Calcium signalling mediated through ��7 and non-��7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release

BACKGROUND AND PURPOSE

Ca²⁺ signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the α7 nAChR, in bovine chromaffin cells are still matters of debate.

EXPERIMENTAL APPROACH

We have used chromaffin cell cultures loaded with Fluo-4 or transfected with aequorins directed to the cytosol or mitochondria, several nAChR agonists (nicotine, 5-iodo-A-85380, PNU282987 and choline), and the α7 nAChR allosteric modulator PNU120596.

KEY RESULTS

Minimal [Ca²⁺]_c transients, induced by low concentrations of selective α7 nAChR agonists and nicotine, were markedly increased by the α7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the α7 nAChR antagonist α-bungarotoxin (α7-modulated-response). Conversely, high concentrations of the α7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca²⁺]_c transients, that were blocked by mecamylamine but were unaffected by α-bungarotoxin (non-α7 response). [Ca²⁺]_c increases mediated by α7 nAChR were related to Ca²⁺ entry through non-L-type Ca²⁺ channels, whereas non-α7 nAChR-mediated signals were related to L-type Ca²⁺ channels; Ca²⁺-induced Ca²⁺-release contributed to both responses. Mitochondrial involvement in the control of [Ca²⁺]_c transients, mediated by either receptor, was minimal. Catecholamine release coupled to α7 nAChRs was more efficient in terms of catecholamine released/[Ca²⁺]_c.

CONCLUSIONS AND IMPLICATIONS

[Ca²⁺]_c and catecholamine release mediated by α7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the α7 nAChR response was lost and the non-α7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of α7 nAChRs.     

Repeated nicotine administration robustly increases bPiDDB inhibitory potency at α6β2-containing nicotinic receptors mediating nicotine-evoked dopamine release

The novel nicotinic receptor (nAChR) antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), and its chemically reduced analog, r-bPiDDB, potently inhibit nicotine-evoked dopamine (DA) release from rat striatal slices. Since tobacco smokers self-administer nicotine repeatedly, animal models incorporating repeated nicotine treatment allow for mechanistic evaluation of therapeutic candidates following neuroadaptive changes. The current study determined the ability of bPiDDB, r-bPiDDB and α-conotoxin MII (α-CtxMII), a peptide antagonist selective for α6β2-containing nAChRs, to inhibit nicotine-evoked [³H]DA release from striatal slices from rats repeatedly administered nicotine (0.4 mg/kg for 10 days) or saline (control). Concomitant exposure to maximally effective concentrations of r-bPiDDB (1 nM) and α-CtxMII (1 nM) resulted in inhibition of nicotine-evoked [³H]DA release no greater than that produced by either antagonist alone, suggesting that r-bPiDDB inhibits α6β2-containing nAChRs. Repeated nicotine treatment increased locomotor activity, demonstrating behavioral sensitization. Concentration-response curves for nicotine-evoked [³H]DA release were not different between nicotine-treated and control groups. Maximal inhibition for α-CtxMII was greater following repeated nicotine compared to control (I_max = 90% vs. 62%), with no change in potency. bPiDDB was 3-orders of magnitude more potent in inhibiting nicotine-evoked [³H]DA release in nicotine-treated rats compared to control rats (IC₅₀ = 5 pM vs. 6 nM), with no change in maximal inhibition. Neither a shift to the left in the concentration response nor a change in maximal inhibition was observed for r-bPiDDB following repeated nicotine. Thus, repeated nicotine treatment may differentially regulate the stoichiometry, conformation and/or composition of α6β2-containing nAChRs mediating nicotine-evoked striatal DA release. Therefore, bPiDDB and r-bPiDDB appear to target different α6β2-containing nAChR subtypes.     

