Iono- and metabotropically induced purinergic calcium signalling in rat neocortical neurons

ATP receptor-mediated Ca²⁺ concentration changes were recorded from neocortical neurones in brain slices from 2 week-old rats. To measure the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i) slices were incubated with fura-2/AM, and the microfluorimetry system was focused on an individual cell. During transients the intracellular level of [Ca²⁺]_i in the majority of neocortical neurones (98 of 102) varied in the concentration range of ATP 5–2000 μM between 41.3±5 and 163±7 nM. The rank order of efficacy for purinoreceptor agonists in concentration 100 μM was: ATPγS>ATP>ADPAMP≈Adenosine≈α,β-methylene ATP>UTP. 10 μM PPADS, a P₂-purinoreceptor antagonist, reduced the ATP-induced [Ca²⁺]_i response by 26%±4%. After elimination of calcium from extracellular solution the first ATP-induced [Ca²⁺]_i transient decreased to 65±8%, suggesting the participation of metabotropic P_2y triggered Ca-release in the generation of the transient. Elevation of cytosolic Ca²⁺ by activation of plasmalemmal Ca²⁺ channels failed to potentiate such release indicating the absence of effective reloading of the corresponding stores. No Ca²⁺-induced Ca²⁺-release has been observed in the investigated neurons.     

Dibutyryl cGMP raises cytosolic concentrations of Ca in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca²⁺]_i in 42% of neurons (n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca²⁺ abolished the dbcGMP effect. Application of Ni²⁺ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca²⁺ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca²⁺ channel antagonist, or L-type Ca²⁺ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.     

Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.     

Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV (n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba²⁺ and increasing the extracellular K⁺ to 30 mM from 5 mM. In the absence of extracellular Ca²⁺ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl₃, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl₃ also inhibited [Ca²⁺]_i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca²⁺]_i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca²⁺ influx. In turn, this [Ca²⁺]_i increase leads to an activation of the Ca²⁺-activated K⁺ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K⁺ efflux.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca 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 μM), 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 μM) 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.     

Effects of ginsenosides on Ca channels and membrane capacitance in rat adrenal chromaffin cells

We investigated the effects of ginseng total saponins (GTS) and five ginsenosides on voltage-dependent Ca²⁺ channels and membrane capacitance using rat adrenal chromaffin cells. In this study, cells were voltage-clamped in a whole-cell recording mode and a perforated patch-clamp technique was used. The inward Ca²⁺ currents (I_Ca) was elicited by depolarization and the change in cell membrane capacitance (ΔC_m) was monitored. The application of GTS (100 μg/ml) induced rapid and reversible inhibition of the Ca²⁺ current by 38.8 ± 3.6% (n = 16). To identify the particular single component that seems to be responsible for Ca²⁺ current inhibition, the effects of five ginsenosides (ginsenoside Rb₁, Rc, Re, Rf, and Rg₁) on the Ca²⁺ current were examined. The inhibitions to the Ca²⁺ current by Rb₁, Rc, Re, Rf, and Rg₁ were 15.3 ± 2.2% (n = 5); 36.9 ± 2.4% (n = 7); 28.1 ± 1.9% (n = 12); 19.0 ± 2.5% (n = 10); and 16.3 ± 1.6% (n = 15), respectively. The order of inhibitory potency (100 μM) was Rc > Re > Rf > Rg₁ > Rb₁. A software based phase detector technique was used to monitor membrane capacitance change (ΔC_m). The application of GTS (100 μg/ml) induced inhibitory effects on ΔC_m by 60.8 ± 9.7% (n = 10). The inhibitions of membrane capacitance by Rb₁, Rc, Re, Rf, and Rg₁ were 35.3 ± 5.5% (n = 7); 41.8 ± 7.0% (n = 8); 40.5 ± 5.9% (n = 9); 51.2 ± 7.6% (n = 9); and 35.9 ± 5.1% (n = 10), respectively. The inhibitory potencies of the ginsenosides on ΔC_m were Rf > Rc > Re > Rg₁ > Rb₁. Therefore, we found that GTS and ginsenosides exerted inhibitory effects on both Ca²⁺ currents and ΔC_m in rat adrenal chromaffin cells. These results suggest that ginseng saponins regulate catecholamine secretion from adrenal chromaffin cells and this regulation could be the cellular basis of antistress effects induced by ginseng.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Activity-evoked extracellular pH shifts in slices of rat dorsal lateral geniculate nucleus

