Evidence for cadmium mobilization of intracellular calcium through a divalent cation receptor in renal distal epithelial A6 cells

The effect of Cd(2+) on intracellular Ca(2+) homeostasis was examined in renal epithelial A6 cells loaded with Fura-2. Cd(2+) (10 microM to 1 mM) produced a transient spike in cytosolic Ca(2+) in a dose-dependent manner. The phospholipase C inhibitor U73122 and the cation receptor agonist, neomycin, both diminish Cd(2+)-evoked increase in intracellular Ca(2+) ([deltaCa(2+)](Cd)). Further, thapsigargin, an inhibitor of intracellular Ca(2+)-ATPases, significantly reduced [deltaCa(2+)](Cd). Extending these observations, inositol-3-phosphate (IP(3)) binding studies showed that the resting level of intracellular IP(3) underwent a 1.45-fold increase when exposed to Cd(2+). Furthermore, we found that the Cd(2+)-related heavy metals, Zn(2+) and Ni(2+), were even more potent inducers of Ca(2+) mobilization and IP(3) generation than Cd(2+). It can be concluded that Cd(2+), and possibly Zn(2+) and Ni(2+), may act as agonists of a cation-sensing receptor (CSR) belonging to G-protein receptors capable of mediating IP(3) release of Ca(2+) from intracellular stores. The CSR receptor in A6 epithelia could not be stimulated with neomycin or Gd(3+), suggesting that the receptor is different from the calcium-sensing receptor.     

Extracellular pH changes and accompanying cation shifts during ouabain-induced spreading depression

Interstitial ionic shifts that accompany ouabain-induced spreading depression (SD) were studied in rat hippocampal and cortical slices in the presence and absence of extracellular Ca(2+). A double-barreled ion-selective microelectrode specific for H(+), K(+), Na(+), or Ca(2+) was placed in the CA1 stratum radiatum or midcortical layer. Superfusion of 100 microM ouabain caused a rapid, negative, interstitial voltage shift (2-10 mV) after 3-5 min. The negativity was accompanied by a rapid alkaline transient followed by prolonged acidosis. In media containing 3 mM Ca(2+), the alkalosis induced by ouabain averaged 0.07 +/- 0.01 unit pH. In media with no added Ca(2+) and 2 mM EGTA, the alkaline shift was not significantly different (0.09 +/- 0.02 unit pH). The alkaline transient was unaffected by inhibiting Na(+)-H(+) exchange with ethylisopropylamiloride (EIPA) or by blocking endoplasmic reticulum Ca(2+) uptake with thapsigargin or cyclopiazonic acid. Alkaline transients were also observed in Ca(2+)-free media when SD was induced by microinjecting high K(+). The late acidification accompanying ouabain-induced SD was significantly reduced in Ca(2+)-free media and in solutions containing EIPA. The ouabain-induced SD was associated with a rapid but relatively modest increase in [K(+)](o). In the presence of 3 mM external Ca(2+), the mean peak elevation of [K(+)](o) was 12 +/- 0.62 mM. In Ca(2+)-free media, the elevation of [K(+)](o) had a more gradual onset and reached a significantly larger peak value, which averaged 22 +/- 1.1 mM. The decrease in [Na(+)](o) that accompanied ouabain-induced SD was somewhat greater. The [Na(+)](o) decreased by averages of 40 +/- 7 and 33 +/- 3 mM in Ca(2+) and Ca(2+)-free media, respectively. In media containing 1.2 mM Ca(2+), ouabain-induced SD was associated with a substantial decrease in [Ca(2+)](o) that averaged 0.73 +/- 0. 07 mM. These data demonstrate that in comparison with conventional SD, ouabain-induced SD exhibits ion shifts that are qualitatively similar but quantitatively diminished. The presence of external Ca(2+) can modulate the phenomenon but is irrelevant to the generation of the SD and its accompanying alkaline pH transient. Significance of these results is discussed in reference to the propagation of SD and the generation of interstitial pH changes.     

