Acidic regulation of junction channels between glomus cells in the rat carotid body. Possible role of [Ca]i

The purpose of this work was to characterize the gap junctions between cultured glomus cells of the rat carotid body and to assess the effects of acidity and accompanying changes in [Ca²⁺]_i on electric coupling. Dual voltage clamping of coupled glomus cells showed a mean macrojunctional conductance (G_j) of 1.16 nS±0.6 (S.E.), range 0.15–4.86 nS. At normal pH_o (7.43), a steady transjunctional voltage (ΔV_j=100.1±10.9 mV) showed multiple junction channel activity with a mean microconductance (g_j) of 93.98±0.6 pS, range 0.3–324.5 pS. Single-channel conductances, calculated as variance/mean g_j, gave a mean value of 16.7±0.2 pS, range 5.13–39.38 pS. Manual measurements of single-channel activity showed a mean g_j of 22.03±0.2 pS, range 1.3–160 pS. Computer analysis of the noise spectral density distribution gave a channel mean open time of 12.7±1.5 ms, range 6.37–23.42 ms. The number of junction channels, estimated in each experiment from G_j/single-channel g_j, showed a range of 7 to 258 channels (mean, 107.2). Optical measurements of [Ca²⁺]_i gave a mean value of 80.2±4.27 nM at pH_o of 7.43. Acidification of the medium with lactic acid (1 mM, pH 6.3) induced: 1) Variable changes in G_j (decreases and increases); 2) A significant decrease in mean g_j (to 80.36±0.34 pS) and in single-channel conductance (g_j=12.8±0.2 pS in computer analyses and 17.23±0.2 pS when measured by hand); 3) Variable changes in open times, resulting in a similar mean (12.8±1.5 ms) and 4) No change in the number of junction channels. When pH_o was lowered to 6.3 [Ca²⁺]_i did not change significantly (there were increases and decreases). However, when pH_o was lowered to 4.4, [Ca²⁺]_i increased significantly to 157.1±8.1 nM. It is concluded that saline acidification to pH 6.3 depresses the conductance of junction channels and this effect may be either a direct effect on channel proteins or synergistically enhanced by increases in [Ca²⁺]_i. However, there are no studies correlating changes of [Ca²⁺]_i and intercellular coupling in glomus cells. Stronger acidification (pH_o 4.4), producing much larger changes in [Ca²⁺]_i, may enhance this synergism. But, again, there are no studies correlating these effects.     

Capsaicin modulates K+ currents from dissociated rat taste receptor cells

Chili pepper is one of most widely used spices. The main active component of chili pepper is the capsaicin. The effects of capsaicin on sensory nerve endings are well known; however, little is known regarding the direct effect of capsaicin on taste receptor cells (TRCs). In this study, patch clamp methods were used to study the effects of capsaicin on the K(+) currents in TRCs isolated from the rat circumvallate papilla. Fura-2 microspectrofluorimetry was also used to determine the effects of capsaicin on the intracellular Ca(2+) concentration ([Ca(2+)](i)). In the resting state, whole-cell experiments identified outward-rectifying K(+) currents, which were inhibited by 5 mM tetraethylammonium (TEA(+)) chloride. Voltage-dependent K(+) channels with a conductance of 55+/-4 pS (mean+/-S.E.M.; n=3), were observed in cell-attached patches. Capsaicin (500 nM) completely inhibited the outward-rectifying K(+) current in the whole-cell recordings. In cell-attached patches 500 nM capsaicin significantly reduced the open probability (P(o)) of the K(+) channels from 0.401+/-0.052 (n=3) in the resting state, to 0.018+/-0.002 (n=3, P<0.05 by unpaired t-test). In the fura-2-loaded TRCs, micromolar concentrations of capsaicin increased [Ca(2+)](i) in a dose-dependent manner, e.g., 100 microM capsaicin consistently increased the 340:380 fluorescence ratio from 1.04+/-0.05 in the resting state to 1.40+/-0.05 (n=28). These results suggest that capsaicin can enhance or modify the gustatory sensation by inhibiting the K(+) currents of the TRCs directly.     