Activity-dependent extracellular pH shifts were studied in slices of the rat dorsal lateral geniculate nucleus (dLGN) using double-barreled pH-sensitive microelectrodes. In 26 mM HCO₃⁻-buffered media, afferent activation (10 Hz, 5 s) elicited an early alkaline shift of 0.04±0.02 pH units associated with a later, slow acid shift of 0.05±0.03 pH units. Extracellular pH shifts in the ventral lateral geniculate nucleus were rare, and limited to acidifications of approximately 0.02 pH units. The alkaline shift in the dLGN increased in the presence of benzolamide (1–2 μM), an extracellular carbonic anhydrase inhibitor. The mean alkaline shift in benzolamide was 0.10±0.05 pH units. In 26 mM HEPES-buffered saline, the alkaline response averaged 0.09±0.03 pH units. The alkaline shifts persisted in 100 μM picrotoxin (PiTX) but were blocked by 25 μM CNQX/50 μM APV. If stimulation intensity was raised in the presence of CNQX/APV, a second alkalinization arose, presumably due to direct activation of dLGN neurons. The direct responses were amplified by benzolamide, and blocked by either 0 Ca²⁺/EGTA, Cd²⁺ or TTX. In 0 Ca²⁺, addition of 500 μM–5 mM Ba²⁺ restored the alkalosis. Alkaline shifts evoked with extracellular Ba²⁺ were larger and faster than those elicited by equimolar Ca²⁺. In summary, synchronous activation in the dLGN results in an extracellular H⁺ sink, via a Ca²⁺-dependent mechanism, similar to activity-dependent alkaline shifts in hippocampus.     

NMDA induces a biphasic change in intracellular pH in rat hippocampal slices

As alterations in intracellular pH (pH_i) tend to exert a profound effect on the properties of cells, this study was undertaken to examine NMDA-induced changes in pH_i in rat hippocampal slices using the BCECF fluorescent technique. The ‘resting' pH_i in the CA1 pyramidal cell layers was 6.93±0.07 (mean±S.D., n=72 slices) in 25 mM HCO₃⁻/5% CO₂-buffered solution at 37°C. Exposure of hippocampal slices to NMDA in the range of 10–1000 μM produced a biphasic change in pH_i: an initial transient alkaline shift was followed by a long-lasting acid shift. Dizocilpine (10 μM) but not CNQX (40 μM) blocked the NMDA-induced changes in pH_i. In 0 Ca medium (0 mM Ca²⁺ supplemented 1 mM EGTA, referred to as 0 Ca), pH_i acid shift caused by NMDA (20 μM) declined by about 11%, whereas the initial alkaline shift almost completely disappeared. In an independent experiment, the NMDA-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) was reduced by more than 80% in 0 Ca medium. Glucose substitution using equimolar pyruvate (as an energy-yielding substrate) suppressed this NMDA-induced pH_i acid shift by two-thirds, while the NMDA-induced pH_i alkaline shift was enhanced. Fluoride (10 mM), a glycolytic inhibitor, abolished NMDA-induced pH_i acid shift. Furthermore, the lactate content of hippocampal slices was markedly increased following exposure to NMDA. In conclusion, activation of NMDA receptors in rat hippocampal slices evokes a biphasic change in pH_i. The initial alkaline shift is suggested to be associated with calcium influx, and the following acid shift may be caused by an increase in lactate production through the acceleration of glycolysis, as well as the increased [Ca²⁺]_i. The pH_i acid shift produced by the increased lactate may contribute to proton modulation of the NMDA receptor and NMDA-induced cell injury or death.     

