astrocytes to 1 microM veratridine or 50 mM KCl produced a pronounced increase in a calmodulin-dependent NOS activity estimated from cGMP formation. The functional expression of voltage-gated Na(+) channel, which is estimated by the response to veratridine, appeared to be earlier (at second day in culture) than that of voltage-gated Ca(2+) channels, which are estimated by the response to the KCl stimulation (at fourth day in culture). The KCl-evoked NO synthesis was totally reversed by L-type Ca(2+) channel blockers such as nifedipine and verapamil, but not by omega-conotoxin GVIA, an N-type Ca(2+) channel blocker, or omega-agatoxin IVA, a P/Q-type Ca(2+) channel blocker. In addition, verapamil abolished the KCl-induced increase in the intracellular free Ca(2+) concentration. RT-PCR analysis revealed that mRNA for neuronal and endothelial NOS was expressed in human astrocytes. In addition, Western blot analysis and double labeling of NOS and glial fibrillary acidic protein (GFAP) showed that cultured human astrocytes expressed neuronal NOS and endothelial NOS as well as the alpha(1) subunit of Ca(2+) channel. These results suggest that human astrocytes express constitutive NOS that are regulated by voltage-gated L-type Ca(2+) channel as well as Na(+) channel.     

Effects of doxapram on ionic currents recorded in isolated type I cells of the neonatal rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of the respiratory stimulant doxapram on K+ and Ca2+ currents in isolated type I cells of the neonatal rat carotid body. Doxapram (1-100 microM) caused rapid, reversible and dose-dependent inhibitions of K+ currents recorded in type I cells (IC50 approximately 13 microM). Inhibition was voltage-dependent, in that the effects of doxapram were maximal at test potentials where a shoulder in the current-voltage relationship was maximal. These K+ currents were composed of both Ca(2+)-activated and Ca(2+)-independent components. Using high [Mg2+], low [Ca2+] solutions to inhibit Ca(2+)-activated K+ currents, doxapram was also seen to directly inhibit Ca(2+)-independent K+ currents. This effect was voltage-independent and was less potent (IC50 approximately 20 microM) than under control conditions, suggesting that doxapram was a more potent inhibitor of the Ca(2+)-activated K+ currents recorded under control conditions. Doxapram (10 microM) was without effect on L-type Ca2+ channel currents recorded under conditions where K+ channel activity was minimized and was also without significant effect on K+ currents recorded in the neuronal cell line NG-108 15, suggesting a selective effect on carotid body type I cells. The effects of doxapram on type I cells show similarities to those of the physiological stimuli of the carotid body, suggesting that doxapram may share a similar mechanism of action in stimulating the intact organ.     

A tyrosine residue in TM6 of the Vanilloid Receptor TRPV1 involved in desensitization and calcium permeability of capsaicin-activated currents

The Vanilloid Receptor TRPV1 is a non-selective cation channel with a high relative Ca(2+) permeability. TRPV1 exhibits slow desensitization, a potential mechanism regulating adaptation of peripheral sensory neurons to noxious stimuli. The predicted folding pattern of TRPV1 resembles that of voltage-gated channels. Sequence alignment of segments 6 of TRPV1 and voltage-gated Na(+) channels reveals a conserved aromatic amino acid that in Na(+) channels is involved in fast inactivation and pharmacological block. We found that replacing this tyrosine Y671 by positively charged lysine (K) completely abrogated Ca(2+)-dependent desensitization. Y671K also exhibited significant reduction in Ca(2+) permeability that was not responsible for the lack in desensitization. Substitution of Y671 with negatively charged aspartate or uncharged alanine slightly altered desensitization but left Ca(2+) permeability unchanged. Substitution of Y671 with positively charged arginine produced a phenotype similar to Y671K. We propose that residue Y671 is critical for the high relative Ca(2+) permeability of TRPV1 and participates in the structural rearrangements of the channel protein leading to Ca(2+)-dependent desensitization.     