Characterisation of large-conductance calcium-activated potassium channels (BK(Ca)) in human NT2-N cells

Large-conductance calcium-activated potassium (BK(Ca)) channels were studied in inside-out patches of human NTERA2 neuronal cells (NT2-N). In symmetrical (140 mM) K(+) the channel mean conductance was 265 pS, the current reversing at approximately 0 mV. It was selective (P(K)/P(Na)=20:1) and blocked by internal paxilline and TEA. The open probability-voltage relationship for BK(Ca) was fitted with a Boltzmann function, the V((1/2)) being 76.3 mV, 33.6 mV and -14.1 mV at 0.1 muM, 3.3 muM and 10 muM [Ca(2+)](i), respectively. The relationship between open probability and [Ca(2+)](i) was fitted by the Hill equation (Hill coefficient 2.7, half maximal activation at 2.0 muM [Ca(2+)](i)). Open and closed dwell time histograms were fitted by the sum of two and three voltage-dependent exponentials, respectively. Increasing [Ca(2+)](i) produced both an increase in the longer open time constant and a decrease in the longest closed time constant, so increasing mean open time. "Intracellular" ATP evoked a concentration-dependent increase in NT2-N BK(Ca) activity. At +40 mV half-maximum activation occurred at an [ATP](i) of 3 mM (30 nM [Ca(2+)](i)). ADP and GTP were less potent, and AMP-PNP was inactive. This is the first characterisation of a potassium channel in NT2-N cells showing that it is similar to the BK(Ca) channel of other preparations.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca(2+)-activated potassium (BK(Ca)) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK(Ca) channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K(+) in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P(o)) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P(o) was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca(2+)](i) required to half activate BK(Ca) channels was only 1 microM in post-ischemic whereas 2 microM in control neurons, indicating an increase in [Ca(2+)](i) sensitivity after ischemia; and (4) BK(Ca) channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK(Ca) channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Intracellular calcium handling in rat olfactory ensheathing cells and its role in axonal regeneration

Intracellular calcium handling by rat olfactory ensheathing cells (OECs) is implicated in their support for regrowth of adult CNS neurites in a coculture model of axonal regeneration. Pretreatment of OECs with BAPTA-AM to sequester glial intracellular calcium ([Ca(2+)](i)) reduces significantly the numbers of cocultured neurons regrowing neurites. The mean resting [Ca(2+)](i) of OECs cultured alone or with neurons was 300 nM in an external solution containing 2.5 mM calcium ([Ca(2+)](o)). In high [K(+)](o) or zero [Ca(2+)](o), resting [Ca(2+)](i) significantly decreased. [Ca(2+)](i) significantly increased when [Ca(2+)](o) was increased to 20 mM, lonomycin, thapsigargin, and thimerosal increased [Ca(2+)](i), and caffeine, ryanodine, and cyclopiazonic acid were without effect. Of the receptor agonists tested, none induced a change in [Ca(2+)](i). The calcium influx induced by high [Ca(2+)](o) was blocked by La(3+) and SKF96365, partially inhibited by Cd(2+), and insensitive to Ni(2+) and nifedipine. Pretreatment of OECs with La(3+) reduced neurite regrowth in cocultures in a concentration-dependent manner over the range that blocked the non-voltage-gated calcium flux through a putative TRP-like channel, which, we propose, is activated in OEC-mediated axonal regeneration.     

Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons

The characterization of acid-sensing ion channel (ASIC)-like currents has been reported in hippocampal neurons in primary culture. However, it is suggested that the profile of expression of ASICs changes in culture. In this study, we investigated the properties of proton-activated current and its modulation by extracellular Ca(2+) and Zn(2+) in neurons acutely dissociated from the rat hippocampal CA1 using conventional whole-cell patch-clamp recording. A rapidly decaying inward current and membrane depolarization was induced by exogenous application of acidic solution. The current was sensitive to the extracellular proton with a response threshold of pH 7.0-6.8 and the pH(50) of 6.1, the reversal potential close to the Na(+) equilibrium potential. It had a characteristic of acid-sensing ion channels (ASICs) as demonstrated by its sensitivity to amiloride (IC(50)=19.6+/-2.1 microM). Either low [Ca(2+)](o) or high [Zn(2+)](o) increased the amplitude of the current. All these characteristics are consistent with a current mediated through a mixture of homomeric ASIC1a and heteromeric ASIC1a+2a channels and closely replicate many of the characteristics that have been previously reported for hippocampal neurons cultured for a week or more, indicating that culture artifacts do not necessarily flaw the properties of ASICs. Interestingly, we found that high [Zn(2+)](o) (>10(-4) M) slowed the decay time constant of the ASIC-like current significantly in both acutely dissociated and cultured hippocampal neurons. In addition, the facilitating effects of low [Ca(2+)](o) and high [Zn(2+)](o) on the ASIC-like current were not additive. Since tissue acidosis, extracellular Zn(2+) elevation and/or Ca(2+) reduction occur concurrently under some physiological and/or pathological conditions, the present observations suggest that hippocampal ASICs may offer a novel pharmacological target for therapeutic invention.     

