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.     

Possible involvement of transient receptor potential ankyrin 1 in Ca2+ signaling via T‐type Ca2+ channel in mouse sensory neurons

T‐type Ca²⁺ channels and TRPA1 are expressed in sensory neurons and both are associated with pain transmission, but their functional interaction is unclear. Here we demonstrate that pharmacological evidence of the functional relation between T‐type Ca²⁺ channels and TRPA1 in mouse sensory neurons. Low concentration of KCl at 15 mM (15K) evoked increases of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which were suppressed by selective T‐type Ca²⁺ channel blockers. RT‐PCR showed that mouse sensory neurons expressed all subtypes of T‐type Ca²⁺ channel. The magnitude of 15K‐induced [Ca²⁺]_i increase was significantly larger in neurons sensitive to allylisothiocyanate (AITC, a TRPA1 agonist) than in those insensitive to it, and in TRPA1^?/? mouse sensory neurons. TRPA1 blockers diminished the [Ca²⁺]_i responses to 15K in neurons sensitive to AITC, but failed to inhibit 40 mM KCl‐induced [Ca²⁺]_i increases even in AITC‐sensitive neurons. TRPV1 blockers did not inhibit the 15K‐induced [Ca²⁺]_i increase regardless of the sensitivity to capsaicin. [Ca²⁺]_i responses to TRPA1 agonist were enhanced by co‐application with 15K. These pharmacological data suggest the possibility of functional interaction between T‐type Ca²⁺ channels and TRPA1 in sensory neurons. Since TRPA1 channel is activated by intracellular Ca²⁺, we hypothesize that Ca²⁺ entered via T‐type Ca²⁺ channel activation may further stimulate TRPA1, resulting in an enhancement of nociceptive signaling. Thus, T‐type Ca²⁺ channel may be a potential target for TRPA1‐related pain.     

The Riluzole Derivative 2-Amino-6-trifluoromethylthio-benzothiazole (SKA-19), a Mixed KCa2 Activator and NaV Blocker,is a Potent Novel Anticonvulsant

Inhibitors of voltage-gated sodium channels (Na_v) have been used as anticonvulsants since the 1940s, while potassium channel activators have only been investigated more recently. We here describe the discovery of 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19), a thioanalog of riluzole, as a potent, novel anticonvulsant, which combines the two mechanisms. SKA-19 is a use-dependent Na_V channel blocker and an activator of small-conductance Ca²⁺-activated K⁺ channels. SKA-19 reduces action potential firing and increases medium afterhyperpolarization in CA1 pyramidal neurons in hippocampal slices. SKA-19 is orally bioavailable and shows activity in a broad range of rodent seizure models. SKA-19 protects against maximal electroshock-induced seizures in both rats (ED₅₀ 1.6 mg/kg i.p.; 2.3 mg/kg p.o.) and mice (ED₅₀ 4.3 mg/kg p.o.), and is also effective in the 6-Hz model in mice (ED₅₀ 12.2 mg/kg), Frings audiogenic seizure-susceptible mice (ED₅₀ 2.2 mg/kg), and the hippocampal kindled rat model of complex partial seizures (ED₅₀ 5.5 mg/kg). Toxicity tests for abnormal neurological status revealed a therapeutic index (TD₅₀/ED₅₀) of 6–9 following intraperitoneal and of 33 following oral administration. SKA-19 further reduced acute pain in the formalin pain model and raised allodynic threshold in a sciatic nerve ligation model. The anticonvulsant profile of SKA-19 is comparable to riluzole, which similarly affects Na_V and KCa2 channels, except that SKA-19 has a ~4-fold greater duration of action owing to more prolonged brain levels. Based on these findings we propose that compounds combining KCa2 channel-activating and Na_v channel-blocking activity exert broad-spectrum anticonvulsant and analgesic effects.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0305-y) contains supplementary material, which is available to authorized users.     

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.     

In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca²⁺ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca²⁺ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca²⁺ response because of degeneration of primary sensory nerve fibers in the skin.SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca²⁺ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca²⁺ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca²⁺ activity and postoperative pain.     

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.     

Regulation of T-type Ca<Superscript>2+</Superscript> channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca²⁺ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca²⁺ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca²⁺ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca²⁺ current densities. Increased T-type Ca²⁺ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca²⁺ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca²⁺ channels is mediated by several factors, including decreased expression of Ca_v3.2 T-type Ca²⁺ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca²⁺ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca²⁺ channels, which can results in morphological and functional changes in electrical excitability.     

Regionally specific expression of high-voltage-activated calcium channels in thalamic nuclei of epileptic and non-epileptic rats

The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca^2 + channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca^2 + channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca²⁺ currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca^2 + current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca²⁺ channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Ca_v1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca²⁺ channels. Moreover, pharmacological inactivation of L-type Ca^2 + channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca^2 + channels influence tonic and burst firing in ACI and WAG/Rij differently, it is discussed that increased Ca_v1.3 expression may indirectly contribute to increased robustness of burst firing and thereby the epileptic phenotype of absence epilepsy.     

