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.     

Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

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.     

l-DOPA induces concentration-dependent facilitation and inhibition of presynaptic acetylcholine release in the guinea-pig submucous plexus

Neurotransmitter- or neuromodulator-like actions ofl-DOPA were investigated with intracellular recordings from submucous plexus neurons of the guinea-pig caecum.l-DOPA at 30 nM augmented the amplitude of fast EPSPs, but did not affect depolarizations elicited by puff application of acetylcholine (ACh). The augmenting effect ofl-DOPA on the fast EPSPs was counteracted byl-DOPA methyl ester. The fast EPSPs were depressed by 10 μMl-DOPA, but transiently augmented after rinsing the drug.l-DOPA methyl ester did not affect the inhibitory action ofl-DOPA on the fast EPSPs, but antagonized the potentiation following the inhibition. The depolarization elicited by exogenously applied. ACh was inhibited by 10 μMl-DOPA. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of the neuronal soma were measured with fura-2 microfluorophotometry. The transient increase in the [Ca²⁺]_i evoked by the somatic action potential (Δ[Ca²⁺]_AP) was facilitated by 30 nMl-DOPA, but decreased by the drug at 10 μM. It is concluded thatl-DOPA at low concentrations enhances the Δ[Ca²⁺]_AP, increasing the neurotransmitter release, but at high dose diminishes the Δ[Ca²⁺]_AP, inhibiting the neurotransmission.     

The cannabinoid agonist Win55,212-2 inhibits calcium channels by receptor-mediated and direct pathways in cultured rat hippocampal neurons

The effects of the cannabinoid receptor agonist Win55,212 on Ca²⁺ channels were studied in rat hippocampal neurons grown in primary culture. Win55,212-2 inhibited whole-cell Ba²⁺ currents through Ca²⁺ channels by both CB1 receptor-mediated and direct mechanisms. The concentration dependent inhibition of the current showed two clear phases, a high-affinity receptor-mediated phase (IC₅₀=14±2 nM) that was stereoselective and sensitive to a CB1 receptor antagonist, 300 nM SR141716, and a non-saturating phase that was neither stereoselective nor inhibited by SR141716. These concentration-dependent effects were paralleled by Win55212-induced inhibition of glutamatergic synaptic transmission. Win55,212-2 (100 nM) inhibited both ω-agatoxin IVA- and ω-conotoxin GVIA-sensitive currents. Thus, activation of cannabinoid receptors inhibits N- and P/Q-type Ca²⁺ channels. Activation of cannabinoid receptors inhibited only a fraction of the whole-cell Ca²⁺ channel current (17±2%) even though more than half of the whole-cell Ba²⁺ current was carried by N- and P/Q-type Ca²⁺ channels. Concentrations of agonist greater than 1 μM inhibited Ca²⁺ channels directly.     

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.     

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.     

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.     

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.     

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg²⁺, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca²⁺-high Mg²⁺ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence ofd-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg²⁺. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg²⁺ in the solution and in 2 of 17 cells with 4 mM Mg²⁺, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg²⁺ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.     

NMDA-induced increase in [Ca]i andCa uptake in acutely dissociated brain cells derived from adult rats

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-d-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca²⁺]_i measured with fluo3 and⁴⁵Ca²⁺ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 μM) and N-methyl-dl-aspartate (200 μM) increased [Ca²⁺]_i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated⁴⁵Ca²⁺ uptake about 16–10% in the same regions. The increases in [Ca²⁺]_i and⁴⁵Ca²⁺ uptake were inhibited by 40% in the presence of 1 mM MgCl₂ and by 90–50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca²⁺]_i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca²⁺]_i increase in adult age.     

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.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca²⁺ channels or Na⁺-Ca²⁺ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca²⁺ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca²⁺ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

Synthetic ω-conotoxin: a potent calcium channel blocking neurotoxin

The Ca²⁺ channel blocking action of synthetic ω-conotoxin (ωCTX) was studied on isolated frog dorsal root ganglion neurons using a ‘concentration clamp’ technique which enabled internal perfusion and rapid external solution change. At 100 nM, ωCTX showed a time-dependent depression of Ca²⁺ current (I_Ca). At higher concentrations, ωCTX exhibited a dose-dependent depression of I_Ca amplitude without changing the current-voltage relationship. Increases in external Ca²⁺ concentration partly overcame the inhibitory action of ωCTX on the I_Ca amplitude. At 10 μM ωCTX totally blocked I_Ca without effect on the Na⁺ current. It was likely that ωCTX had high selectivity for the Ca²⁺ channel.     