Effects of anandamide and noxious heat on intracellular calcium concentration in nociceptive drg neurons of rats

As an endogenous agonist at the cannabinoid receptor CB1 and the capsaicin-receptor TRPV1, anandamide may exert both anti- and pronociceptive actions. Therefore we studied the effects of anandamide and other activators of both receptors on changes in free cytosolic calcium ([Ca(2+)](i)) in acutely dissociated small dorsal root ganglion neurons (diameter: < or =30 microm). Anandamide (10 microM) increased [Ca(2+)](i) in 76% of the neurons. The EC(50) was 7.41 microM, the Hill slope was 2.15 +/- 0.43 (mean +/- SE). This increase was blocked by the competitive TRPV1-antagonist capsazepine (10 microM) and in Ca(2+)-free extracellular solution. Neither exclusion of voltage-gated sodium channels nor additional blockade of voltage-gated calcium channels of the L-, N-, and/or T-type, significantly reduced the anandamide-induced [Ca(2+)](i) increase or capsaicin-induced [Ca(2+)](i) transients (0.2 microM). The CB1-agonist HU210 (10 microM) inhibited the anandamide-induced rise in [Ca(2+)](i). Conversely, the CB1-antagonist AM251 (3 microM) induced a leftward shift of the concentration-response relationship by approximately 4 microM (P < 0.001; Hill slope, 2.17 +/- 0.75). Intracellular calcium transients in response to noxious heat (47 degrees C for 10 s) were highly correlated with the anandamide-induced [Ca(2+)](i) increases (r = 0.84, P < 0.001). Heat-induced [Ca(2+)](i) transients were facilitated by preincubation with subthreshold concentrations of anandamide (3 microM), an effect that was further enhanced by 3 microM AM251. Although anandamide acts on both TRPV1 and CB1 receptors in the same nociceptive DRG neurons, its pronociceptive effects dominate. Anandamide triggers an influx of calcium through TRPV1 but no intracellular store depletion. It facilitates the heat responsiveness of TRPV1 in a calcium-independent manner. These effects of anandamide differ from those of the classical exogenous TRPV1-agonist capsaicin and suggest a primarily modulatory mode of action of anandamide.     

Involvement of the ryanodine-sensitive Ca2+ store in GLP-1-induced Ca2+ oscillations in insulin-secreting HIT cells

We investigated intracellular Ca(2+) ([Ca(2+)](i)) oscillations evoked by glucagon-like peptide 1 (GLP-1) in relation to the ryanodine receptor (RyR) and Ca(2+)-induced Ca(2+)release (CICR) mechanism in pancreatic B cell HIT. GLP-1 produced [Ca(2+)](i) oscillations in the cells, both in media with and without Ca(2+), an effect inhibited by ruthenium red and mimicked by 8-Br-cAMPS. In addition, the GLP-1-evoked [Ca(2+)](i) rise was initiated at the local intercellular peripheral cytoplasm, and a resultant expansion of the intercellular space was also observed. Caffeine induced [Ca(2+)](i) elevation in the medium with or without Ca(2+), an effect inhibited by ruthenium red. GLP-1-evoked [Ca(2+)](i) oscillations were also enhanced by IBMX, and eliminated by Rp-8-Br-cAMPS or 20 microM H-89 treatments whereas they were unaffected by 2 microM H-89 treatment. Forskolin caused a transient elevation in [Ca(2+)](i) that was reduced by Rp-8-Br-cAMPS, 2 microM or 20 microM H-89. Our results indicate that GLP-1 initially generated a local [Ca(2+)](i) elevation at the peripheral cytoplasm, subsequently producing [Ca(2+)](i) oscillations that were inhibited by ruthenium red, involving ryanodine-sensitive and cAMP-activated CICR mechanisms. The cytoplasmic levels of cAMP as well as local Ca(2+) might be responsible for [Ca(2+)](i) oscillations.     

Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.     

'Pressure-flow'-triggered intracellular Ca2+ transients in rat cardiac myocytes: possible mechanisms and role of mitochondria