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ to ouabain in carotid body glomus cells of adult rabbits.

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ ([Ca2+]i) to ouabain, an Na+/K+ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca2+]i (mean+/-S.E.M.; 38+/-5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca2+]i increase was abolished by the removal of extracellular Na+. D600 (50 microM), an L-type voltage-gated Ca2+ channel antagonist, inhibited the [Ca2+]i increase by 57+/-7% (n=4). Removal of extracellular Ca2+ eliminated the [Ca2+]i increase, but subsequent washing out of ouabain in Ca2+-free solution produced a rise in [Ca2+]i (62+/-8% increase, n=6, P<0.05), referred to as a [Ca2+]i rise after Ca2+-free/ouabain. The magnitude of the [Ca2+]i rise was larger than that of ouabain-induced [Ca2+]i increase. D600 (5 microM) inhibited the [Ca2+]i rise after Ca2+-free/ouabain by 83+/-10% (n=4). These results suggest that ouabain-induced [Ca2+]i increase was due to Ca2+ entry involving L-type Ca2+ channels which could be activated by cytosolic Na+ accumulation. Ca2+ removal might modify the [Ca2+]i response, resulting in the occurrence of a rise in [Ca2+]i after Ca2+-free/ouabain which mostly involved L-type Ca2+ channels.     

Desensitization of somatostatin-induced inhibition of low extracellular magnesium concentration-induced calcium spikes in cultured rat hippocampal neurons

Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg(2+) concentration ([Mg(2+)](o))-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg(2+)](o) to 0.1 mM elicited repetitive [Ca(2+)](i) spikes. These [Ca(2+)](i) spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18-24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca(2+)](i) spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 microM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCepsilon translocation inhibitor peptide (1 microM). In addition, suramin (3 microM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 microM) completely blocked the low [Mg(2+)](o)-induced [Ca(2+)](i) spikes, glutamate-induced [Ca(2+)](i) transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg(2+)](o)-induced [Ca(2+)](i) spikes through the activation of GRK2 and partly a novel PKCepsilon in cultured rat hippocampal neurons.     

Interferon-gamma acutely induces calcium influx in human microglia

The acute actions of the cytokine, interferon-gamma (IFN-gamma), on intracellular calcium [Ca(2+)](i) levels in human microglia were investigated. In the presence of a calcium-containing physiological solution (Ca(2+)-PSS), IFN-gamma caused a progressive increase in [Ca(2+)](i) to a plateau level with a mean rate of increase of 0.81 +/- 0.17 nM/s and mean amplitude of 102 +/- 12 nM (n = 67 cells). Washout of the cytokine did not alter the plateau established with IFN-gamma in Ca(2+)-PSS; however, introduction of a Ca(2+)-free PSS diminished [Ca(2+)](i) to baseline levels. The decrease in [Ca(2+)](i) with Ca(2+)-free PSS would indicate that the response to IFN-gamma was mediated by an influx pathway. This result was confirmed in separate experiments showing the lack of an induced change in [Ca(2+)](i) with IFN-gamma applied in Ca(2+)-free PSS. The increase in [Ca(2+)](i) induced in Ca(2+)-PSS was reduced to near baseline levels when the external solution contained low Cl(-) in the maintained presence of IFN-gamma suggesting that cellular depolarization inhibited the cytokine mediated entry pathway. The compound SKF96365, which blocks store operated influx of Ca(2+) in human microglia, was ineffective in altering the increase in [Ca(2+)](i), however, La(3+) completely inhibited the Ca(2+) response induced by IFN-gamma. Whole-cell patch clamp studies showed no effect of IFN-gamma to alter outward currents and inward rectifier K(+) currents. The influx of Ca(2+) may serve a signaling role in microglia linking IFN-gamma to functional responses of the cells to infiltrating T lymphocytes into the central nervous system (CNS) during inflammatory processes.     

