A dihydropyridine-sensitive calcium channel in rodent osteoblastic cells

Summary Rat osteogenic sarcoma cells (UMR 106-01) and normal rat trabecular bone osteoblasts (ROB) were studied using the whole cell version of the patch clamp technique to determine the existence of calcium (Ca²⁺) channels. Pipette and bath solutions were designed to separate Ca²⁺ channel currents from other voltage-dependent currents, and Ba²⁺ was used as the charge carrier. In both UMR 106-01 and ROB cells, a Ba²⁺ current was measured, which expressed the characteristics of an L-channel, such as activation range, dihydropyridine sensitivity, and little or no inactivation. In some cases, this channel was detectable only with BAY-K-8644 in the bath solution. The dihydropyridine agonist increased the current intensity and shifted the peak inward current to more negative potentials. This study, confirming previous observations, demonstrates the existence of a Ca²⁺ channel in both transformed and normal osteoblastic cells.     

Ryanodine-sensitive intracellular Ca2+ stores in isolated rabbit penile erectile tissue

Norepinephrine release from adrenergic nerve terminals leads to a rise in intracellular Ca²⁺, which promotes penile smooth muscle contraction and detumescence. Ca²⁺ sources are the extracellular space and sarcoplasmic Ca²⁺ stores. To elucidate the role of intracellular stores strips from rabbit erectile tissue were investigated in an organ bath study. Contractions were elicited by phenylephrine (PE) and electrical stimulation. Incubation in Ca²⁺-free solution as well as exposure to nifedipine did not abolish electrical or PE-induced contraction. Ryanocline (10^-5 mol/l), a functional blocker of sarcoplasmic Ca²⁺ channels, significantly reduced PE response. In the presence of caffeine (10^-3 mol/l) the effect was significantly enhanced. Addition of nifedipine nearly abolished the contraction. These results provide evidence for intracellular Ca²⁺ pools in cavernosal tissue and indicate that the ₁-adrenoceptor-induced contraction requires the opening of voltage-gated Ca²⁺ channels and the release of Ca²⁺ from intracellular stores.     

Volatile Anesthetics Reduce Low-voltage-activated Calcium Currents in a Thyroid C-Cell Line

Background: Volatile anesthetics may act in part by inhibiting voltage-dependent calcium channels. The effects of several volatile agents on three types of calcium channels in a thyroid C-cell line were examined.

Methods: Whole-cell calcium currents were recorded using standard patch clamp techniques. Current-voltage relationships were derived before, during, and after application of isoflurane, enflurane, or halothane. Low-voltage-activated (LVA; T type) calcium currents were isolated based on the voltage range of activation. High-voltage-activated (HVA) calcium currents were separated into L and N types using omega-conotoxin GVIA (omega-CTX) and nicardipine.

Results: All three agents reversibly decreased both LVA and HVA currents at clinically relevant concentrations. Isoflurane and enflurane both reduced peak LVA current more than peak HVA current: -33 +/- 6% (mean +/- SE) versus -22 +/- 4% for 0.71 mM isoflurane (n = 6), and -46 +/- 6% versus -35 +/- 5% for 1.21 mM enflurane (n = 6). In contrast, halothane depressed LVA and HVA currents to a similar extent: -22 +/- 4% versus -29 +/- 3% for 0.65 mM halothane (n = 6). Isoflurane had no effect on LVA whole-cell current kinetics. Pretreatment with either omega-CTX (400 nM) or nicardipine (1 micro Meter) did not change the sensitivity of HVA current to isoflurane.     

Diabetic visceral hypersensitivity is associated with activation of mitogen-activated kinase in rat dorsal root ganglia

OBJECTIVE

Diabetic patients often experience visceral hypersensitivity and anorectal dysfunction. We hypothesize that the enhanced excitability of colon projecting dorsal root ganglia (DRG) neurons observed in diabetes is caused by a decrease in the amplitude of the transient A-type K⁺ (I_A) currents resulting from increased phosphorylation of mitogen-activated protein kinases (MAPK) and reduced opening of K_v4.2 channels.