Cardiac myocytes, in the intact heart, are exposed to shear/fluid forces during each cardiac cycle. Here we describe a novel Ca(2+) signalling pathway, generated by 'pressurized flows' (PFs) of solutions, resulting in the activation of slowly developing ( approximately 300 ms) Ca(2+) transients lasting approximately 1700 ms at room temperature. Though subsequent PFs (applied some 10-30 s later) produced much smaller or undetectable responses, such transients could be reactivated following caffeine- or KCl-induced Ca(2+) releases, suggesting that a small, but replenishable, Ca(2+) pool serves as the source for their activation. PF-triggered Ca(2+) transients could be activated in Ca(2+)-free solutions or in solutions that block voltage-gated Ca(2+) channels, stretch-activated channels (SACs), or the Na(+)-Ca(2+) exchanger (NCX), using Cd(2+), Gd(3+), or Ni(2+), respectively. PF-triggered Ca(2+) transients were significantly smaller in quiescent than in electrically paced myocytes. Paced Ca(2+) transients activated at the peak of PF-triggered Ca(2+) transients were not significantly smaller than those produced normally, suggesting functionally separate Ca(2+) pools for paced and PF-triggered transients. Suppression of nitric oxide (NO) or IP(3) signalling pathways did not alter the PF-triggered Ca(2+) transients. On the other hand, mitochondrial metabolic uncoupler FCCP, in the presence of oligomycin (to prevent ATP depletion), reversibly suppressed PF-triggered Ca(2+) transients, as did the mitochondrial Ca(2+) uniporter (mCU) blocker, Ru360. Reducing agent DTT and reactive oxygen species (ROS) scavenger tempol, as well as mitochondrial NCX (mNCX) blocker CGP-37157, inhibited PF-triggered Ca(2+) transients. In rhod-2 AM-loaded and permeabilized cells, confocal imaging of mitochondrial Ca(2+) showed a transient increase in Ca(2+) on caffeine exposure and a decrease in mitochondrial Ca(2+) on application of PF pulses of solution. These signals were strongly suppressed by either Na(+)-free or CGP-37157-containing solutions, implicating mNCX in mediating the Ca(2+) release process. We conclude that subjecting rat cardiac myocytes to pressurized flow pulses of solutions triggers the release of Ca(2+) from a store that appears to access mitochondrial Ca(2+).     

Relative distribution of Ca2+ channels at the crayfish inhibitory neuromuscular junction

We investigated the Ca(2+) channel-synaptic vesicle topography at the inhibitor of the crayfish (Procambarus Clarkii) neuromuscular junction (NMJ) by analyzing the effect of different modes of Ca(2+) channel block on transmitter release. Initial identification of Ca(2+) channels revealed the presence of two classes, P and non-P-type with P-type channels governing approximately 70% of the total Ca(2+) influx. The remaining Ca(2+) influx was completely blocked by Cd(2+) but not by saturating concentrations of omega-conotoxins MVIIC and GVIA, or nifedipine and SNX-482. To examine the relative spatial distribution of Ca(2+) channels with respect to synaptic vesicles, we compared changes in inhibitory postsynaptic current amplitude and synaptic delay resulting from different spatial profiles of [Ca(2+)](i) around release sites. Specifically, addition of either [Mg(2+)](o), which decreases single-channel current, or omega-Aga IVA, which completely blocks P-type channels, prolonged synaptic delay by a similar amount when Ca(2+) influx block was <40%. Because non-P-type channels are able to compensate for blocked P-type channels, it suggests that these channels overlap considerably in their distribution. However, when Ca(2+) influx was blocked by approximately 50%, omega-Aga IVA increased delay significantly more than Mg(2+), suggesting that P-type channels are located closer than non-P-type channels to synaptic vesicles. This distribution of Ca(2+) channels was further supported by the observations that non-P-type channels are unable to trigger release in physiological saline and EGTA preferentially prolongs synaptic delay dominated by non-P-type channels when transmitter release is evoked with broad action potentials. We therefore conclude that although non-P-type channels do not directly trigger release under physiological conditions, their distribution partially overlaps with P-type channels.     

Dual effect of Zn2+ on multiple types of voltage-dependent Ca2+ currents in rat palaeocortical neurons