BK channels in human glioma cells have enhanced calcium sensitivity

We have previously demonstrated the expression of large-conductance, calcium-activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside-out patches were activated at -60 mV by 2.1 x 10(-6) M cytosolic Ca(2+), were highly K(+)-selective, and had a slope conductance of approximately iqual 210 pS. We characterized the Ca(2+) sensitivity of these channels in detail by isolating BK currents in outside-out patches with different free [Ca(2+)](i). The half-maximal voltage for channel activation, V(0.5), of glioma BK currents in outside-out patches was +138 mV with 0 Ca(2+)/10 EGTA. V(0.5) was shifted to +81 mV and -14 mV with free [Ca(2+)](i) of 1.5 x 10(-7) M and 2.1 x 10(-6) M, respectively. These results suggest that glioma BK channels have a higher Ca(2+) sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary beta-subunits by glioma cells. Expression of the auxiliary beta-subunit of BK channels by glioma cells may relate to the high Ca(2+) sensitivity of glioma BK channels.     

Selective measurement of endothelial or smooth muscle [Ca(2+)](i) in pressurized/perfused cerebral arteries with fura-2

Despite a critical role for calcium in endothelial regulation of cerebrovascular tone, endothelial intracellular calcium ([Ca(2+)](i)) has never been measured in the context of an intact pressurized cerebral vessel. The purpose of the present study was to selectively measure endothelial or smooth muscle [Ca(2+)](i) and diameter in a pressurized/perfused cerebral vessel. In a pressurized rat middle cerebral artery, fura-2 AM was administered selectively to either the luminal (endothelium) or abluminal (smooth muscle) side of the vessel. Selectivity of loading was determined by measuring fura-2 fluorescence before and after removal of the endothelium. Removal of the endothelium virtually eliminated fura-2 fluorescence. In addition, 2-methylthioadenosine triphosphate (2MeS-ATP, a selective endothelial P2 receptor agonist) was used to infer the selectivity of fura-2 loading. It was reasoned that 2MeS-ATP should produce a decrease in smooth muscle [Ca(2+)](i) and an increase in endothelial [Ca(2+)](i) in selectively loaded vessels, consistent with its role as an NO-dependent dilator. In smooth muscle loaded vessels, [Ca(2+)](i) went from 252+/-8 to 82+/-9 nM following luminal administration of 2MeS-ATP, whereas in endothelial loaded vessels, [Ca(2+)](i) went from 137+/-11 to 271+/-20 nM. Thus, a method is provided which allows for selective measurement of endothelial or smooth muscle [Ca(2+)](i) with simultaneous measurement of diameter in a pressurized cerebral vessel.     

Antioxidants prevent elevation in [Ca(2+)](i) induced by low extracellular magnesium in cultured canine cerebral vascular smooth muscle cells: possible relationship to Mg(2+) deficiency-induced vasospasm and stroke

Low serum concentrations of Mg(2+) ions have been reported, recently, in patients with coronary disease, atherosclerosis and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [alpha-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Ca(2+) ([Ca(2+)](i)) overload associated with cerebral vascular injury induced by low extracellular free Mg(2+) ([Mg(2+)](o)). Exposure of cultured canine cerebral vascular smooth muscle cells to low [Mg(2+)](o) (0.15-0.6 mM) vs. normal [Mg(2+)](o) (1.2 mM) for either 10 min or 2 h induced concentration-dependent rises in [Ca(2+)](i). Treatment of the cultured cells with either PDTC (0.1 microM) or alpha-tocopherol (15 microM) for 24 h, alone, failed to interfere with basal [Ca(2+)](i) levels. However, preincubation of the cells with either alpha-tocopherol or PDTC for 24 h completely inhibited the elevation of [Ca(2+)](i) induced by exposure to low [Mg(2+)](o), not only for 10 min, but also for 2 h. These results indicate that alpha-tocopherol and PDTC prevent rises in [Ca(2+)](i) produced by low [Mg(2+)](o), which probably result from low [Mg(2+)](o)-induced lipid peroxidation of cerebral vascular smooth muscle cell membranes. Moreover, these new results suggest that such protective effects of alpha-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in cerebral vascular injury associated with low [Mg(2+)](o) and accumulation of [Ca(2+)](i).     