A NMDA receptor glycine site partial agonist, GLYX-13, simultaneously enhances LTP and reduces LTD at Schaffer collateral-CA1 synapses in hippocampus

N-methyl-d-aspartate glutamate receptors (NMDARs) are a key route for Ca²⁺ influx into neurons important to both activity-dependent synaptic plasticity and, when uncontrolled, triggering events that cause neuronal degeneration and death. Among regulatory binding sites on the NMDAR complex is a glycine binding site, distinct from the glutamate binding site, which must be co-activated for NMDAR channel opening. We developed a novel glycine site partial agonist, GLYX-13, which is both nootropic and neuroprotective in vivo. Here, we assessed the effects of GLYX-13 on long-term synaptic plasticity and NMDAR transmission at Schaffer collateral-CA1 synapses in hippocampal slices in vitro. GLYX-13 simultaneously enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission, while reducing long-term depression (LTD). GLYX-13 reduced NMDA receptor-mediated synaptic currents in CA1 pyramidal neurons evoked by low frequency Schaffer collateral stimulation, but enhanced NMDAR currents during high frequency bursts of activity, and these actions were occluded by a saturating concentration of the glycine site agonist d-serine. Direct two-photon imaging of Schaffer collateral burst-evoked increases in [Ca²⁺] in individual dendritic spines revealed that GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca²⁺ influx. Examining the rate of MK-801 block of synaptic versus extrasynaptic NMDAR-gated channels revealed that GLYX-13 selectively enhanced activation of burst-driven extrasynaptic NMDARs, with an action that was blocked by the NR2B-selective NMDAR antagonist ifenprodil. Our data suggest that GLYX-13 may have unique therapeutic potential as a learning and memory enhancer because of its ability to simultaneously enhance LTP and suppress LTD.     

Angiotensin AT1 and AT2 receptor antagonists modulate nicotine-evoked [H]dopamine and [H]norepinephrine release

Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4β2* and α7* nAChRs using [³H]nicotine and [³H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [³H]dopamine and [³H]norepinephrine release (IC₅₀: 3.9 ± 1.2 and 2.2 ± 0.7 μM; I_max: 82 ± 3 and 89 ± 6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [³H]nicotine or [³H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4β2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of nicotine on dopamine and norepinephrine release in brain regions involved in nicotine reward and hypertension.     

Unusual properties of α7 nicotinic acetylcholine receptor ion channels in B lymphocyte-derived SP-2/0 cells

This study demonstrates the presence of α7 nicotinic acetylcholine receptors (nAChR) in B lymphocyte-derived SP-2/0 cells by means of flow cytometry and immunocytochemistry. According to lectin and sandwich ELISA, the α7 subunits expressed in SP-2/0 cells are more glycosylated compared to those expressed in the brain or normal B lymphocytes and are combined with β2 subunits. At zero and negative pipette potentials, either acetylcholine or α7-specific agonist PNU282987 stimulated the ion channel activity in SP-2/0 cells revealed by single channel patch-clamp recordings. The conductivity was within the range of 19 to 39 pS and reversal potential was between −17 mV and +28 mV, the currents were potentiated by α7-specific positive allosteric modulator PNU120596 and were partially blocked by α7-specific antagonist methyllicaconitine (MLA). However, they were oriented downwards suggesting that the channels mediated the cation outflux rather than influx. As shown by Ca²⁺ imaging studies, PNU282987 did not stimulate immediate Ca²⁺ influx into SP-2/0 cells. Instead, Ca²⁺ influx through Ca-release-activated channels (CRACs) was observed within minutes after either PNU282987 or MLA application. It is concluded that SP-2/0 express α7β2 nAChRs, which mediate the cation outflux under negative pipette potentials applied, possibly, due to depolarized membrane or negative surface charge formed by carbohydrate residues. In addition, α7β2 nAChRs may influence CRACs in ion-independent way.     