The involvement of nitric oxide synthase neurons in enteric neuropathies

Nitric oxide (NO), produced by the neural nitric oxide synthase enzyme (nNOS) is a transmitter of inhibitory neurons supplying the muscle of the gastrointestinal tract. Transmission from these neurons is necessary for sphincter relaxation that allows the passage of gut contents, and also for relaxation of muscle during propulsive activity in the colon. There are deficiencies of transmission from NOS neurons to the lower esophageal sphincter in esophageal achalasia, to the pyloric sphincter in hypertrophic pyloric stenosis and to the internal anal sphincter in colonic achalasia. Deficits in NOS neurons are observed in two disorders in which colonic propulsion fails, Hirschsprung's disease and Chagas' disease. In addition, damage to NOS neurons occurs when there is stress to cells, in diabetes, resulting in gastroparesis, and following ischemia and reperfusion. A number of factors may contribute to the propensity of NOS neurons to be involved in enteric neuropathies. One of these is the failure of the neurons to maintain Ca(2+) homeostasis. In neurons in general, stress can increase cytoplasmic Ca(2+), causing a Ca(2+) toxicity. NOS neurons face the additional problem that NOS is activated by Ca(2+). This is hypothesized to produce an excess of NO, whose free radical properties can cause cell damage, which is exacerbated by peroxynitrite formed when NO reacts with oxygen free radicals.     

Regulation of the on bipolar cell mGluR6 pathway by Ca2+.

Glutamate produces a hyperpolarizing synaptic potential in On bipolar cells by binding to the metabotropic glutamate receptor mGluR6, leading to closure of a cation channel. Here it is demonstrated that this cation channel is regulated by intracellular Ca(2+). Glutamate-evoked currents were recorded from On bipolar cells in light-adapted salamander retinal slices in the presence of 2 mm external Ca(2+). When glutamate was applied almost continuously, interrupted only briefly to measure the size of the response, the glutamate response remained robust. However, currents elicited by intermittent and brief applications of glutamate exhibited time-dependent run down. Run down of the glutamate response was also voltage dependent, because it was accelerated by membrane hyperpolarization. Run down was triggered, at least in part, by a rise in intracellular Ca(2+); measured as a function of time or voltage, it was attenuated by intracellular buffering of Ca(2+) with BAPTA or by omitting Ca(2+) from the bathing solution. Current-voltage measurements demonstrated that Ca(2+) induced run down of the glutamate response by downregulating cation channel function, rather than by preventing closure of the channel by glutamate and mGluR6. A major source of the Ca(2+) that mediated this inhibition is the cation channel itself, which was found to be permeable to Ca(2+), accounting for the use dependence of the run down. These results suggest that Ca(2+) influx through the cation channel during background illumination could provide a signal to close the cation channel and repolarize the membrane toward its dark potential, an adaptive mechanism for coping with changes in ambient light.     

Spatial and temporal distribution of N-type Ca(2+) channels in gerbil global cerebral ischemia

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (alpha(1A)), N-type (alpha(1B)), and L-type (alpha(1C) and alpha(1D)) Ca(2+) channels were observed in the hippocampus at days 1-5 after ischemic injury. However, increased expression of N-type Ca(2+) channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca(2+) channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca(2+) concentration ([Ca(2+)](i)) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca(2+) channels may prove useful for future investigations.     

Effects of methylmercury on perineurial Na+ and Ca(2+)-dependent potentials at neuromuscular junctions of the mouse.