Effects of inhibition of fatty acid amide hydrolase vs. the anandamide membrane transporter on TRPV1-mediated calcium responses in adult DRG neurons; the role of CB receptors

The aim of the present study was to investigate the relationship between TRPV1 stimulation and endocannabinoid-driven CB(1) receptor-mediated inhibition of activity in adult rat dorsal root ganglion (DRG) neurons, a model of primary afferent nociceptors. Calcium-imaging studies were performed to compare the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 microm) vs. the anandamide (AEA) uptake inhibitor UCM707 (1 microm) on capsaicin (100 nm) and N-arachidonoyl dopamine (NADA; 1 microm)-evoked changes in intracellular calcium [Ca(2+)](i) in DRG neurons. The ability of the CB(1) receptor antagonist AM251 (1 microm) to modulate the effects of URB597 and UCM707 was also determined. Suprafusion of NADA and capsaicin evoked robust increases in [Ca(2+)](i) in DRG neurons (89 +/- 4% and 132 +/- 6% of the depolarizing KCl response, respectively). Co-incubation with URB597 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (39 +/- 3% and 79 +/- 4% of KCl response, respectively). Similarly, co-incubation with UCM707 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (59 +/- 7% and 72 +/- 4% of KCl response, respectively). The CB(1) receptor antagonist AM251 significantly attenuated the effects of URB597 on NADA-evoked increases in [Ca(2+)](i) but not the effects of URB597 on capsaicin-evoked increases in [Ca(2+)](i). By contrast, AM251 significantly attenuated the inhibitory effects of UCM707 on both NADA and capsaicin-evoked increases in [Ca(2+)](i.) These data suggest that transport of both NADA and capsaicin into DRG neurons and the subsequent activation of TRPV1 is partly governed by FAAH-dependent mechanisms as well as via the putative AEA membrane transporter.     

Regulation of transmitter release by Ca(2+) and synaptotagmin: insights from a large CNS synapse

Transmitter release at synapses is driven by elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) near the sites of vesicle fusion. [Ca(2+)](i) signals of profoundly different amplitude and kinetics drive the phasic release component during a presynaptic action potential, and asynchronous release at later times. Studies using direct control of [Ca(2+)](i) at a large glutamatergic terminal, the calyx of Held, have provided significant insight into how intracellular Ca(2+) regulates transmitter release over a wide concentration range. Synaptotagmin-2 (Syt2), the major isoform of the Syt1/2 Ca(2+) sensors at these synapses, triggers highly Ca(2+)-cooperative release above 1μM [Ca(2+)](i), but suppresses release at low [Ca(2+)](i). Thus, neurons utilize a highly sophisticated release apparatus to maximize the dynamic range of Ca(2+)-evoked versus spontaneous release.     

Raising intracellular calcium attenuates neuronal apoptosis triggered by staurosporine or oxygen-glucose deprivation in the presence of glutamate receptor blockade

The relationship between intracellular Ca(2+) ([Ca(2+)](i)) regulation and programmed cell death is not well-defined; both increases and decreases in [Ca(2+)](i) have been observed in cells undergoing apoptosis. We determined [Ca(2+)](i) in cultured murine cortical neurons undergoing apoptosis after exposure to staurosporine or following oxygen-glucose deprivation in the presence of glutamate receptor antagonists. Neuronal [Ca(2+)](i) was decreased 1-4 h after exposure to staurosporine (30 nM). A [Ca(2+)](i) decrease was also observed 1 h after the end of the oxygen-glucose deprivation period when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were added to the bathing medium during the deprivation period. A similar decrease in [Ca(2+)](i) produced by reducing extracellular Ca(2+) or chelating intracellular Ca(2+) was sufficient to induce neuronal apoptosis. Raising [Ca(2+)](i) either by activating voltage-sensitive Ca(2+) channels with (-) Bay K8644 or by application of low concentrations of kainate attenuated both staurosporine and oxygen-glucose deprivation-induced apoptosis.     