α7 nAChRs expressed on antigen presenting cells are insensitive to the conventional antagonists α-bungarotoxin and methyllycaconitine

Expression of α7 nicotinic acetylcholine receptors (nAChRs) on antigen presenting cells (APCs), such as macrophages and dendritic cells, is now well established. We have shown that GTS-21, a selective α7 nAChR agonist, downregulates APC-dependent CD4⁺ T cell differentiation into regulatory T cells (Tregs) and effector Th1, Th2 and Th17 cells by inhibiting antigen processing, thereby interfering with antigen presentation. α7 nAChRs on Jurkat human leukemic T cells require functional T cell receptors (TCRs)/CD3 and leukocyte-specific tyrosine kinase to mediate nicotine-induced Ca²⁺-signaling via Ca²⁺ release from intracellular stores, and are insensitive to two conventional α7 nAChR antagonists, α-bungarotoxin (α-BTX) and methyllycaconitine (MLA). We investigated the effects of GTS-21, α-BTX and MLA on ovalbumin (OVA)-induced Th cytokine release from spleen cells isolated from OVA-specific TCR transgenic DO11.10 mice. We found that: (1) GTS-21 dose-dependently suppresses OVA-induced IFN-γ, IL-4 and IL-17 release, but neither α-BTX nor MLA alone affected the OVA-induced cytokine release. (2) Neither α-BTX nor MLA abolished the suppressive effects of GTS-21 on IFN-γ and IL-17 release from OVA-activated DO11.10 spleen cells. (3) GTS-21 significantly suppressed OVA-induced APC-dependent CD4⁺ T cell differentiation into Tregs. Neither MLA nor mecamylamine, a non-specific nAChR antagonist, abolished the suppressive effect of GTS-21 on Treg differentiation. These results suggest that α7 nAChRs on APCs involved in cytokine synthesis and T cell differentiation are insensitive to the conventional α7 nAChR antagonists, α-BTX and MLA, and that α7 nAChRs on APCs differ pharmacologically from those in neurons.     

Activation of G Proteins by Aluminum Fluoride Enhances RANKL-Mediated Osteoclastogenesis

Receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca²⁺ mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca²⁺/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca²⁺ mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca²⁺ mobilization induced by aluminum fluoride (AlF₄^-), a G-protein activator, with or without RANKL and the effects of AlF₄^- on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF₄^- induces intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations, which is dependent on extracellular Ca²⁺ influx. Notably, co-stimulation of AlF₄^- with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF₄^-. Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca²⁺]_i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.     

Introduction of unsaturation into theN-n-alkyl chain of the nicotinic receptor antagonists, NONI and NDNI: Effect on affinity and selectivity

N-n-octylnicotinium iodide (NONI) and N-n-decylnicotinium iodide (NDNI) are selective nicotinic receptor (nAChR) antagonists mediating nicotine-evoked striatal dopamine (DA) release, and inhibiting [3H]nicotine binding, respectively. This study evaluated effects of introducing unsaturation into the N-n-alkyl chains of NONI and NDNI on inhibition of [3H]nicotine and [3H]methyllycaconitine binding (alpha4beta2* and alpha7* nAChRs, respectively), (86)Rb+ efflux and [3H]DA release (agonist or antagonist effects at alpha4beta2* and alpha6beta2*-containing nAChRs, respectively). In the NONI series, introduction of a C3-cis- (NONB3c), C3-trans- (NONB3t), C7-double-bond (NONB7e), or C3-triple-bond (NONB3y) afforded a 4-fold to 250-fold increased affinity for [3H]nicotine binding sites compared with NONI. NONB7e and NONB3y inhibited nicotine-evoked 86Rb+ efflux, indicating alpha4beta2* antagonism. NONI analogs exhibited a 3-fold to 8-fold greater potency inhibiting nicotine-evoked [3H]DA overflow compared with NONI (IC50 = 0.62 microM; Imax = 89%), with no change in Imax, except for NONB3y (Imax = 50%). In the NDNI series, introduction of a C4-cis- (NDNB4c), C4-trans-double-bond (NDNB4t), or C3-triple-bond (NDNB3y) afforded a 4-fold to 80-fold decreased affinity for [3H]nicotine binding sites compared with NDNI, whereas introduction of a C9 double-bond (NDNB9e) did not alter affinity. NDNB3y and NDNB4t inhibited nicotine-evoked 86Rb+ efflux, indicating antagonism at alpha4beta2* nAChRs. Although NDNI had no effect, NDNB4t and NDNB9e potently inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.02-0.14 microM, Imax = 90%), as did NDNB4c (IC50 = 0.08 microM; Imax = 50%), whereas NDNB3y showed no inhibition. None of the analogs had significant affinity for alpha7* nAChRs. Thus, unsaturated NONI analogs had enhanced affinity at alpha4beta2*- and alpha6beta2*-containing nAChRs, however a general reduction of affinity at alpha4beta2* and an uncovering of antagonist effects at alpha6beta2*-containing nAChRs were observed with unsaturated NDNI analogs.     

Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons

Context: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities.

Objective: The effects of fucoidan on Ca²⁺ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined.

Materials and methods: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca²⁺ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5?mg/mL or 1.5?mg/mL was applied for 3?min to determine its effects on Ca²⁺ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5?mg/mL for 3?h.

Results: The Ca²⁺ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca²⁺ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca²⁺ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca²⁺ channels, PR1/PR2.

Discussion and conclusions: Our data indicate that fucoidan suppresses the intracellular Ca²⁺ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca²⁺ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.     

Evaluation of N-type Ca2+ channel currents in cultured rat superior cervical ganglion neurons

Electrophysiological studies of the effects of test compounds on ion channel currents have been useful in the identification of novel therapeutic agents. Here, we examined the use of cultured superior cervical ganglion (SCG) neurons as a model system for the electrophysiological evaluation of N-type Ca²⁺ channels in vitro. As previously reported, Ca²⁺ channel currents in acutely dissociated preparations of SCG neurons were mainly N-type, as defined by inhibition of Ca²⁺ channel current with the specific N-type Ca²⁺ channel blocker, ω-conopeptides GVIA or MVIIA. However, a cultured preparation that could be used over an extended period of time would be more useful for drug discovery since acutely dissociated preparations require daily dissections. We found that with extended time in culture the amplitudes of Ca²⁺ channel currents increased with time. While there was a reduction in the percentage of N-type Ca²⁺ channel component, the majority (70%) of Ca²⁺ channel currents in long-term cultured SCG neurons remained N-type. A portion of the MVIIA-resistant Ca²⁺ channel current component was blocked by the ω-conopeptide MVIIC and ω-agatoxin IIIA, but not by ω-agatoxin IVA, a pharmacological profile similar to Q-type Ca²⁺ channel current. These studies suggest that cultured SCG neurons would be useful for the study of N-type Ca²⁺ currents even after prolonged time in culture, and may also be used to indicate the selectivity of test compounds for other Ca²⁺ channel subtypes. Drug Dev. Res. 41:85–90, 1997. © 1997 Wiley-Liss, Inc.     

bPiDI: a novel selective α6β2* nicotinic receptor antagonist and preclinical candidate treatment for nicotine abuse

BACKGROUND AND PURPOSE

Nicotinic acetylcholine receptors (nAChRs) containing α6β2 subunits expressed by dopamine neurons regulate nicotine-evoked dopamine release. Previous results show that the α6β2* nAChR antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB) inhibits nicotine-evoked dopamine release from dorsal striatum and decreases nicotine self-administration in rats. However, overt toxicity emerged with repeated bPiDDB treatment. The current study evaluated the preclinical pharmacology of a bPiDDB analogue.

EXPERIMENTAL APPROACH

The C₁₀ analogue of bPiDDB, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), was evaluated preclinically for nAChR antagonist activity.