The ability of acute application of the neurotoxicant methylmercury (MeHg) to disrupt the function of presynaptic Ca2+ and Na+ channels at intact neuromuscular junctions was examined using mouse triangularis sterni motor nerves. In Ba(2+)-containing solutions, potential changes arising from Na+ and Ca2+ channel function could be recorded from the perineurial sheath surrounding motor neurons when K+ channels were blocked by tetraethylammonium chloride and 3,4-diaminopyridine. MeHg (100 microM) reduced both Na(+)- and Ba(2+)-dependent components to block within 3-5 min at apparently equivalent rates. Time to block was approximately 7 min after exposure to 50 microM MeHg. In 2 of 5 preparations exposed to 50 microM MeHg, the Ca2+ channel-mediated component was blocked prior to the Na+ channel-mediated component. In the remaining three preparations, Na(+)- and Ba(2+)-dependent potentials were blocked at similar times. Following block by MeHg, neither perfusing the preparation in MeHg-free solutions nor increasing the intensity and/or duration of stimulus to the intercostal nerves resulted in recovery of Na+ or Ca2+ potentials. In the presence of K+ channel blockers, repetitive firing of nerves in response to a single stimulus was observed in 20-30% of the triangularis preparations; in the two preparations treated with MeHg in which repetitive firing was observed, it decreased prior to block of the stimulus-induced Na+/Ba2+ potentials. These results corroborate the results obtained in isolated synaptosomes and pheochromocytoma cells, and suggest that MeHg decreases motor nerve excitability by disrupting Na+ channel function and may block neurotransmitter release by disrupting Na+ and Ca2+ channel function.     

Role of nitric oxide on ATP-induced Ca2+ signaling in outer hair cells of the guinea pig cochlea

Recently, a negative feedback effect of nitric oxide (NO) on the adenosine 5'-triphosphate (ATP)-induced Ca2+ response has been described in cochlear inner hair cells. We here investigated the role of NO on the ATP-induced Ca2+ response in outer hair cells (OHCs) of the guinea pig cochlea using the NO-sensitive dye DAF-2 and Ca2+ -sensitive dye fura-2. Extracellular ATP induced NO production in OHCs, which was inhibited by L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase (NOS) inhibitor, and suramin, a P2 receptor antagonist. ATP failed to induce NO production in the Ca2+ -free solution. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, enhanced the ATP-induced increase of the intracellular Ca2+ concentrations ([Ca2+]i), while L-NAME inhibited it. SNAP accelerated ATP-induced Mn2+ quenching in fura-2 fluorescence, while L-NAME suppressed it. 8-Bromoguanosine-cGMP, a membrane permeable analog of cGMP, mimicked the effects of SNAP. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase inhibited the ATP-induced [Ca2+]i increase. Selective neuronal NOS inhibitors, namely either 7-nitro-indazole or 1-(2-trifluoromethylphenyl) imidazole, mimicked the effects of L-NAME regarding both ATP-induced Ca2+ response and NO production. Immunofluorescent staining of neuronal nitric oxide synthase (nNOS) in isolated OHCs showed the localization of nNOS in the apical region of OHCs. These results suggest that the ATP-induced Ca2+ influx via a direct action of P2X receptors may be the principal source for nNOS activity in the apical region of OHCs. Thereafter, NO can be produced while conversely enhancing the Ca2+ influx via the NO-cGMP-PKG pathway by a feedback mechanism.     

Cholinergic inhibition of short (outer) hair cells of the chick's cochlea.

Cochlear hair cells are thought to be inhibited by the release of ACh from efferent neurons. Several studies have implicated Ca2+ as a postsynaptic intermediary in hair cell inhibition, but its role remains unproven. We have made whole-cell, tight-seal recordings from single short hair cells (the avian analog of outer hair cells in the mammalian cochlea), isolated from the chick's cochlea, to determine the mechanism of cholinergic inhibition. These cells hyperpolarized upon exposure to ACh, although a brief depolarization preceded the much larger, longer-lasting hyperpolarization. In voltage clamp ACh evoked an outward current that reversed in sign near the K+ equilibrium potential. A small, transient inward current preceded the predominant outward current. The ACh-evoked K+ current depended on Ca2+ in the external saline, or could be prevented when the cell was dialyzed with the rapid Ca2+ buffer BAPTA. In BAPTA-loaded cells a residual inward current was seen. This activated with very little delay upon exposure of the cell to ACh and reversed near 0 mV membrane potential. Thus, the hair cell ACh receptor appears to be a nonspecific cation channel through which Ca2+ enters and triggers the opening of nearby Ca(2+)-activated K+ channels. However, the ACh-evoked K+ channels are not the same as the "maxi" K+ channels activated by Ca2+ influx through voltage-gated Ca2+ channels in these same cells.     

