Nonselective Cation Current Research Articles

Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells

Transient receptor potential channels (TRPs) regulate tumor growth via calcium-dependent mechanisms. The (thermosensitive) capsaicin receptor TRPV1 is overexpressed in numerous highly aggressive cancers. TRPV1 has potent antiproliferative activity and is therefore potentially applicable in targeted therapy of malignancies. Recently, we characterized TRPM8 functions in pancreatic neuroendocrine tumors (NETs), however, the role of TRPV1 is unknown. Here, we studied the expression and the role of TRPV1 in regulating intracellular Ca2+ and chromogranin A (CgA) secretion in pancreatic NET BON-1 cell line and in primary NET cells (prNET). TRPV1 expression was detected by RT-PCR, Western blot and immunofluorescence. Intracellular free Ca2+ ([Ca2+]i) was measured by fura-2; TRPV1 channel currents by the planar patch-clamp technique. Nonselective cation currents were analyzed by a color-coded plot method and CgA secretion by ELISA. Pancreatic BON-1 cells and NETs express TRPV1. Pharmacological blockade of TRPs by La3+ (100μM) or by ruthenium-red (RuR) or by capsazepine (CPZ) (both at 10μM) suppressed the capsaicin (CAP)- or heat-stimulated increase of [Ca2+]i in NET cells. CAP (20μM) also increased nonselective cation channel currents in BON-1 cells. Furthermore, CAP (10μM) stimulated CgA secretion, which was inhibited by CPZ or by RuR (both 10μM). La3+ potently reduced both stimulated and the basal CgA secretion. Our study shows for the first time that TRPV1 is expressed in pancreatic NETs. Activation of TRPV1 translates into changes of intracellular Ca2+, a known mechanism triggering the secretion of CgA. The clinical relevance of TRPV1 activation in NETs requires further investigations.

SOC and now also SIC: Store‐operated and store‐inhibited channels

There is a specialized form of calcium influx that involves a close communication between endoplasmic reticulum and the channels at the plasma membrane. In one side store depletion activates channels known as store-operated channels (SOC), which are responsible of the well-studied store-operated calcium entry (SOCE). SOC comprises two different types of channels. Orai, which is exclusively activated by store depletion being the channel responsible of the calcium release-activated calcium current, and transient receptor potential canonical channel, which in contrast, is activated by store depletion only under specific conditions and carries nonselective cationic currents. On the other hand, it has been recently shown that store depletion also inhibits calcium channels. The first member identified, of what we named as store-inhibited channels (SIC), is the L-type voltage-gated calcium channel. Stores control both SOC and SIC by means of the multifunctional protein STIM1. The identification of SOC and SIC opens a new scenario for the role of store depletion in the modulation of different calcium entry pathways, which may satisfy different cellular processes.

Plasma membrane insertion of TRPC5 channels contributes to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons

In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2-APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I(tail)) and the associated dendritic Ca(2+) influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I(tail). In addition, pretreatment with the calmodulin (CaM) inhibitor W-7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I(tail)) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3-kinase (PI(3)K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I(tail) and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy.

Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells

Hydrogen sulfide (H2S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H2S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca2+ imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H2S donor, activates cytosolic calcium (Cac) increase, as well as potassium and nonselective cationic currents, in B-TECs. Stimulation with NaHS in the same concentration range (1nM–200μM) evoked Cac signals also in “normal” human microvascular endothelial cells (HMVECs), but the amplitude was significantly lower. Moreover, although NaHS failed to promote either migration or proliferation on HMVECs, B-TEC migration was enhanced at low-micromolar NaHS concentrations (1–10μM). Remarkably H2S mediates tumor proangiogenic signaling triggered by vascular endothelial growth factor (VEGF). B-TECs pretreated with dl-propargylglycine (5mM, 30min), an inhibitor of the H2S-producing enzyme cystathionine γ-lyase, showed drastically reduced migration and Cac signals induced by VEGF (20ng/ml). We conclude that H2S plays a role in proangiogenic signaling of tumor-derived but not normal human ECs. Furthermore the ability of this gasotransmitter to interfere with B-TEC responsiveness to VEGF suggests that it could be an interesting target for antiangiogenic strategies in tumor treatment.

Molecular determinants of PKA-dependent inhibition of TRPC5 channel

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gα(s). Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gα(s) [Gα(s) (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gα(s) (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by G(s)/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β(2)-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.