The effects of Zn(2+) were evaluated on high-voltage-activated Ca(2+) currents expressed by pyramidal neurons acutely dissociated from rat piriform cortex. Whole-cell, patch-clamp experiments were carried out using Ba(2+) (5 mM) as the charge carrier. Zn(2+) blocked total high-voltage-activated Ba(2+) currents with an IC(50) of approximately 21 microM. In addition, after application of non-saturating Zn(2+) concentrations, residual currents activated with substantially slower kinetics than control Ba(2+) currents. Both of the above-mentioned effects of Zn(2+) were also observed in high-voltage-activated currents recorded in the presence of nearly-physiological concentrations of extracellular Ca(2+) (1 and 2 mM) rather than Ba(2+). Under the latter conditions, 30 microM Zn(2+) inhibited high-voltage-activated currents somewhat less than observed in extracellular Ba(2+) (approximately 47% and approximately 41%, respectively, vs. approximately 59%), but slowed Ca(2+)-current activation to very similar degrees. All of the pharmacological components in which Ba(2+) currents could be dissected (L-, N-, P/Q-, and R-type) were inhibited by Zn(2+), the percentage of current blocked by 30 microM Zn(2+) ranging from 34 to 57%. Moreover, the activation kinetics of all pharmacological Ba(2+) current components were slowed by Zn(2+). Hence, the lower activation speed observed in residual Ba(2+) currents after Zn(2+) block is due to a true slowing of macroscopic Ca(2+)-current activation kinetics and not to the preferential inhibition of a fast-activating current component. The inhibitory effect of Zn(2+) on Ba(2+) current amplitude was voltage-independent over the whole voltage range explored (-60 to +30 mV), hence the Zn(2+)-dependent decrease of Ba(2+) current activation speed is not the consequence of a voltage- and time-dependent relief from block. Zn(2+) also caused a slight, but significant, reduction of Ba(2+) current deactivation speed upon repolarization, which is further evidence against a depolarization-dependent unblocking mechanism. Finally, the slowing effect of Zn(2+) on Ca(2+)-channel activation kinetics was found to result in a significant, extra reduction of Ba(2+) current amplitude when action-potential-like waveforms, rather than step pulses, were used as depolarizing stimuli. We conclude that Zn(2+) exerts a dual action on multiple types of voltage-gated Ca(2+) channels, causing a blocking effect and altering the speed at which channels are delivered to conducting states, with mechanism(s) that could be distinct.     

Hypocretin/orexin peptide signaling in the ascending arousal system: elevation of intracellular calcium in the mouse dorsal raphe and laterodorsal tegmentum

Dysfunction of the hypocretin/orexin (Hcrt/Orx) peptide system is closely linked to the sleep disorder narcolepsy, suggesting that it is also central to the normal regulation of sleep and wakefulness. Indeed, Hcrt/Orx peptides produce long-lasting excitation of arousal-related neurons, including those in the laterodorsal tegmentum (LDT) and the dorsal raphe (DR), although the mechanisms underlying these actions are not understood. Since Hcrt/Orx mobilizes intracellular calcium ([Ca(2+)](i)) in cells transfected with orexin receptors and since receptor-mediated Ca(2+) transients are ubiquitous signaling mechanisms, we investigated whether Hcrt/Orx regulates [Ca(2+)](i) in the LDT and DR. Changes in [Ca(2+)](i) were monitored by fluorescence changes of fura-2 AM loaded cells in young mouse brain slices. We found Hcrt/Orx (Orexin-A, 30-1,000 nM) evoked long-lasting increases in [Ca(2+)](i) with differing temporal profiles ranging from spiking to smooth plateaus. A fragment of Hcrt/Orx (16-33) failed to evoke changes in [Ca(2+)](i) and changes were not blocked by TTX or ionotropic glutamate receptor antagonists, suggesting they resulted from specific activation of postsynaptic orexin receptors. Unlike orexin receptor-transfected cells, Hcrt/Orx-responses were not attenuated by depletion of Ca(2+) stores with cyclopiazonic acid (CPA; 3-30 microM), thapsigargin (3 microM), or ryanodine (20 microM), although store-depletion by either CPA or ryanodine blocked Ca(2+) mobilization by the metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 30 microM). In contrast, Hcrt/Orx responses were strongly attenuated by lowering extracellular Ca(2+) ( approximately 20 microM) but were not inhibited by concentrations of KB-R7943 (10 microM) selective for blockade of sodium/calcium exchange. Nifedipine (10 microM), inhibited Hcrt/Orx responses but was more effective at abolishing spiking than plateau responses. Bay K 8644 (5-10 microM), an L-type calcium channel agonist, potentiated responses. Finally, responses were attenuated by inhibitors of protein kinase C (PKC) but not by inhibitors of adenylyl cyclase. Collectively, our findings indicate that Hcrt/Orx signaling in the reticular activating system involves elevation of [Ca(2+)](i) by a PKC-involved influx of Ca(2+) across the plasma membrane, in part, via L-type calcium channels. Thus the physiological release of Hcrt/Orx may help regulate Ca(2+)-dependent processes such as gene expression and NO production in the LDT and DR in relation with behavioral state. Accordingly, the loss of Hcrt/Orx signaling in narcolepsy would be expected to disrupt calcium-dependent processes in these and other target structures.     