Ca-Channels Involved in Neostriatal Glutamatergic Transmission

The actions of peptidic toxins that work as Ca²⁺-channel antagonists were investigated on neostriatal glutamatergic transmission. Both intracellularly recorded excitatory postsynaptic potentials (EPSPs) and extracellularly recorded population spikes (PS) evoked by afferent stimulation were evaluated in the presence of 10 μM bicuculline. Percentage of block (mean ± SEM; n = 4) for these events (EPSP and PS, respectively) was: ω-AgTxIVA (100–200 nM): 35 ± 2 and 54 ± 4%; ω-CgTxGVIA (1 μM): 37 ± 3 and 63 ± 6%; ω-CgTxMVIIC (500 nM): 40 ± 4 and 50 ± 2%; and calciseptine (500 nM): 5 ± 4 and 9 ± 6%. When given together, toxins had additive effects. The calciseptine effects were nonsignificant. The toxins were also tested on Ca²⁺-dependent random synaptic responses induced by 100 μM 4-AP. Each toxin reduced the frequency of spontaneous EPSPs by more than 60% (n = 2). The summed actions of individual toxins yields more than 100% block (superadditivity); suggesting that several terminals may possess more than one channel type. The reduction in frequency was not accompanied by a reduction in amplitude confirming that toxins’ actions were presynaptic. It is concluded that at least three different Ca²⁺-channel subtypes are involved in glutamate release in neostriatal afferents: N-type, P/Q-type, and a type resistant to the toxins used. The L-type Ca²⁺-channel had little, if any, participation.     

Effect of lead on intracellular free calcium ion concentration in a presynaptic neuronal model: F-NMR study of NG108-15 cells

The intracellular free calcium ion concentration ([Ca²⁺]_i) of the neuroblastoma × glioma hybrid cell line, NG108-15, was measured using the ¹⁹F-nuclear magnetic resonance divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (5F-BAPTA). The basal [Ca²⁺]_i was measured to be 106 ± 14 nM. Treatment with 5 μM lead (Pb) for 2 h produced a 2-fold increase in [Ca²⁺]_i to 200 ± 24 nM and a measurable intracellular free Pb²⁺ concentration ([Pb²⁺]_i) of 30 ± 10 pM. Intracellular free Zn²⁺ concentrations ([Zn²⁺]_i) were also observed in the presence of Pb. This represents the first direct demonstration that Pb elevates the [Ca²⁺]_i in neurons, thus providing evidence for a role of [Ca²⁺]_i in mediating the neurotoxicity of Pb.     

Dorsal root ganglion neurons of embryonic chicks contain nitric oxide synthase and respond to nitric oxide

We investigated the function of nitric oxide (NO) in dorsal root ganglion (DRG) neurons from 10 day embryonic chicks and adult birds. NADPH-diaphorase activity, a histochemical marker for nitric ooxide synthase (NOS) in paraformaldehyde-fixed neurons, and NOS-like immunoreactivity were localized in all neurons in thoracic and lumbar ganglia from embros. However, only a subset of neurons from adults contained NOS-like immunoreactivity and NADPH-diaophorase activity. Thus, embryonic chick DRG neurons have the potential to synthesize NO in response to elevated cytoplasmic Ca²⁺. We also investigated the ability of dissociated embryonic chick DRG neurons to respond to NO by examining the effects of NO donors and 8-bromoguasonine 3′,5′-cyclic monophosphate (8-Br-cGMP) on Ca²⁺ current (I_Ca) using the amphotericin-permeabilized patch-clamp technique: sodium nitroprusside (5 μM) reduced I_Ca to 0.68 ± 0.06 (mean±S.D., n = 5) of control, S-nitroso-N-acetylpenicillamine) (1 μM) reduced I_Cato0.44 ± 0.06 (n = 4) of control, while 8-Br-cGMP (1 mM) reduced I_{Cato0.58 ± 0.22 (n = 5)} of control. I_Ca was reduced in every neuron tested and this effect was partially reversed after ≈ 10min of washing. Thus, I_Ca of embryonic chick DRG neurons is inhibited by NO, possibly by a cGMP-dependent mechanism. These results indicate that all DRG neurons in embryonic chicks contain NOS-like immunoreactivity and respond to NO. Further, the percentage of NADPH-diaphorase positive neurons is reduced during development.     