Calcium‐activated non‐selective cation currents are involved in generation of tonic and bursting activity in dopamine neurons of the substantia nigra pars compacta

Nigral dopamine neurons are transiently activated by high frequency glutamatergic inputs relaying reward-predicting sensory information. The tonic firing pattern of dopamine cells responds to these inputs with a transient burst of spikes that requires NMDA receptors. Here, we show that NMDA receptor activation further excites the cell by recruiting a calcium-activated non-selective cation current (ICAN) capable of generating a plateau potential. Burst firing in vitro is eliminated after blockade of ICAN with flufenamic acid, 9-phenanthrol, or intracellular BAPTA. ICAN is likely to be mediated by a transient receptor potential (TRP) channel, and RT-PCR was used to confirm expression of TRPM2 and TRPM4mRNA in substantia nigra pars compacta.We propose that ICAN is selectively activated during burst firing to boost NMDA currents and allow plateau potentials. This boost mechanism may render DA cells vulnerable to excitotoxicity.

Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K(+), suggesting that it is regulated not only by K(+) conductances but by inward conductances such as Na(+) and/or Ca(2+). We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na(+) with equimolar tetraethylammonium or Ca(2+) with Mn(2+) inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na(+) with N-methyl-D-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bI(NSCC)) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bI(NSCC). In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle.

Contribution of Rho‐kinase to membrane excitability of murine colonic smooth muscle

The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents. The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus.

TRPM2 Channel Membrane Currents in Primary Rat Megakaryocytes Were Activated by the Agonist ADP-Ribose but Not Oxidative Stress

Melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition mechanisms in response to ADP-ribose (ADPR), oxidative stress, flufenamic acid (FFA) and 2-aminoethoxydiphenyl borate (2-APB) are not clear. We tested the effects of FFA and 2-APB on ADPR-induced TRPM2 cation channel currents in rat native bone marrow megakaryocytes. Megakaryocyte cells were freshly isolated from rat bone marrow and studied with the conventional whole-cell patch-clamp technique. Extracellular H2O2, FFA and 2-APB were added through the patch chamber, while intracellular ADPR was applied through the pipette. Nonselective cation currents were consistently induced by ADPR but not H2O2. Current density of ADPR in the cells was significantly (P<0.001) higher than in control. The time courses of ADPR effects in the megakaryocytes were characterized by a delay of 2.24±0.73. The ADPR-induced Ca2+ gate was not blocked by either the IP3 receptor inhibitor 2-APB or the PLC inhibitor FFA. In conclusion, TRPM2 channels were constitutively activated by intracellular ADPR, although the channel currents in rat native megakaryocytes were not affected by extracellular H2O2, 2-APB or FFA. Activation of TRPM2 channels in megakaryocytes seems to be intracellular and ADPR-dependent.

Thermosensitive transient receptor potential channels in human corneal epithelial cells

Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-TRPV4 are all heat-activated non-selective cation channels that are modestly permeable to Ca(2+). TRPV1, TRPV3, and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca(2+)](i) transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT-PCR and TRPV2 transcripts were also detected. In fura2-loaded HCEC, a TRPV1-3 selective agonist, 100 µM 2-aminoethoxydiphenyl borate (2-APB), induced intracellular Ca(2+) transients and an increase in non-selective cation outward currents that were suppressed by ruthenium-red (RuR) (10-20 µM), a non-selective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 to over 40 °C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine CPZ (10 µM) or another related blocker, lanthanum chloride (La(3+)) (100 µM) suppressed these temperature-induced Ca(2+) increases. Planar patch-clamp technique was used to characterize the currents underlying Ca(2+) transients. Increasing the temperature to over 40 °C induced reversible rises in non-selective cation currents. Moreover, a hypotonic challenge (25%) increased non-selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermosensitive TRPV3 activity TRPV1, TRPV2, and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress.

Characterization of TRPM8-Like Channels Activated by the Cooling Agent Icilin in the Macrophage Cell Line RAW 264.7