IgG-mediated signal transduction in canine mastocytoma-derived cells

BACKGROUND: We have reported canine cutaneous mastocytoma-derived cells named CM-MC sensitized with monomeric IgG released histamine upon anti-IgG stimulation. However, IgG or IgE-mediated signal transduction in the cells remains to be examined. METHODS: Monomeric IgG-binding to cells was measured by flow cytometry using FITC-anti-IgG. IgG-mediated protein tyrosine phosphorylation was studied by Western blotting using anti-phosphotyrosine antibody. We monitored the intracellular Ca(2+) concentration ([Ca(2+)](i)) when IgG-primed cells were activated with anti-canine IgG. Release of Ca(2+) from intracellular stores was analyzed with thapsigargin in the absence of extracellular Ca(2+). The Ca(2+) entry via store-operated Ca(2+) channel from the external environment was characterized using Ba(2+), Ni(2+) and EGTA. Cells sensitized with canine serum abundant in IgG and IgE or heat-inactivated serum were activated by anti-canine IgG or anti-canine IgE. The effect of extracellular Ca(2+) and reaction time on IgG-mediated histamine release was examined. Staurosporine and ER-27319 were used to clarify the IgG-mediated protein tyrosine phosphorylation. RESULTS: Abundant IgG-binding sites on the cell were detected by FACS analysis. Anti-IgG induced rapid protein tyrosine phosphorylation and [Ca(2+)](i) elevation. When extracellular Ca(2+) was excluded by EGTA, a mild and transient increase in [Ca(2+)](i) was observed, indicating the release of Ca(2+) from anti-IgG-sensitive intracellular Ca(2+) stores. The constant Ba(2+) entry from external environment proved the Ca(2+) influx occurred mainly via a store-operated Ca(2+) channel which was inhibited by Ni(2+) and EGTA. Canine serum-sensitized cells showed a rapid and sustained increase in [Ca(2+)](i) upon both anti-IgG and anti-IgE stimulation. The [Ca(2+)](i) elevation induced by anti-IgE was decreased in the cells sensitized with heat-inactivated serum. Histamine release from CM-MCs was absolutely dependent on extracellular Ca(2+), and reached equilibrium within 5 min. Staurosporine inhibited the tyrosine phosphorylation of 38-, 65-, 70-, 80-kD proteins. ER-27319 inhibited the tyrosine phosphorylation of 38- and 70-kD proteins. Staurosporine also inhibited IgG-mediated [Ca(2+)](i) elevation and histamine release in a dose-dependent manner. CONCLUSIONS: Canine cutaneous mastocytoma-derived (CM-MC) cells were activated by both IgG- and IgE-mediated mechanisms. IgG-mediated protein tyrosine phosphorylation and Ca(2+) influx were similar to those mediated by IgE. CM-MC cells are useful for the study of allergic inflammation caused by IgG-dependent mechanisms.     

Modulation of spreading depression by changes in extracellular pH

Spreading depression (SD) and related phenomena have been implicated in hypoxic-ischemic injury. In such settings, SD occurs in the presence of marked extracellular acidosis. SD itself can also generate changes in extracellular pH (pH(o)), including a pronounced early alkaline shift. In a hippocampal slice model, we investigated the effect of interstitial acidosis on the generation and propagation of SD in the CA1 stratum radiatum. In addition, a carbonic anhydrase inhibitor (benzolamide) was used to decrease buffering of the alkaline shift to investigate its role in the modulation of SD. pH(o) was lowered by a decrease in saline HCO(3)(-) (from 26 to 13 to 6.5 mM at 5% CO(2)), or by an increase in the CO(2) content (from 5 to 15% in 26 mM HCO(3)(-)). Recordings with pH microelectrodes revealed respective pHo values of 7.23 +/- 0. 13, 6.95 +/- 0.10, 6.67 +/- 0.09, and 6.97 +/- 0.12. The overall effect of acidosis was an increase in the threshold for SD induction, a decrease in velocity, and a shortened SD duration. This inhibition was most pronounced at the lowest pH(o) (in 6.5 mM HCO(3)(-)) where SD was often blocked. The effects of acidosis were reversible on return to control saline. Benzolamide (10 microM) caused an approximate doubling of the early alkaline shift to an amplitude of 0.3-0.4 U pH. The amplified alkalosis was associated with an increased duration and/or increased velocity of the wave. These effects were most pronounced in acidic media (13 mM HCO(3)(-)/5% CO(2)) where benzolamide increased the SD duration by 55 +/- 32%. The initial velocity (including time for induction) and propagation velocity (measured between distal electrodes) were enhanced by 35 +/- 25 and 26 +/- 16%, respectively. Measurements of [Ca(2+)](o) demonstrated an increase in duration of the Ca(2+) transient when the alkaline shift was amplified by benzolamide. The augmentation of SD caused by benzolamide was blocked in media containing the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid. These data indicate that the induction and propagation of SD is inhibited by a fall in baseline pH characteristic of ischemic conditions and that the early alkaline shift can remove this inhibition by relieving the proton block on NMDA receptors. Under ischemic conditions, the intrinsic alkalosis may therefore enable SD and thereby contribute to NMDA receptor-mediated injury.     