Serotonin-induced platelet intracellular Ca2+ response in patients with anorexia nervosa

INTRODUCTION: Serotonin (5-HT) is involved in the regulation of food intake. In anorexia nervosa there is a disturbance in 5-HT function. The stimulation of 5-HT(2)-receptors in platelets is a useful peripheral model to investigate the cascade of signal transduction and neuronal functioning. METHODS: 25 anorexic female patients between the ages of 11 and 18 years with a mean body mass index (BMI) of 13.9+/-1.3 kg/m(2) participated in this study. The 21 healthy female controls revealed a mean BMI of 20.5+/-2.7 kg/m(2). 5-HT stimulated intracellular Ca(2+) response of the platelets was obtained using the Fura-2 method at the time of admission, during therapy and when the target BMI was reached. RESULTS: We found a significant (p<0.01) decrease in 5-HT-induced Delta[Ca(2+)](i) at admission and a significant (p<0.05) increase of Delta[Ca(2+)](i) during treatment in patients with anorexia nervosa. Anorexic patients with and without comorbid depression had a comparable Ca(2+) release. However, low and high Ca(2+) responders showed a different course of Delta[Ca(2+)](i). The treatment with antidepressants led to a significant increase of Delta[Ca(2+)](i) in those patients with concomitant depression. DISCUSSION: Since the course of Delta[Ca(2+)](i) is not related to BMI or the presence of comorbid depression, we conclude that serotonergic transmission or signaling pathways could be disturbed in patients suffering from anorexia nervosa. One inference of this preliminary study is that administration of antidepressants may be more effective in patients with concomitant depression.     

Hypoxic and hypercapnic responses of [Ca]0 and [K]0 in the cat carotid body in vitro

Measurements of extracellular Ca2+ and K+ activities [( Ca2+]o, [K+]o) in the superfused cat carotid body in vitro with triple-barrelled ion-selective electrodes have shown that hypoxia induced a decrease in [Ca2+]o of 0.035 +/- 0.17 mM (mean +/- S.D.; n = 17) and a biphasic change in [K+]o which consisted of an increase of 2.3 +/- 1.8 mM followed by an undershoot of -0.52 +/- 0.34 mM (mean +/- S.D.; n = 17). Hypercapnia induced a monophasic upward deflection increase of both [Ca2+]o and [K+]o of about 0.037 +/- 0.013 mM and 0.33 +/- 0.15 mM, respectively (n = 17). During hypoxia, lowering [Ca2+] in the medium to 0.1 mM resulted in a reversed [Ca2+]o response, attenuated [K+]o increase and absence of chemosensory nerve discharges. TTX generally did not affect the hypoxic and hypercapnic induced ionic changes, although the [K+]o undershoot was reduced by 30%. Co2+ competitively blocked the changes in [Ca2+]o and the increase in the sensory nerve discharge elicited by hypoxia and, not competitively, the changes of [K+]o. The ionic changes to hypercapnia were less affected by Co2+. Ouabain inhibited the [K+]o undershoot induced by hypoxia, as did the removal of Na+ from medium. It is concluded that changes in extracellular free Ca2+ and K+ ions concentration induced by hypoxia and hypercapnia represent ionic fluxes related to the transduction process of carotid body cells (glomus and/or sustentacular).     