Icilin is recognized as a chemical agonist of nociceptors and can activate TRPM8 channels. However, whether this agent has any effects on immune cells remains unknown. In this study, the effects of icilin on ion currents were investigated in RAW 264.7 murine macrophage-like cells. Icilin (1-100 μM) increased the amplitude of nonselective (NS) cation current (INS) in a concentration-dependent manner with an EC50 value of 8.6 μM. LaCl3 (100 μM) or capsazepine (30 μM) reversed icilin-induced INS; however, neither apamin (200 nM) nor iberiotoxin (200 nM) had any effects on it. In cell-attached configuration, when the electrode was filled with icilin (30 μM), a unique population of NS cation channels were activated with single-channel conductance of 158 pS. With the use of a long-lasting ramp pulse protocol, increasing icilin concentration produced a left shift in the activation curve of NS channels, with no change in the slope factor of the curve. The probability of channel opening enhanced by icilin was increased by either elevated extracellular Ca2+ or application of ionomycin (10 μM), while it was reduced by BAPTA-AM (10 μM). Icilin-stimulated activity is associated with an increase in mean open time and a reduction in mean closed time. Under current-clamp conditions, icilin caused membrane depolarization. Therefore, icilin interacts with the TRPM8-like channel to increase INS and depolarizes the membrane in these cells.

Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

In a previous study we proposed that the depolarized state of the wake-promoting hypocretin/orexin (hcrt/orx) neurons was independent of synaptic inputs as it persisted in tetrodotoxin and low calcium/high magnesium solutions. Here we show first that these cells are hyperpolarized when external sodium is lowered, suggesting that non-selective cation channels (NSCCs) could be involved. As canonical transient receptor channels (TRPCs) are known to form NSCCs, we looked for TRPCs subunits using single-cell RT-PCR and found that TRPC6 mRNA was detectable in a small minority, TRPC1, TRPC3 and TRPC7 in a majority and TRPC4 and 5 in the vast majority (∼90%) of hcrt/orx neurons. Using intracellular applications of TRPC antibodies against subunits known to form NSCCs, we then found that only TRPC5 antibodies elicited an outward current, together with hyperpolarization and inhibition of the cells. These effects were blocked by co-application of a TRPC5 antigen peptide. Voltage-clamp ramps in the presence or absence of TRPC5 antibodies indicated the presence of a current with a reversal potential close to −15 mV. Application of the non-selective TRPC channel blocker, flufenamic acid, had a similar effect, which could be occluded in cells pre-loaded with TRPC5 antibodies. Finally, using the same TRPC5 antibodies we found that most hcrt/orx cells show immunostaining for the TRPC5 subunit. These results suggest that hcrt/orx neurons are endowed with a constitutively active non-selective cation current which depends on TRPC channels containing the TRPC5 subunit and which is responsible for the depolarized and active state of these cells.

The modulation of TRPM7 currents by nafamostat mesilate depends directly upon extracellular concentrations of divalent cations

Concentrations of extracellular divalent cations (Ca2+ and Mg2+) fall substantially during intensive synaptic transmission as well as during some pathophysiological conditions such as epilepsy and brain ischemia. Here we report that a synthetic serine protease inhibitor, nafamostat mesylate (NM), and several of its analogues, block recombinant TRPM7 currents expressed in HEK293T cells in inverse relationship to the concentration of extracellular divalent cations. Lowering extracellular Ca2+ and Mg2+ also evokes a divalent-sensitive non-selective cation current that is mediated by TRPM7 expression in hippocampal neurons. In cultured hippocampal neurons, NM blocked these TRPM7-mediated currents with an apparent affinity of 27 μM, as well as the paradoxical Ca2+ influx associated with lowering extracellular Ca2+. Unexpectedly, pre-exposure to NM strongly potentiated TRPM7 currents. In the presence of physiological concentrations of extracellular divalent cations, NM activates TRPM7. The stimulating effects of NM on TRPM7 currents are also inversely related to extracellular Ca2+ and Mg2+. DAPI and HSB but not netropsin, blocked and stimulated TRPM7. In contrast, mono-cationic, the metabolites of NM, p-GBA and AN, as well as protease inhibitor leupeptin and gabexate failed to substantially modulate TRPM7. NM thus provides a molecular template for the design of putative modulators of TRPM7.

Mechanoelectrical coupling enhances initiation and affects perpetuation of atrial fibrillation during acute atrial dilation