α2-Adrenoceptors couple to inhibition of R-type calcium currents in myenteric neurons

Alpha2-adrenoceptors inhibit Ca2+ influx through voltage-gated Ca2+ channels throughout the nervous system and Ca2+ channel function is modulated following activation of some G-protein coupled receptors. We studied the specific Ca2+ channel inhibited following alpha2-adrenoceptor activation in guinea-pig small intestinal myenteric neurons. Ca2+ currents (I(Ca2+)) were studied using whole-cell patch-clamp techniques. Changes in intracellular Ca2+ (delta[Ca2+]i) in nerve cell bodies and varicosities were studied using digital imaging where Ca2+ influx was evoked by KCl (60 mmol L(-1)) depolarization. The alpha2-adrenoceptor agonist, UK 14 304 (0.01-1 micromol L(-1)) inhibited I(Ca2+) and delta[Ca2+]i; maximum inhibition of I(Ca2+) was 40%. UK 14 304 did not affect I(Ca2+) in the presence of SNX-482 or NiCl2 (R-type Ca2+ channel antagonists). UK 14 304 inhibited I(Ca2+) in the presence of nifedipine, omega-agatoxin IVA or omega-conotoxin, inhibitors of L-, P/Q- and N-type Ca2+ channels. UK 14 304 induced inhibition of I(Ca2+) was blocked by pertussis toxin pretreatment (1 microg mL(-1) for 2 h). Alpha2-adrenoceptors couple to inhibition of R-type Ca2+ channels via a pertussis toxin-sensitive pathway in myenteric neurons. R-type channels may be a target for the inhibitory actions of noradrenaline released from sympathetic nerves on to myenteric neurons.     

Exertional rhabdomyolysis in a patient with calcium adenosine triphosphatase deficiency.

A patient with exertional rhabdomyolysis and continuously elevated serum creatine kinase (CK) was investigated. The known causes of recurrent attacks of rhabdomyolysis were ruled out by appropriate histochemical and biochemical investigations. During ischaemic exercise tests an abnormal K(+)-efflux from exercising muscles was observed. The patient was found to have a deficiency of muscular Ca(2+)-ATPase. Dantrolene sodium therapy gave relief of muscle symptoms and improved the exercise tolerance. Both the CK level and the K(+)-efflux in ischaemic forearm testing became normal on this therapy.     

Molecular and electrophysiological evidence for the expression of BK channels in oligodendroglial precursor cells

Changes in intracellular Ca(2+) play a key role in regulating gene expression and developmental changes in oligodendroglial precursor cells (OPCs). However, the mechanisms by which Ca(2+) influx in OPCs is controlled remains incompletely understood. Although there are several mechanisms that modulate Ca(2+) influx, in many systems the large-conductance, voltage- and Ca(2+) -activated K(+) channel (BK channel) plays an important role in regulating both membrane excitability and intracellular Ca(2+) levels. To date, the role of the BK channel in the regulation of intracellular Ca(2+) in oligodendroglial lineage cells is unknown. Here we investigated whether cells of the oligodendroglial lineage express BK channels and what potential role they play in regulation of Ca(2+) influx in these cells. In oligodendrocytes derived from differentiated adult neural precursor cells (NPCs, obtained from C57bl6 mice) we observed outward currents that were sensitive to the BK channel blocker iberiotoxin (IbTx). Further confirmation of the expression of the BK channel was obtained utilizing other blockers of the BK channel and by confocal immunofluoresence labelling of the BK channel on oligodendroglia. Using Fura-2AM to monitor intracellular Ca(2+) , it was observed that inhibition of the BK channel during glutamate-induced depolarization led to an additive increase in intracellular Ca(2+) levels. Electrophysiological difference currents demonstrated that the expression levels of the BK channel decrease with developmental age. This latter finding was further corroborated via RT-PCR and Western blot analysis. We conclude that the BK channel is involved in regulating Ca(2+) influx in OPCs, and may potentially play a role during differentiation of oligodendroglial lineage cells.     