Properties of NMDA receptors in rat spinal cord motoneurons

Postnatal development and properties of N-methyl-d-aspartate (NMDA) receptors were studied with whole-cell and outside-out patch-clamp techniques in interneurons and fluorescence-labelled motoneurons in rat spinal cord slices. Both the absolute amplitude of NMDA-induced currents and currents normalized with respect to the motoneuron capacitance increased significantly at postnatal days 10-13 when compared to the responses evoked at postnatal days 2-3. The mean amplitude of the responses to kainate also increased in motoneurons of postnatal days 10-13. Single-channel currents induced by low concentrations of glutamate, exhibited four distinct amplitude levels corresponding to 19.2 +/- 2.4 pS, 38.4 +/- 3.5 pS, 56.3 +/- 2. 4 pS and 69.6 +/- 3.7 pS. In contrast, the conductance of single channels, recorded under identical conditions, in rat spinal cord interneurons was less, 15.3 +/- 3.2 pS, 29.9 +/- 5.4 pS, 46.7 +/- 4. 8 pS and 62.4 +/- 3.9 pS. The high (56/70 pS) conductance single-channel openings in motoneuron patches were sensitive to NMDA receptor inhibitors D-2-amino-5-phosphonovalerate, 7-chlorokynurenic acid and ifenprodil. Whole-cell NMDA-evoked currents were blocked in a voltage-dependent manner by extracellular Mg2+ with an apparent dissociation constant for Mg2+ binding at 0 mV of 1.8 +/- 0.5 mm. The conductance and relative distribution of NMDA receptor channel openings induced by 1 micrometer glutamate in patches isolated from the motoneurons were independent of age from postnatal day 4 to 14. The results suggest that the properties of NMDA receptor channels in motoneurons differ from those in spinal cord interneurons and cells transfected with NR1/NR2 subunits.     

Chlorotoxin-sensitive Ca2+-activated Cl- channel in type R2 reactive astrocytes from adult rat brain

Astrocytes express four types of Cl(-) or anion channels, but Ca(2+)-activated Cl(-) (Cl(Ca)) channels have not been described. We studied Cl(-) channels in a morphologically distinct subpopulation ( approximately 5% of cells) of small (10-12 micro m, 11.8 +/- 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, -57.1 +/- 4.0 mV, polarized to -72.7 +/- 4.5 mV with BAPTA-AM, an intracellular Ca(2+) chelator, and depolarized to -30.7 +/- 6.1 mV with thapsigargin, which mobilizes Ca(2+) from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl(-) reversal potential, which was blocked by Cl(-) channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn(2+), by I(-) (10 mM), and by chlorotoxin (EC(50) = 47 nM). With conventional whole-cell clamp, NPPB- and Zn(2+)-sensitive currents became larger with increasing [Ca(2+)](i) (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca(2+) sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive Cl(Ca) channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive Cl(Ca) channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte.     

Effects of hypoxia and putative transmitters on [Ca2+]i of rat glomus cells

Dissociated rat glomus cells were loaded with Fura-2 AM to study the effects of hypoxia, and carotid body transmitters on intracellular calcium, [Ca2+]i. The mean control [Ca2+]i was 55 nM in isolated cells and 67 nM in clusters. The following procedures changed [Ca2+]i:0[Ca2+]o+EGTA reduced [Ca2+]i by about 50%, suggesting that the remaining calcium originated from intracellular organelles. [Ca2+]i increased when [Ca2+]o was doubled.Hypoxia by sodium dithionite (Na2S2O4) induced large [Ca2+]i increases in clustered and isolated cells. Smaller rises occurred with 100% N2 hypoxia. The augmented [Ca2+]i, induced by Na2S2O4, was reduced (not eliminated) in 0[Ca2+]o+EGTA, suggesting that some calcium was intracellularly released. Nifedipine depressed (did not block) the Na2S2O4-induced calcium increase, implying some inflow via other (N, T or P/Q) voltage-dependent or voltage-independent calcium channels.Cholinergic agents (ACh, nicotine, muscarine, bethanechol and pilocarpine) increased [Ca2+]i. The ACh effect was produced exclusively by calcium inflow since it was eliminated in 0[Ca2+]o+EGTA. Cholinergic effects were depressed (not obliterated) by D-tubocurarine (D-TC), hexamethonium (C6) and atropine.ACh, nicotine and pilocarpine potentiated the excitatory effect of Na2S2O4 on [Ca2+]i. Bethanechol depressed this excitation whereas muscarine had inconsistent effects.Atropine and C6 depressed [Ca2+]i increases elicited by Na2S2O4 but the effects of D-TC were variable.Dopamine (DA) had variable effects. It increased [Ca2+]i in 75% of cases, and reduced the Na2S2O4 -induced calcium increase.Thus, calcium increases during Na2S2O4 occur by direct effects on the glomus cells and feedback action through released ACh and DA.