Characteristics of Ca Currents in Rabbit Basilar Arterial Smooth Muscle Cells

In order to determine the exact nature of Ca channels involved in various cerebrovascular contractile behaviour including vasospasm, we performed experiments to identify and characterize the types of Ca channels in rabbit basilar arterial smooth muscle cells by using kinetic and pharmacologic tools. Sngle smooth muscle cells were enzymatically isolated from rabbit basilar artery. Single cells were voltageclamped, and membrane currents were recorded using the whole-cell configuration of patch clamp technique. The measured cell capacitance (Cm) was 19.2 ± 0.65 pF ( n 21) and input resistance (Rmpu, ) was 2.04 ± 0.12 Gn(n 12), These passive membame properties are similar to other cerebraovascular smooth muscle cells. Inward Ca2 ' -channel current was recorded. Replacement of external Ca2+( 2 mM) with Ba" (10 mM) increased the amplitude of the current and did not shift the I-V relationship for lBa in comparison with that for Ie.. Changing the holding potential from -SO to -40 mV decreased the current amplitude but did not shift the voltage dependence. No detectable low-threshold, rapid inactivating inward current was observed. Steady-state activation and inactivation curves for lea(V, 210c') -4.4 mV; V, 2(inac,) -22.3 mV) and lBa(v, 2(oc') -7.5 mV; VI 2 {Inact } -20.5 mV) were determined. The theoritical 'window current's amplitude was calculated for lea and lBa• Calcium channel current was almost completely inhibited by l,uM nicardipine and enhanced by Bay K 8644, suggesting this is carried by 'L-type' but not by 'T-type' calcium channel. Bay K 8644 significantly shifted activation curve to the negative potential. Both 8-br-cAMPW.l ~ 1 mM) and 8-br-cGMP( 0.1 ~ 1 mM), a membrane permeable cyclic nucleotides, decreased the current amplitude. From the above results, it is suggested that only 'L-type' Ca-current(ICa_d exists in rabbit basilar arterial smooth muscle cells

The Possible Role of Nitric Oxide on Enteric Nerves-Mediated Relaxation of the Gastric Smooth Muscle of the Guinea Pig

The influence of the enteric nerves stimulation on the contractility of gastric circular muscle was studied in guinea pig stomachs. The enteric nerves were activated by electric field stimulation(EFS; 90 V, 1 rns, 32 Hz square pulses for 1 s), EFS produced initial transient contraction followed by relaxation and slow recovery. The initial contraction was sensitively blocked by treatment with atropine. The following relaxation still occurred in nonadrenergic noncholinergidNANC) state. EFSinduced relaxation was reduced by LG-nitro-L-arginine(L-NNA), a nitric oxide(NO) synthase inhibitor. The relaxation was restored from the suppressed state after the application of L-arginine(L-arg), a substrate of nitric oxide synthase. With conventional intracellular recording, slow wave and inhibitory junction potential(IJP) were recorded. L-NNA had no effect on IJP, while apamin blocked it. In conclusion, it is suggested that NO may playa major role in EFS-induced relaxation. The exact mechanism of this relaxation is unknown, but the relaxation does not result from the IJP induced by the activation of apamin-sensitive potassium channels

Loss of Autophagy Diminishes Pancreatic β Cell Mass and Function with Resultant Hyperglycemia

SummaryAutophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic β cells and glucose homeostasis has not been described. We produced mice with β cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. β cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of β cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient β cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in β cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic β cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles

Mechanosensitive activation of K+ channel via phospholipase C-induced depletion of phosphatidylinositol 4,5-bisphosphate in B lymphocytes