Ionic dependence of depolarization-mediated adipokinetic hormone release from the locust corpus cardiacum

The locust corpus cardiacum (CC) is a peripheral neurohemal organ in which are clustered a prodigious array of neurosecretory cells (NSCs), nearly all of which synthesize and release adipokinetic hormones (AKHs). We have examined the extracellular requirements for Na+ and Ca2+ in the process of AKH release following NSC depolarization by high extracellular K+ or veratridine. Na+ is not required for release mediated by high external K+ although Ca2+ is. The Ca2+ channel antagonists cobalt and lanthanum prevent release and support the hypothesis that depolarization with K+ leads to Ca2+ channel activation and subsequent AKH release. Tetrodotoxin does not block K+-mediated release suggesting that Na+ channel activation and Na+ influx are not required for K+-mediated release. The alkaloid veratridine leads to cobalt- and tetrodotoxin-sensitive release and this suggests that cell depolarization by Na+ channel activation is nevertheless capable of opening Ca2+ channels and initiating release. Release mediated by high external K+ is reduced by nifedipine but is not significantly reduced by methoxyverapamil, however veratridine-mediated release is slightly reduced by methoxyverapamil. Glandular lobes accumulated greater amounts of 45Ca2+ following high K+-mediated depolarization compared to glands incubated in normal saline and this enhanced accumulation was blocked by the Ca2+ channel antagonist lanthanum. During prolonged exposure to high K+ saline the release of AKHs and the uptake of 45Ca2+ reach a maximum and then gradually decline. The temporal pattern of the reduction in AKH release is similar to that of 45Ca2+ accumulation by the glandular lobe. This reduction in AKH release and 45Ca2+ uptake may result from inactivation of Ca2+ channels associated with the release process. These results indicate that Ca2+ influx into the NSCs by way of voltage-sensitive Ca2+ channels plays a critical role in the process of depolarization-mediated AKH release.     

Expression of ion channels and mutational effects in giant Drosophila neurons differentiated from cell division-arrested embryonic neuroblasts.

A culture system of "giant" Drosophila neurons derived from cytokinesis-arrested embryonic neuroblasts was developed to overcome the technical difficulties usually encountered in studying small Drosophila neurons. Cytochalasin B-treated neuroblasts differentiated into giant multinucleated cells that displayed neuronal morphology and neuron-specific markers (Wu et al., 1990). Here, we report that these giant neurons express different excitability patterns and membrane channels similar to those reported in excitable tissues of Drosophila. Individual neurons exhibited distinct all-or-none or graded voltage responses upon current injection. Both current- and voltage-clamp recordings could be performed on the same neuron because of the large cell size, thus making it possible to elucidate the functional role of the individual types of channels. By using pharmacological agents and ion substitution, the following currents were identified in these giant neurons: inward Na+ and Ca2+ currents and outward voltage-activated (the A-type and delayed rectifier) and Ca(2+)-activated K+ currents. In addition, we found a tetrodotoxin (TTX)-sensitive, Na(+)-dependent outward K+ current and a persistent component of an inward Na+ current, which have not been reported in Drosophila previously. This culture system can be used to analyze the mutational perturbations in ion channels and the resultant alterations in membrane excitability. Neurons from the mutant slowpoke (slo), which is known to lack a component of the Ca(2+)-activated K+ currents in muscles, exhibited prolonged action potentials associated with defects in the Ca(2+)-activated K+ current. This abnormality appeared to be more severe in the neurites than in the soma.     