Acute atrial dilation increases the susceptibility to atrial fibrillation (AF). However, the mechanisms by which atrial stretch may contribute to the initiation and perpetuation of AF remain to be determined. The purpose of this study was to use a novel multiscale model of atrial electromechanics and mechanoelectrical feedback to test the hypothesis that acute stretch increases vulnerability to AF by heterogeneous activation of stretch-activated channels. Human atria were represented by a triangular mesh obtained from magnetic resonance imaging data. Atrial trabecular bundle structure was incorporated by varying thicknesses of the atrial wall. Atrial membrane behavior was modeled by the Courtemanche-Ramirez-Nattel model with the addition of a nonselective stretch-activated cation current (I(sac)). Mechanical behavior was modeled by a series elastic, a contractile, and a parallel elastic element in which contractile force was related to intracellular concentration of free calcium and sarcomere length. Acute atrial dilation was simulated by increasing stretch throughout the atrial wall. Stimulation near the pulmonary vein ostia at an interval of 600 ms induced AF at an overall stretch ratio of 1.10. Initiation and perpetuation of AF in our model were related to increased dispersion of effective refractory period, conduction slowing, and local conduction block, all related to heterogeneous activation of I(sac). Upon local contraction, mechanoelectrical coupling affects perpetuation of AF by temporarily changing local excitability. During acute atrial dilation, heterogeneous activation of I(sac) enhances initiation and can affect perpetuation of AF.

Macrolide Antibiotics Inhibit Mucus Secretion and Calcium Entry in Swine Airway Submucosal Mucous Gland Cells

Macrolide antibiotics such as erythromycin (EM) and azithromycin (AZM) are beneficial in the treatment of mucus hypersecretion in inflammatory pulmonary diseases. Several indirect and direct mechanisms of action have been proposed. This study investigates the direct effect of macrolides on secretory function of isolated submucosal mucous gland cells (SMGCs). We hypothesize that macrolides inhibit the calcium influx necessary for evoked mucus secretion. To test this, we quantified mucin protein release using enzyme-linked immunosorbent assay, calcium-activated K(+) (K(Ca)), and calcium-activated Cl(-) (Cl(Ca)) currents. We measured nonselective cation current (NSCC) using whole-cell patch-clamp techniques; intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 Ca(2+) imaging. We found that both EM and AZM are agonists at muscarinic receptors. EM (10 μM) evoked a small but significant increase in mucin release but inhibited the mucin release induced by subsequent acetylcholine (ACh) treatment. Both EM and AZM (10 μM) evoked K(Ca) and Cl(Ca) whole-cell currents, which were blocked by atropine. EM and AZM also accelerated the decay of inositol trisphosphate-induced K(Ca) and Cl(Ca) currents without changing the peak current amplitudes. Likewise, internal application of AZM (10 μM) enhanced the decay rate of ACh-induced K(Ca) and Cl(Ca) currents. EM (1-10 μM) and AZM (0.1-10 μM) slowly (over 25-30 min) inhibited thapsigargin (TG)-induced Ca(2+) entry when applied during the plateau phase of Ca(2+) entry but blunted TG-induced Ca(2+) entry by 70% after a 5-min pretreatment before initiating calcium entry. EM blocked TG-induced NSCC. We conclude that macrolide antibiotics are partial agonists at muscarinic receptors but inhibit stimulated mucus release by inhibiting calcium entry in SMGCs.

β-Nicotinamide Adenine Dinucleotide Is an Enteric Inhibitory Neurotransmitter in Human and Nonhuman Primate Colons

An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5'-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K(+) channels. In murine colon β-nicotinamide adenine dinucleotide (β-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here β-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. A small-volume superfusion assay and high-pressure liquid chromatography with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of β-NAD, ATP, and metabolites. Postjunctional responses to nerve stimulation, β-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of β-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts were assayed by reverse transcription polymerase chain reaction. In contrast to ATP, overflow of β-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by the neurotoxins, tetrodotoxin, and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous β-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179), and (1R,2S,4S,5S)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS 2500). β-NAD activated nonselective cation currents in SMCs, but failed to activate outward currents. β-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current.

GPR55-dependent and -independent ion signalling in response to lysophosphatidylinositol in endothelial cells.