T-type Ca2+ channels contribute to IBMX/forskolin- and K(+)-induced Ca(2+) transients in porcine olfactory receptor neurons

T-type Ca(2+) channels are low-voltage-activated Ca(2+) channels that control Ca(2+) entry in excitable cells during small depolarization above resting potentials. Using Ca(2+) imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca(2+) channels in IBMX/forskolin- and sparingly elevated extracellular K(+)-induced Ca(2+) transients in freshly isolated porcine olfactory receptor neurons (ORNs). In the presence of mibefradil (10microM) or Ni(2+) (100microM), the selective T-type Ca(2+) channel inhibitors, IBMX/forskolin-induced Ca(2+) transients in the soma were either strongly (>60%) inhibited or abolished completely. However, the Ca(2+) transients in the knob were only partially (<60%) inhibited. Ca(2+) transients induced by 30mM K(+) were also partially ( approximately 60%) inhibited at both the knob and soma. Furthermore, ORNs responded to as little as a 2.5mM increase in the extracellular K(+) concentration (7.5mM K(+)), and such responses were completely inhibited by mibefradil or Ni(2+). These results reveal functional expression of T-type Ca(2+) channels in porcine ORNs, and suggest a role for these channels in the spread Ca(2+) transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs.     

Carbonic anhydrase-dependent bicarbonate reabsorption in the rat proximal tubule.

The extent to which bicarbonate reabsorption in the rat proximal convoluted tubule depends on carbonic anhydrase has been examined by in vivo microperfusion and the measurement of total CO2 concentration by microcalorimetry. Tubules were perfused with an ultrafiltrate-like solution at 13 nl/min, and volume reabsorptive rate (JV) was measured using [14C]inulin. Addition of either 800 or 100 microM acetazolamide to the perfusion solution completely inhibited the reabsorption of total CO2. The control total CO2 reabsorptive rate (JtCO2) was 147 +/- 23 pmol/mm.min, and acetazolamide reduced JtCO2 to -3 +/- 5 pmol/mm.min. Acetazolamide reduced JV by 65% from a control of 2.3 +/- 0.4 to 0.8 +/- 0.1 nl/mm.min. The dose-response curve for acetazolamide showed that the I50 for inhibition of JtCO2 was 4 microM. The inactive congener of acetazolamide, t-butyl acetazolamide, did not reduce JV or inhibit bicarbonate reabsorption, indicating that the effect of acetazolamide on JtCO2 was specific for carbonic anhydrase inhibition. Since bicarbonate reabsorption was completely blocked by carbonic anhydrase inhibition, there is no need to postulate either carbonic acid recycling or carbonic anhydrase-independent bicarbonate reabsorption.     

Axonal surface charges: binding or screening by divalent cations governed by external pH

1. The effect of different extracellular alkaline-earth cations (Ca(2+), Mg(2+), Sr(2+), Ba(2+)) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular micro-electrodes. This was done at the following pHs of the bathing saline: 6.00, 5.35, 5.00, and 4.65 +/- 0.05.2. At pH 6.00, the four alkaline earths had essentially the same effect upon the threshold membrane potential.3. At pH 5.35 or lower, it was found that equal concentrations of the alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different pHs were: pH 5.35, Ca(2+) > Mg(2+) > Sr(2+) >/= Ba(2+); pH 5.00, Ca(2+) approximately Ba(2+) > Sr(2+) >/= Mg(2+); pH 4.65, Ba(2+), Sr(2+) > Ca(2+) > Mg(2+).4. It is shown that the individual selectivity sequences are predicted rather closely by the equilibrium selectivity theory for alkaline-earth cations.5. It is concluded that the only difference between excitable cells which show screening and those which bind divalent cations is the net density of ionized, surface acidic groups in the region of the sodium gates.     