Cyclosporine A modulation of Ca activated K channels in cardiac sensory afferent neurons

Whole-cell and single channel recordings were used to characterize the effects of the immunosuppressant cyclosporine A (CsA) on cardiac sensory neurons (CSN) of the nodose ganglia. Application of 10 nM CsA resulted in a 29.1% decrease in CSN input resistance and an average −8±3 mV hyperpolarization of membrane potential. Application of 10 nM CsA had no effect on evoked Ca⁺⁺ currents but increased evoked K⁺ currents by 158.9±24%. Application of 10 nM CsA significantly increased the open probability of KCa channels by 183±9%. These results suggest that application of CsA results in the activation of KCa channels in cardiac sensory neurons and this effect may contribute to the cellular mechanisms underlying CsA modulation of vagal afferent neurons.     

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.     

Antinociception induced by 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), an N-methyl-d-aspartate (NMDA) competitive antagonist, plus 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA antagonist, differs from that induced by MK-801 plus DNQX

Excitatory amino acid receptors have been implicated in mediating pain. 3-((±)-2-Car☐ypiperazin4-yl)-propyl-1-phosphonic acid (CPP), a competitive N-methyl-d-aspartate (NMDA) antagonist and MK-801, a phencyclidine (PCP) ligand and non-competitive NMDA antagonist, were injected intrathecally in mice alone or in combination with 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA antagonist. When tested in the formalin model of pain, antinociception following CPP plus DNQX was greater than that after MK-801 plus DNQX in both the acute and tonic phases. These dissimilarities are not consistent with activity of CPP and MK-801 at the same sites in the spinal cord.     

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.     

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.     

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.     

Differential localization of Ca2+ channel α1 subunits in the enteric nervous system: Presence of α1B channel-like immunoreactivity in intrinsic primary afferent neurons

Immunocytochemistry was employed to locate calcium (Ca²⁺) channel proteins in the enteric nervous system (ENS) of the rat and guinea pig. Anti-peptide antibodies that specifically recognize the α₁ subunits of class A (P/Q-type), B (N-type), C and D (L-type) Ca²⁺ channels were utilized. α_1B channel-like immunoreactivity was abundant in both enteric plexuses, the mucosa, and circular and longitudinal muscle layers. Immunoreactivity was predominantly found in cholinergic varicosities, supporting a role for Ca²⁺ channels, which contain the α_1B subunit, in acetylcholine release. Immunoreactivity was also associated with the cell soma of calbindin-immunoreactive submucosal and myenteric neurons, cells that have been proposed to be intrinsic primary afferent neurons. α_1C channel-like immunoreactivity was distributed diffusely in the cell membrane of a large subset of neuronal cell bodies and processes, whereas α_1D was found mainly in the cell soma and proximal dendrites of vasoactive intestinal polypeptide-immunoreactive neurons in the guinea pig gut. α_1A channel-like immunoreactivity was found in a small subset of cell bodies and processes in the rat ENS. The differential localization of the α₁ subunits of Ca²⁺ channels in the ENS implies that they serve distinct roles in neuronal excitation and signaling within the bowel. The presence of α_1B channel-like immunoreactivity in putative intrinsic primary afferent neurons suggested that class B Ca²⁺ channels play a role in enteric sensory neurotransmission; therefore, we determined the effects of the N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CTx GVIA), on the reflex-evoked activity of enteric neurons. Demonstrating the phosphorylation of cyclic AMP (cAMP)-responsive element-binding protein (pCREB) identified neurons that became active in response to distension. Distension elicited hexamethonium-resistant pCREB immunoreactivity in calbindin-immunoreactive neurons in each plexus; however, in preparations stimulated in the presence of ω-CTx GVIA, pCREB immunoreactivity was found only in calbindin-immunoreactive neurons in the submucosal plexus and not in myenteric ganglia. These data confirm that intrinsic primary afferent neurons are located in the submucosal plexus and that N-type Ca²⁺ channels play a role in sensory neurotransmission. J. Comp. Neurol. 409:85–104, 1999. © 1999 Wiley-Liss, Inc.     

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.     

Nifedipine blocks apamin-induced bursting activity in nigral dopamine-containing neurons

Intrinsic sinusoidal oscillations in membrane potential, characteristic of nigral dopamine cells, are converted to plateau potentials following application of apamin, a potent antagonist of SK-type Ca²⁺-activated K⁺ channels. Blockade of these channels also changes neuronal firing pattern from a single-spike pacemaker discharge to a multiple spike bursting pattern. Nifedipine, a selective antagonist of L-type Ca²⁺ channels, blocks plateau potential generation; however, its effects on firing pattern have yet to be determined. In the present study, extracellular single unit recording techniques were used in conjunction with a brain slice preparation to determine whether nifedipine, in a concentration known to block plateau potential generation, also affects bursting activity. Nifedipine (30 μM) was equipotent in inhibiting the firing rate of control (51.2±10.8%) and apamin-treated (44.9±5.4%) neurons. Slow firing neurons (<2 Hz) were particularly sensitive to the inhibitory effects of the drug. Apamin-induced bursting was completely suppressed by nifedipine and accompanied by a significant increase in the regularity of firing. By contrast, pacemaker-like activity exhibited by control neurons was unaffected by the drug. These data demonstrate that the intrinsic plateau properties exhibited by DA neurons are responsible for the generation of phasic activity induced following blockade of apamin-sensitive Ca²⁺-activated K⁺ channels and provide further support for the involvement of an L-type Ca²⁺ conductance in mediating this type of activity.     

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.     

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.