8-Azido-Nucleotides as Substrates of Torpedo Electric Organ Apyrase. Effect of Photoactivation on Apyrase Activity

Ecto-apyrase is a widespread enzymatic activity that hydrolyses tri- and diphosphonucleotides and consequently controls the amount of available extracellular ATP and ADP. In the nervous system, purines have important neuromodulatory actions, acting at pre- and postsynaptic sites, and consequently, ecto-apyrase may play an indirect role in the modulation of nucleotide- and nucleoside-mediated processes. The azido-nucleotides have been largely employed to characterize the nucleotide binding sites of several proteins. In the present work the azido-nucleotides are described as putative substrates for apyrase activity in a presynaptic plasma membrane preparation (PSPM) from the Torpedo electric organ. Both 8-N₃-ATP and 8-N₃-ADP were hydrolyzed in a calcium-dependent manner showing V_max of 23.8 ± 4.8 and 14.5 ± 3 U/mg of protein, and K_m values (in μM) of 116 ± 39 and 119 ± 4, respectively. V_max for calcium-dependent hydrolysis of ATP and ADP were significantly higher: 59.2 ± 3.9 and 32.9 ± 3.5 U/mg of protein respectively, while K_m values did not show any significant differences regarding azido-nucleotides: 83.8 ± 12 μM for Ca²⁺-ATP and 121 ± 34 μM for Ca²⁺-ADP. The photoactivation of the PSPM in the presence of the azido-derivatives results in an irreversible inactivation of apyrase activity, showing an IC₅₀ of 10 μM and a maximal inhibitory effect of 38 and 60% on Ca²⁺-ATPase and Ca²⁺-ADPase activities. Apyrase was protected from inactivation by nucleotides that are natural substrates for this enzymatic activity and also by AMP while adenosine did not protect from apyrase inhibition.     

Effects of Na gradient on the intracellular Ca oscillation in the sympathetic ganglion cell: Na---Ca exchange in the neurone cell soma?

(+)-Tubocurarine ((+)-Tc:10–100 μM) reduced the duration of the afterhyperpolarization, which was induced by the activation of Ca²⁺-dependent K⁺-conductance (G_K,Ca) following an action potential in the bullfrog sympathetic ganglion cell, but did not affect the maximum rates of rise and fall of Na⁺- and Ca²⁺-dependent action potentials. The amplitudes of slow rhythmic membrane hyperpolarizations produced by rhythmic rises in the G_K,Ca were also decreased by (+)-Tc without a change in their intervals. Thus, (+)-Tc appears to block the Ca²⁺-dependent K⁺-channel of the bullfrog sympathetic ganglion cell.     

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.     

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.     

Protective effects of vasoactive intestinal peptide against delayed glutamate neurotoxicity in cultured retina

The effects of vasoactive intestinal peptide (VIP) on glutamate-induced delayed death were examined using the primary cultures of rat retinal neurons. Effects of VIP on glutamate-induced neurotoxicity were evaluated by double staining with fluorescein diacetate and propidium iodide. Glutamate (1 mM) was applied to the culture for 10 min in the presence and absence of VIP, and visible cells enumerated 24 h after culture in normal medium. Effects of VIP on increase in the intracellular Ca²⁺ concentration and currents induced by glutamate in retinal neurons were investigated using the Ca²⁺ image analyzing system with fura-2 and whole-cell patch-clamp recording, respectively. The cAMP contents in retinal cultures were measured by radioimmunoassay. VIP (10 nM–1 μM) dose-dependently protected against glutamate-induced neurotoxicity in cultured retinal neurons. Protection by VIP (100 nM) against glutamate (1 mM)-induced neurotoxicity was antagonized by VIP6-28 (1 μM), a VIP antagonist, and H-89 (100 nM and 1 μM), a protein kinase A inhibitor. However, VIP had no effect on glutamate-induced inward currents nor glutamate-induced increase in the intracellular Ca²⁺ concentration. A 10-min exposure of VIP (100 nM) with glutamate (1 mM) resulted in an increase in the cAMP level to 446±58 from 22±1 pmol/mg protein. These findings suggest that VIP protects against the glutamate-induced neurotoxicity in retinal cultures by elevating the cAMP level via VIP receptors and thereby activating protein kinase A.