Serotonergic inhibition of action potential evoked calcium transients in NOS-containing mesopontine cholinergic neurons

Nitric oxide synthase (NOS)-containing mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT) are hypothesized to drive the behavioral states of waking and REM sleep through a tonic increase in firing rate which begins before and is maintained throughout these states. In principle, increased firing could elevate intracellular calcium levels and regulate numerous cellular processes including excitability, gene expression, and the activity of neuronal NOS in a state-dependent manner. We investigated whether repetitive firing, evoked by current injection and N-methyl-D-aspartate (NMDA) receptor activation, produces somatic and proximal dendritic [Ca(2+)](i) transients and whether these transients are modulated by serotonin, a transmitter thought to play a critical role in regulating the state-dependent firing of MPCh neurons. [Ca(2+)](i) was monitored optically from neurons filled with Ca(2+) indicators in guinea pig brain slices while measuring membrane potential with sharp microelectrodes or patch pipettes. Neither hyperpolarizing current steps nor subthreshold depolarizing steps altered [Ca(2+)](i). In contrast, suprathreshold currents caused large and rapid increases in [Ca(2+)](i) that were related to firing rate. TTX (1 microM) strongly attenuated this relation. Addition of tetraethylammonium (TEA, 20 mM), which resulted in Ca(2+) spiking on depolarization, restored the change in [Ca(2+)](i) to pre-TTX levels. Suprathreshold doses of NMDA also produced increases in [Ca(2+)](i) that were reduced by up to 60% by TTX. Application of 5-HT, which hyperpolarized LDT neurons without detectable changes in [Ca(2+)](i), suppressed both current- and NMDA-evoked increases in [Ca(2+)](i) by reducing the number of evoked spikes and by inhibiting spike-evoked Ca(2+) transients by approximately 40% in the soma and proximal dendrites. This inhibition was accompanied by a subtle increase in the spike repolarization rate and a decrease in spike width, as expected for inhibition of high-threshold Ca(2+) currents in these neurons. NADPH-diaphorase histochemistry confirmed that recorded cells were NOS-containing. These findings indicate the prime role of action potentials in elevating [Ca(2+)](i) in NOS-containing MPCh neurons. Moreover, they demonstrate that serotonin can inhibit somatic and proximal dendritic [Ca(2+)](i) increases both indirectly by reducing firing rate and directly by decreasing the spike-evoked transients. Functionally, these data suggest that spike-evoked Ca(2+) signals in MPCh neurons should be largest during REM sleep when serotonin inputs are expected to be lowest even if equivalent firing rates are reached during waking. Such Ca(2+) signals may function to trigger Ca(2+)-dependent processes including cfos expression and nitric oxide production in a REM-specific manner.     

Ca(2+)-dependent Ca(2+) clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals

Ca(2+) clearance in frog motor nerve terminals was studied by fluorometry of Ca(2+) indicators. Rises in intracellular Ca(2+) ([Ca(2+)](i)) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca(2+) transient) reached a peak or plateau within 6-20 stimuli and decayed at least in three phases with the time constants of 82-87 ms (81-85%), a few seconds (11-12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca(2+) pumps at the cell membrane by external Li(+) and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li(+) alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca(2+) transients. Blocking Ca(2+) pumps at Ca(2+) stores by thapsigargin did not affect 100 stimuli-induced Ca(2+) transients but increased the initial decay time constant of 200 stimuli-induced Ca(2+) transients with no change in other parameters. Inhibiting mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca(2+) transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca(2+) transients against [Ca(2+)](i) yielded the supralinear [Ca(2+)](i) dependence of Ca(2+) efflux out of the cytosol. Blocking Ca(2+) extrusion or mitochondrial Ca(2+) uptake significantly reduced this [Ca(2+)](i)-dependent Ca(2+) efflux. Thus Ca(2+)-dependent mitochondrial Ca(2+) uptake and plasmalemmal Ca(2+) extrusion clear out a small Ca(2+) load in frog motor nerve terminals, while thapsigargin-sensitive Ca(2+) pump boosts the clearance of a heavy Ca(2+) load. Furthermore, the activity of plasmalemmal Ca(2+) pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.     