L-NAME对大鼠局灶性脑缺血灌注损伤Fos蛋白表达的影响

目的观察一氧化氮含量的变化对缺血再灌注损伤后Fos蛋白表达的影响。方法采用线拴法制作大鼠局灶性脑缺血再灌注损伤模型,利用NADPH组化和Fos蛋白免疫组化双标技术研究NOS抑制剂L－NAME对大鼠局灶性脑缺血再灌注损伤脑皮层Fos蛋白表达的影响。结果缺血60min再灌注3h后损伤侧脑组织皮质一氧化氮合酶阳性神经元较正常增多并深染,Fos蛋白表达增加,L－NAME（3mg／kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组减少,L－NAME（10mg/kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组明显减少,同时也可见给予L－NAME后脑组织皮质内NOS阳性神经元无论在数量上还是在细胞着色、胞体突起均明显减少。结论c－fos基因表达也可能部分参与了NO的致神经细胞损伤过程。     

Calcium-activated chloride channels in müller cells acutely isolated from tiger salamander retina

Ca(2+)-activated chloride channels were identified with whole-cell patch-clamp recording techniques in salamander retinal Müller cells. Cl(Ca) channels were activated by membrane depolarizations that elicited Ca2+ influx or the application of the Ca2+ ionophore, ionomycin. The Ca channel blocker, Cd2+, abolished the Cl(Ca) channel tail currents. Increasing the duration of the depolarizing pulse resulted in enhancement of the Cl(Ca) channel tail current. Repetitive depolarizations with rapid pulses to +20 mV produced a buildup of I(Cl(Ca)), which reversed at 0 mV in symmetrical [Cl-] and at -40 mV when intracellular [Cl-] was reduced to 10% of the external concentration. I(Cl(Ca)) was blocked by the Cl channel blocker niflumic acid, while niflumic acid had no effect on voltage-gated Ca channels. These results offer the first demonstration of Cl(Ca) channels in a nonastrocytic glial cell and expand our understanding of the functional capacities of retinal glial cells.     

Involvement of the nitric oxide-cyclic GMP pathway and neuronal nitric oxide synthase in ATP-induced Ca2+ signalling in cochlear inner hair cells

We recently demonstrated that extracellular adenosine 5'-triphosphate (ATP) induced nitric oxide (NO) production in the inner hair cells (IHCs) of the guinea pig cochlea, which inhibited the ATP-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) by a feedback mechanism [Shen, J., Harada, N. & Yamashita, T. (2003) Neurosci. Lett., 337, 135-138]. We herein investigated the role of the NO-cGMP pathway and neuronal NO synthase (nNOS) in the ATP-induced Ca(2+) signalling in IHCs using the Ca(2+)-sensitive dye fura-2 and the NO-sensitive dye DAF-2. Fura-2 fluorescence-quenching experiments with Mn(2+) showed that ATP triggered a Mn(2+) influx. L-N(G)-nitroarginine methyl ester (L-NAME), a nonspecific NOS inhibitor, accelerated the ATP-induced Mn(2+) influx while S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, suppressed it. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase, and KT5823, an inhibitor of cGMP-dependent protein kinase, enhanced the ATP-induced [Ca(2+)](i) increase. 8-Bromoguanosine-cGMP, a membrane-permeant analogue of cGMP mimicked the effects of SNAP. Moreover, the effects of 7-nitroindazole, a selective nNOS inhibitor, mimicked the effects of L-NAME regarding both the enhancement of the ATP-induced Ca(2+) response and the attenuation of NO production. Immunofluorescent staining of nNOS using a single IHC revealed that nNOS was distributed throughout the IHCs, but enriched in the apical region of the IHCs as shown by intense staining. In conclusion, the ATP-induced Ca(2+) influx may be the principal source for nNOS activity, which may interact with P2X receptors in the apical region of IHCs. Thereafter, NO can be produced and conversely inhibits the Ca(2+) influx via the NO-cGMP-PKG pathway by a feedback mechanism.