KEY RESULTS

bPiDI inhibits nicotine-evoked [³H]dopamine overflow (IC₅₀ = 150 nM, I_max = 58%) from rat striatal slices. Schild analysis revealed a rightward shift in the nicotine concentration–response curve and surmountability with increasing nicotine concentration; however, the Schild regression slope differed significantly from 1.0, indicating surmountable allosteric inhibition. Co-exposure of maximally inhibitory concentrations of bPiDI (1 µM) and the α6β2* nAChR antagonist α-conotoxin MII (1 nM) produced inhibition not different from either antagonist alone, indicating that bPiDI acts at α6β2* nAChRs. Nicotine treatment (0.4 mg·kg⁻¹·day⁻¹, 10 days) increased more than 100-fold the potency of bPiDI (IC₅₀ = 1.45 nM) to inhibit nicotine-evoked dopamine release. Acute treatment with bPiDI (1.94–5.83 µmol·kg⁻¹, s.c.) specifically reduced nicotine self-administration relative to responding for food. Across seven daily treatments, bPiDI decreased nicotine self-administration; however, tolerance developed to the acute decrease in food-maintained responding. No observable body weight loss or lethargy was observed with repeated bPiDI.

CONCLUSIONS AND IMPLICATIONS

These results are consistent with the hypothesis that α6β2* nAChR antagonists have potential for development as pharmacotherapies for tobacco smoking cessation.     

Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition

Aim:

To identify the mechanisms underlying the elevation of intracellular Ca²⁺ level ([Ca²⁺]_i) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons.

Methods:

Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca²⁺]_i was measured using fura-2 AM. Ca²⁺ current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay.

Results:

Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca²⁺]_i in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons depended on extracellular Ca²⁺, and was blocked by P/Q-type Ca²⁺channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca²⁺ channel blocker nifedipine (10 μmol/L) or N-type Ca²⁺channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca²⁺ current in ARC neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L).

Conclusion:

Lowering glucose level enhances the activity of P/Q type Ca²⁺channels and elevates [Ca²⁺]_i level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β.     

An unusual phospholipase A2 from puff adder Bitis arietans venom - a novel blocker of nicotinic acetylcholine receptors

The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A₂ (PLA₂s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca²⁺ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [¹²⁵I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC₅₀ being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC₅₀ of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA₂s from snake venoms. Indeed, the protein possessed high PLA₂ activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA₂ that contains 14 disulphide bonds and the first nAChR blocker possessing PLA₂ activity.     

The epithelial calcium channels TRPV5 and TRPV6: regulation and implications for disease

The epithelial Ca²⁺ channels TRPV5 and TRPV6 represent a new family of Ca²⁺ channels that belongs to the superfamily of transient receptor potential channels. TRPV5 and TRPV6 constitute the apical Ca²⁺ entry mechanism in active Ca²⁺ transport in kidney and intestine. The central role of TRPV5 and TRPV6 in active Ca²⁺ (re)absorption makes it a prime target for regulation to maintain Ca²⁺ balance. This review covers the hormonal regulation, interaction with accessory proteins and (patho)physiological implications of these epithelial Ca²⁺ channels.     

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels

BACKGROUND

Depletion of the Ca²⁺ store by ryanodine receptor (RyR) agonists induces store-operated Ca²⁺ entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE.

EXPERIMENTAL APPROACH

SOCE and ORAI1-3 currents were recorded by Ca²⁺ imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3.

KEY RESULTS

4-CEP induced a significant release of Ca²⁺ in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca²⁺ release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca²⁺ store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3.

CONCLUSION AND IMPLICATIONS

4-CEP not only acts as a RyR agonist to deplete the Ca²⁺ store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists.     

Cellular Ca2+ Dynamics in Urinary Bladder Smooth Muscle From Transgenic Mice Overexpressing Na+-Ca2+ Exchanger

The rise of Ca²⁺ concentration ([Ca²⁺] _i ) by reducing external Na⁺ in urinary bladder smooth muscle cells (UBSMCs) from transgenic mice overexpressing Na⁺/Ca²⁺ exchanger type-1.3 (NCX1.3^tg/tg) was about 4 times as large as that in the wild-type (WT). NCX1 protein expression in UB increased about 4-fold in NCX1.3^tg/tg. The Ca²⁺ release by caffeine in UBSMCs was comparable between NCX1.3^tg/tg and WT, but [Ca²⁺]_i decay was faster in NCX1.3^tg/tg. Contractions induced by acetylcholine, 60 mM K⁺, or electrical stimulation were significantly smaller in UB segments of NCX1.3^tg/tg. NCX worked in Ca²⁺-extrusion mode during these contractions in UBSMCs of both WT and NCX1.3^tg/tg.     