T-Type calcium channel alpha1G and alpha1H subunits in human retinoblastoma cells and their loss after differentiation

Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba(2+)-containing bath solutions, the current has an activation midpoint near -25 mV and appears to be fully inactivated at -20 mV. Sr(2+) and Ca(2+) are preferred charge carriers relative to Ba(2+), and the current vanishes in the absence of these divalent cations. Cd(2+) blocks the current with an IC(50) of 160 microM, and Ni(2+) blocks in a biphasic manner with IC(50)s of 22 and 352 microM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. RT-PCR revealed the presence of alpha 1G and alpha 1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both alpha 1G and alpha 1H mRNA was found, and this was paralleled by the loss of T-type Ca channel currents. alpha 1I subunit mRNA levels were low in undifferentiated and differentiated cells. These results suggest that T-type Ca channels could play a role in undifferentiated retinoblastoma cell physiology since alpha 1G and alpha 1H Ca channel subunit expression is reduced in cells that have differentiated and exited the cell cycle.     

KB-R7943 reveals possible involvement of Na(+)-Ca2+ exchanger in elevation of intracellular Ca2+ in rat carotid arterial myocytes.

A Na(+)/Ca(2+) exchanger (NCX) is one of the major regulators of intracellular Ca(2+) concentration ([Ca(2+)](i)) in cardiac muscle cells. Although vascular smooth muscle myocytes also express NCX proteins, their functional role has not been clear, mainly due to the lack of specific inhibitors of NCX and relatively low levels of expression of NCX. In the present study, we have examined the involvement of NCX in the Na(+) deficient (0 Na(+)) elevation of [Ca(2+)](i) in rat carotid arterial myocytes using KB-R7943, an inhibitor of NCX. Perfusion with a Na(+)-free bathing solution, prepared by replacement of Na(+) with N-methyl-D-glucamine, induced an elevation of [Ca(2+)](i), which was effectively inhibited by KB-R7943 (IC(50)=3.5 microM). This inhibition was reversed by washout of KB-R7943. In contrast, D600, a blocker of voltage dependent L-type Ca(2+) channels (VDCC), did not affect the 0 Na(+)-induced elevation of [Ca(2+)](i). Treatment of myocytes with ryanodine abolished the elevation of [Ca(2+)](i) caused by caffeine but not that caused by 0 Na(+). Application of Cd(2+), which is known to block NCX as well as VDCC, also significantly inhibited the 0 Na(+) induced elevation. These results suggest that KB-R7943 inhibits the extracellular Na(+) dependent ([Na(+)](o)) change in [Ca(2+)](i) in rat carotid arterial myocytes, which is presumably activated by the reverse mode of NCX.     

Somatic Ca(2+) dynamics in response to choline-mediated excitation in histaminergic tuberomammillary neurons

Histaminergic tuberomammillary (TM) neurons of the posterior hypothalamus have been implicated in cognition, alertness and sleep-wakefulness cycles. Spontaneous firing of TM neurons has been associated with histamine release and wakefulness. The expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in TM neurons suggests a role for endogenous choline and for nicotinic drugs in the regulation of intracellular Ca(2+) metabolism, normal TM neuronal activity and histamine release. First, we established the link between TM neuronal spontaneous firing frequency and cytosolic free Ca(2+) concentration ([Ca(2+)](i)). A strong correlation was observed: an onset of spontaneous firing (3-4Hz) was accompanied by a 20-fold increase in [Ca(2+)](i) from 56+/-18 nM to 1.0+/-0.6 microM. The same range of firing frequencies has been observed in TM neurons in vivo and is associated with wakefulness. Secondly, choline-induced activation of alpha7 nAChRs did not elevate [Ca(2+)](i) directly, i.e. in the absence of high-threshold voltage-gated Ca(2+) channel (HVGCC) activation. Cd(2+) (200 microM) completely blocked all Ca(2+) signals, but inhibited only 37+/-16% of alpha7 nAChR-mediated currents. Thirdly, the responsiveness of [Ca(2+)](i) to choline-mediated excitation was inhibited by hyperpolarization and enhanced by depolarization, sensitizing [Ca(2+)](i) at membrane voltages associated with normal TM neuronal activity. These properties of [Ca(2+)](i) define the ability of TM neurons to translate cholinergic stimuli of identical strengths into different cytosolic Ca(2+) effects, providing the physiological substrate for state-specific modulation of incoming cholinergic information and would be expected to play a very important role in determining activity profiles of TM neurons exposed to elevated concentrations of cholinergic agents, such as choline and nicotine.