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons

Intracellular magnesium concentration ([Mg²⁺]_i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg²⁺]_i was 0.48±0.08 mM (mean±SEM, n=23) at rest, and it increased 3-fold by depolarization with a 60-mM K⁺ solution. The [Mg²⁺]_i increase was observed in the absence of extracellular Mg²⁺, but the increase disappeared in the absence of extracellular Ca²⁺. 50 μM cadmium or 100 μM verapamil, a Ca²⁺ channel blocker, also diminished the rise of [Mg²⁺]_i. The additional measurement of an intracellular Ca²⁺ concentration ([Ca²⁺]_i) indicated that the [Mg²⁺]_i rise requires a threshold concentration of [Ca²⁺]_i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca²⁺-influx through voltage dependent Ca channels and this causes the release of Mg²⁺ from intracellular stores into the cytoplasm.     

SnCl(2) reduces voltage-activated calcium channel currents of dorsal root ganglion neurons of rats

Stannous dichloride (SnCl₂) occurs in the environment where it has been especially enriched in aquatic ecosystems. Furthermore, it is used in food manufacturing (e.g. for stabilizing soft drinks or as an anti-corrosive substance) and in nuclear medicine where it is employed as a reducing agent for technecium-99m (99mTc) and therefore is applied intravenously to human beings.SnCl₂ is known to have toxic effects on the nervous system which can be related to alterations of intracellular calcium homeostasis ([Ca²⁺]_i). In this study the whole cell patch clamp technique is used on dorsal root ganglion neurons of 3-week-old “Wistar” rats to evaluate the effects of SnCl₂ on voltage-activated calcium channel currents (I_Ca(V)).I_Ca(V) were reduced concentration-dependently by SnCl₂ (1–50 μM). 1 μM SnCl₂ reduced I_Ca(V) by 8.1 ± 4.5% (peak current) and 19.2 ± 8.9% (sustained current), whereas 50 μM inhibited I_Ca(V) by 50.6 ± 4.3% (peak current) and 55.6 ± 11.3% (sustained current). Sustained currents were slightly but not significantly more reduced than peak currents. The effect appeared not to be reversible. The threshold concentration was below 1 μM.The current–voltage relation did not shift which is an indication that different calcium channel subtypes were equally affected. There was a slight but not significant shift of the activation/inactivation curves towards the depolarizing direction.We conclude that voltage-gated calcium channels are affected by Sn²⁺ similarly to other divalent metal cations (e.g. Pb²⁺ or Zn²⁺).The reduction of I_Ca(V) could be related to the neurotoxic effects of SnCl₂.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

The bradykinin receptor — a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation

Bradykinin (BK) induced [³H]norepinephrine ([³H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [³H]NE release by BK is mediated by activation of BK-B₂-receptors, as determined using type specific BK receptor antagonists. BK induces [³H]NE release with a half maximal effective concentration of30 ± 0.5nM, and reaches maximal net fractional release of9.0 ± 1% with 200 nM BK. The BK-induced release is Ca²⁺ dependent, reaching maximal release at 1.0 mM Ca²⁺, is pertussis toxin insensitive (1 μg/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca²⁺ channel blockers, verapamil (10 μM), nifedipine (10 μM), and ω-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co²⁺ (ED₅₀ = 0.2mM) and Ni²⁺ (ED_50²⁺ = 1mM). These results indicate the involvement of a Ca²⁺ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca²⁺]_ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca²⁺]_ex is similar up to concentration of 1 μM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca²⁺ to BK-stimulated cells in Ca²⁺-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca²⁺ for induction of release. Protein kinase C inhibitors polymyxin B, K_252a, sangivamicin, and Ara-A, do not affect release induced by BK, indicating that also the diacylglycerol pathway activated by phospholipase C is not involved in the BK-mediated release. Since (a) the receptor-mediated release is absolutely calcium-dependent, with no release detected when Ca²⁺ is omitted from the extracellular medium, and (b) the receptor-triggered release of Ca²⁺ from intracellular stores is independent of [Ca²⁺]_ex⁷, it appears that calcium influx, and not Ca²⁺ released from intracellular stores, is the signal for stimulating release. Therefore, it is suggested that the primary signal stimulating release is Ca²⁺ influx via a specific calcium channel, and that the BK receptor may be coupled to this channel, which could be classified as a receptor-operated channel.     