Buspirone-induced antinociception is mediated by <Emphasis Type="SmallCaps">l</Emphasis>-type calcium channels and calcium/caffeine-sensitive pools in mice

Rationale. Previous studies have shown that buspirone, a partial 5-HT_1A receptor agonist, produces antinociceptive effects in rats and mice; Ca²⁺ plays a critical role as a second messenger in mediating nociceptive transmission. 5-HT_1A receptors have been proven to be coupled functionally with various types of Ca²⁺ channels in neurons, including N-, P/Q-, T-, or L-type. It was of interest to investigate the involvement of extracellular/intracellular Ca²⁺ in buspirone-induced antinociception. Objectives. To determine whether central serotonergic pathways participate in the antinociceptive processes of buspirone, and investigate the involvement of Ca²⁺ mechanisms, particularly L-voltage-gated Ca²⁺ channels and Ca²⁺/caffeine-sensitive pools, in buspirone-induced antinociception. Methods. Antinociception was assessed using the hot-plate test (55°C, hind-paw licking latency) in mice treated with either buspirone (1.25–20 mg/kg i.p.) alone or the combination of buspirone and fluoxetine (2.5–10 mg/kg i.p.), 5-HTP (25 mg/kg i.p.), nimodipine (2.5–10 mg/kg i.p.), nifedipine (2.5–10 mg/kg i.p.), CaCl₂ (25–200 nmol per mouse i.c.v.), EGTA (5–30 nmol per mouse i.c.v.), or ryanodine (0.25–2 nmol per mouse i.c.v.). Results. Buspirone dose dependently increased the licking latency in the hot-plate test in mice. This effect of buspirone was enhanced by fluoxetine, 5-HTP, nimodipine, and nifedipine. Interestingly, central administration of Ca²⁺ reversed the antinociceptive effects of buspirone. In contrast to these, ryanodine or EGTA administered centrally potentiated buspirone-induced antinociception. Conclusions. Decreasing neuronal Ca²⁺ levels potentiated buspirone-induced antinociception; conversely, increasing intracellular Ca²⁺ abolished the antinociceptive effects of buspirone. These results suggest that Ca²⁺ influx from extracellular fluid and release of Ca²⁺ from Ca²⁺/caffeine-sensitive microsomal pools may be involved in buspirone-induced antinociception. Electronic Publication     

Endothelin Receptor Overexpression Alters Diastolic Function in Cultured Rat Ventricular Myocytes

The endothelin (ET) signaling pathway controls many physiological processes in myocardium and often becomes upregulated in heart diseases. The aim of the present study was to investigate the effects of ET receptor upregulation on the contractile function of adult ventricular myocytes. Primary cultured adult rat ventricular myocytes were used as a model system of ET receptor overexpression in the heart. Endothelin receptor type A (ET_A) or type B (ET_B) was overexpressed by Adenoviral infection, and the twitch responses of infected ventricular myocytes were measured after ET-1 stimulation. Overexpression of ET_A exaggerated positive inotropic effect (PIE) and diastolic shortening of ET-1, and induced a new twitch response including twitch broadening. On the contrary, overexpression of ET_B increased PIE of ET-1, but did not affect other two twitch responses. Control myocytes expressing endogenous receptors showed a parallel increase in twitch amplitude and systolic Ca²⁺ in response to ET-1. However, intracellular Ca²⁺ did not change in proportion to the changes in contractility in myocytes overexpressing ET_A. Overexpression of ET_A enhanced both systolic and diastolic contractility without parallel changes in Ca²⁺. Differential regulation of this nature indicates that upregulation of ET_A may contribute to diastolic myocardial dysfunction by selectively targeting myofi lament proteins that regulate resting cell length, twitch duration and responsiveness to prevailing Ca²⁺.