Background and purposeThe glycerol-based lysophospholipid lysophosphatidylinositol (LPI) is an endogenous agonist of the G-protein-coupled receptor 55 (GPR55) exhibiting cannabinoid receptor-like properties in endothelial cells. To estimate the contribution of GPR55 to the physiological effects of LPI, the GPR55-dependent and -independent electrical responses in this cell type were investigated.Experimental approachApplying small interference RNA-mediated knock-down and transient overexpression, GPR55-dependent and -independent effects of LPI on cytosolic free Ca2+ concentration, membrane potential and transmembrane ion currents were studied in EA.hy296 cells.Key resultsIn a GPR55-dependent, GDPβS and U73122-sensitive manner, LPI induced rapid and transient intracellular Ca2+ release that was associated with activation of charybdotoxin–sensitive, large conductance, Ca2+-activated, K+ channels (BKCa) and temporary membrane hyperpolarization. Following these initial electrical reactions, LPI elicited GPR55-independent long-lasting Na+ loading and a non-selective inward current causing sustained membrane depolarization that depended on extracellular Ca2+ and Na+ and was partially inhibited by Ni2+ and La3+. This inward current was due to the activation of a voltage-independent non-selective cation current. The Ni2+ and La3+-insensitive depolarization with LPI was prevented by inhibition of the Na/K-ATPase by ouabain.Conclusions and implicationsLPI elicited a biphasic response in endothelial cells of which the immediate Ca2+ signalling depends on GPR55 while the subsequent depolarization is due to Na+ loading via non-selective cation channels and an inhibition of the Na/K-ATPase. Thus, LPI is a potent signalling molecule that affects endothelial functions by modulating several cellular electrical responses that are only partially linked to GPR55.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels in the regulation of midbrain dopamine systems

Hyperpolarization-activated, cyclic nucleotide-gated channels (HCN channels) are expressed widely in the brain and invovled in various neuronal activities, including the control of neuronal rhythmic activity, setting the resting membrane potential, as well as dendritic integration. HCN channels also participate in the regulation of spontaneous activity of midbrain dopamine (DA) neurons to some extent. In slice preparations of midbrain, a hyperpolarization-activated non-selective cation current (Ih) mediated by the channels has been proposed as an electrophysiological marker to identify DA neurons. Recent evidence, however, shows that the functional roles of HCN channels in midbrain DA neurons are obviously underestimated. Here, we review the recent advances in the studies of the functional roles of Ih in midbrain DA neurons and further, their involvement in drug addiction and Parkinson's disease.

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca(2+)](i)) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH(4)Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at -80mV, NH(4)Cl triggered an inward current, the main component of which is a Ca(2+)-dependent chloride current. Calcium imaging confirmed that NH(4)Cl provoked a [Ca(2+)](i) increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I(In)). These currents were stimulated during external addition of NH(4)Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH(4)Cl and extracellular acidic medium, was shown to trigger a [Ca(2+)](i) increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH(4)Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.

Lipopolysaccharides directly decrease Ca2+ oscillations and the hyperpolarization-activated nonselective cation current If in immortalized HL-1 cardiomyocytes

Lipopolysaccharide (LPS) has been implicated in sepsis-mediated heart failure and chronic cardiac myopathies. We determined that LPS directly and reversibly affects cardiac myocyte function by altering regulation of intracellular Ca2+ concentration ([Ca2+]i) in immortalized cardiomyocytes, HL-1 cells. [Ca2+]i oscillated (<0.4 Hz), displaying slow and transient components. LPS (1 microg/ml), derived either from Escherichia coli or from Salmonella enteritidis, reversibly abolished Ca2+ oscillations and decreased basal [Ca2+]i by 30-40 nM. HL-1 cells expressed Toll-like receptors, i.e., TLR-2 and TLR-4. Thus, we differentiated effects of LPS on [Ca2+]i and Ca2+ oscillations by addition of utlrapure LPS, a TLR-4 ligand. Ultrapure LPS had no effect on basal [Ca2+]i, but it reduced the rate of Ca2+ oscillations. Interestingly, Pam3CSK4, a TLR-2 ligand, affected neither Ca2+ parameter, and the effect of ultrapure LPS and Pam3CSK4 combined was similar to that of utlrapure LPS alone. Thus, unpurified LPS directly inhibits HL-1 calcium metabolism via TLR-4 and non-TLR-4-dependent mechanisms. Since others have shown that endotoxin impairs the hyperpolarization-activated, nonselective cationic pacemaker current (I(f)), which is expressed in HL-1 cells, we utilized whole cell voltage-clamp techniques to demonstrate that LPS (1 microg/ml) reduced I(f) in HL-1 cells. This inhibition was marginal at physiologic membrane potentials and significant at very negative potentials (P < 0.05 at -140, -150, and -160 mV). So, we also evaluated effects of LPS on tail currents of fully activated I(f). LPS reduced the slope conductance of the tail currents from 498 +/- 140 pS/pF to 223 +/- 65 pS/pF (P < 0.05) without affecting reversal potential of -11 mV. Ultrapure LPS had similar effect on I(f), whereas Pam3CSK4 had no effect on I(f). We conclude that LPS inhibits activation of I(f), enhances its deactivation, and impairs regulation of [Ca2+]i in HL-1 cardiomyocytes via TLR-4 and other mechanisms.