Warm cells revealed by microfluorimetry of Ca in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca²⁺ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43°C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca²⁺]_i from 52.5±9.5 nM (mean±S.D., n=18) to 171.0±15.6 nM in cells between 15 and 25 μm in diameter. The depletion of extracellular Ca²⁺ diminished the Ca²⁺ elevation. The Na⁺-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Effects of the removal of extracellular Ca on [Ca]i responses to FCCP and acetate in carotid body glomus cells of adult rabbits

The effects of the removal of extracellular Ca²⁺ on the responses of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to acidic stimuli, a protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and an organic acid acetate, were examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2 microfluorometry. Application of FCCP (1 μM) induced an increase in [Ca²⁺]_i (mean±S.E.M., 108±14%). After withdrawal of the protonophore the increased [Ca²⁺]_i returned slowly to a resting level. The [Ca²⁺]_i response was attenuated by an inorganic Ca²⁺ channel antagonist Ni²⁺ (2 mM) by 81±4%, and by an L-type voltage-gated Ca²⁺ channel antagonist D600 (10 μM) by 53±13%. The removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i response in 71% of the tested cells (n=17), and depressed it by 68±6% in the rest. Recovery following stimulation with FCCP in the absence of Ca²⁺ reversibly produced a rapid and large rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/FCCP. The magnitude of a [Ca²⁺]_i rise after Ca²⁺-free/FCCP (285±28%, P<0.05) was larger than that of an increase in [Ca²⁺]_i induced by FCCP in the presence of Ca²⁺ and had a correlation with the intensity of the suppression of the [Ca²⁺]_i response by Ca²⁺ removal. A [Ca²⁺]_i rise after Ca²⁺-free/FCCP was inhibited mostly by D600. Similarly, recovery following exposure to acetate in the absence of Ca²⁺ caused a rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/acetate which was sensitive to D600. The magnitude of the [Ca²⁺]_i rise was larger than that of a change in [Ca²⁺]_i caused by acetate in the presence of Ca²⁺. These results suggest that FCCP-induced increase in [Ca²⁺]_i was, in most cells, due to Ca²⁺ influx via L-type voltage-gated Ca²⁺ channels and, in some cells, due to both Ca²⁺ influx and Ca²⁺ release from internal Ca²⁺ pool. The removal of extracellular Ca²⁺ might modify [Ca²⁺]_i responses to acidic stimuli, causing [Ca²⁺]_i rises after Ca²⁺-free/acidic stimuli which involve mostly L-type Ca²⁺ channels.     

Ryanodine receptor-mediated [Ca]i release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body

The hypothesis that intracellular calcium ([Ca²⁺]_i) release in glomus cells via ryanodine receptor (RyR) activation by caffeine may be independent of natural stimuli and chemosensory discharge was tested in the rat carotid body (CB). CB type I cells were isolated, plated and preloaded with calcium-sensitive fluorescent probe, Indo-1AM. With the increase of caffeine dose (0–50 mM) cytosolic calcium ([Ca²⁺]_c) increased from 85±15 nM to 1933±190 nM (n=6) at normoxia (P ₂=125–130 Torr, P ₂=25–30 Torr, pH 7.30–7.35). Hypoxia (P ₂=10–15 Torr) increased and hypocapnia (P ₂=7–9 Torr) decreased the cytoplasmic calcium [Ca²⁺]_c levels, independent of caffeine. Caffeine-related [Ca²⁺]_c increase was the same in the presence and the absence of extracellular calcium ([Ca²⁺]_o), indicating the source of Ca²⁺ ions is the cellular store. Permeabilization of the cell membrane with saponin (25 μg/ml) retained the caffeine response. Additional treatment of the cells with 50 μM ryanodine (an inhibitor of the caffeine-activated RyR site) abolished caffeine-stimulated response. In vitro CB chemosensory (carotid sinus nerve, CSN) responses to hypoxia (P ₂=35–40 Torr) were not altered by caffeine. These results suggest that [Ca²⁺]_i stores in CB cells, mobilized by RyR activation, do not participate in the CSN responses to natural stimuli.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.