In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5-bisphosphate (PIP(2)) is not known yet. The mouse B cells express large conductance background K(+) channels (LK(bg)) that are inhibited by PIP(2). In inside-out patch clamp studies, the application of MgATP (1 mm) also inhibited LK(bg) due to the generation of PIP(2) by phosphoinositide (PI)-kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LK(bg), which was antagonized by PIP(2) (> 1 microm) or higher concentration of MgATP (5 mm). The inhibition by PIP(2) was partially reversible. However, the application of methyl-beta-cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LK(bg) activity and facilitated the activation by stretch. In cell-attached patches, LK(bg) were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano-activation of LK(bg) was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m-3M3FBS or by cross-linking IgM-type B cell receptors activated LK(bg). Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI-231 induces tyrosine phosphorylation of PLCgamma2 and PIP(2) hydrolysis of plasma membrane. The time dependence of PIP(2) hydrolysis and LK(bg) activation were similar. The presence of LK(bg) and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch-dependent ion channel activation, namely, that the degradation of PIP(2) caused by stretch-activated PLC releases LK(bg) from the tonic inhibition by PIP(2)

Sodium-activated Potassium Current in Guinea pig Gastric Myocytes

This study was designed to identify and characterize Na+-activated K+ current (IK(Na)) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+ ([K+]o). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68±2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steadystate current produced under [K+]o (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+- and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+-selective, since reversal potentials (Erev) of various concentrations of [K+]o-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 µM), a blocker of IK(Na), completely and reversibly inhibited this current. The characteristics of the current coincide with those of IK(Na) of other cells. Our results indicate the presence of IK(Na) in guinea pig gastric myocytes

Activation of rat transient receptor potential cation channel subfamily V member 1 channels by 2-aminoethoxydiphenyl borate

Background: The transient receptor potential cation channel subfamily V member 1 (TRPV1) channel has been proved to be a molecular integrator of inflammatory pain sensation. 2-Aminoethoxydiphenyl borate (2-APB) and its analogs have been noticed as attractive candidates for the development of a selective TRPV1 agonist and/or antagonist. However, selectivity and effectiveness, species dependence, and the binding site(s) of 2-APB on TRPV1 channel protein remain controversial. Methods: The present study aimed to characterize acting sites of 2-APB on heterologously expressed rat TRPV1 (rTRPV1) channels in HEK 293 cells. Rat TRPV1 currents were recorded by cell-free, excised patch clamp techniques. Results: In inside-out and outside-out patch modes, 2-APB applied either side of the membrane dose-dependently activated rTRPV1 channels. 2-APB dose-dependently potentiated rTRPV1 currents, that activated by capsaicin, protons, or noxious heat. 2-APB potentiated the capsaicin-activated rTRPV1 current from both side of the patch membrane. A structural analogue of 2-APB, diphenylboronic anhydride, showed the same potentiation effect on the capsaicin-activated rTRPV1 current. Conclusion: It is suggested that 2-APB directly opens rTRPV1 channels from both sides of the membrane and potentiates the opening of channels by inflammatory stimuli

Stromal interaction molecule 2 regulates C2C12 myoblast differentiation

Background: Enhanced intracellular Ca2+ signaling by stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) is required for skeletal muscle differentiation. However, the contribution of STIM2, STIM1's analogue protein, on muscle cell differentiation has not been clearly elucidated. The present study aimed to explore the contribution of STIM2-mediated SOCE on C2C12 myoblast differentiation. Methods: Changes in STIM2 expression level (reverse transcription-polymerase chain reaction and Western blotting) and SOCE activity ([Ca2+]i measurement) were measured during 3 days of in vitro differentiation of C2C12 skeletal myoblast. Transcriptional regulation of STIM2 by nuclear factor of activated T cells, cytoplasmic (NFATc) overexpression was observed, and the effect of STIM2 knockdown on NFAT transcriptional activity (luciferase assay) and myoblast differentiation was quantified. Results: Increase of STIM2 protein level and enhanced SOCE activity were observed in differentiating myoblasts. Treatment with a SOCE blocker (2-APB) inhibited the differentiation. Overexpression of NFATc1 increased STIM2 expression and SOCE activity. Knockdown of STIM2 decreased NFAT transcriptional activity, SOCE activity, and differentiation of C2C12 myoblast. Conclusion: It is suggested that STIM2-activated SOCE controls C2C12 myoblast differentiation