RESEARCH DESIGN AND METHODS

We performed patch-clamp recordings of colon projecting DRG neurons from control and streptozotocin-induced diabetic (STZ-D) rats. Western blot analyses and immunocytochemistry studies were used to elucidate the intracellular signaling pathways that modulate the I_A current. In vivo studies were performed to demonstrate that abnormal MAPK signaling is responsible for the enhanced visceromotor response to colorectal distention in STZ-D rats.

RESULTS

Patch-clamp studies demonstrated that I_A current was diminished in the colon projecting DRG neurons of STZ-D rats. Western blot analysis of STZ-D DRG neurons revealed increases in phosphorylated MAPK and K_V4.2. In diabetic DRG neurons, increased intracellular Ca²⁺ ([Ca²⁺]_i), protein kinase C (PKC), and MAPK were involved in the regulation of I_A current through modulation of K_v4.2. Hypersensitive visceromotor responses to colorectal distention in STZ-D rats were normalized by administration of MAPK inhibitor U0126.

CONCLUSIONS

We demonstrated that reduction of the I_A current in STZ-D DRG neurons is triggered by impaired [Ca²⁺]_i ion homeostasis, and this in turn activates the PKC-MAPK pathways, resulting in decreased opening of the K_v4.2 channels. Hence, the PKC-MAPK–K_v4.2 pathways represent a potential therapeutic target for treating visceral hypersensitivity in diabetes.Patients with long-standing diabetes often demonstrate visceral hypersensitivity and anorectal dysfunction. This may result in altered bowel habits, rectal urgency, and diarrhea (1–4). The pathophysiology of these conditions remains unclear. Previous studies suggest that diabetes-induced sensory neuropathies may be the consequence of increased activity of primary afferent fibers leading to an increased excitatory tone in the spinal cord (5). The spectrum of interacting ionic currents in different types of neurons appears to be important in determining the excitability of sensory neurons (6,7). The transient A-type K⁺ (I_A) current is an important determinant of neuronal excitability. This current participates in the transduction of graded stimulating currents into graded firing rates (8). K_v4.2 channels, which are the primary molecular correlates of the I_A current in sensory neurons, are prime targets for modulation (9,10).Sensory neuropathies in diabetes are associated with abnormal signaling in the intracellular Ca²⁺ ([Ca²⁺]_i) pathway in the dorsal root ganglia (DRG) neurons. Enhanced influx of Ca²⁺ via multiple high-threshold Ca²⁺ currents and/or abnormal [Ca²⁺]_i uptake by endoplasmic reticulum occurs in sensory neurons of several models of diabetes (11–15). Increased [Ca²⁺]_i signaling has also been implicated in the pathogenesis of a variety of neurodegenerative disorders (16). We hypothesize that diabetes is associated with Ca²⁺-mediated activation of mitogen-activated protein kinases (MAPK) that modulates the I_A current in the DRG neurons. The reduction in I_A current results in enhanced neural excitability, which may cause rectal hypersensitivity. To test this hypothesis, we examined how diabetes-evoked changes in [Ca²⁺]_i homeostasis modulate the excitability of distal colon projecting DRG neurons. We demonstrated that diabetic visceral hypersensitivity in the rectum is mediated by an abnormal I_A current resulting from increased phosphorylation of MAPK in DRG neurons. This decreases the opening of K_v4.2 channels and reduces the amplitude of I_A current. The increased neuronal excitability appears to be responsible for rectal hypersensitivity in diabetes.     

Calcium channel blockers are inadequate for malignant hyperthermia crisis

Purpose

Malignant hyperthermia (MH) results from disordered calcium (Ca²⁺) homeostasis in skeletal muscle during general anesthesia. Although Ca²⁺ channel blockers may be given to treat the tachycardia and circulatory instability, coadministration of Ca²⁺ channel blockers and dantrolene is contraindicated during MH crisis. We evaluated the effect of Ca²⁺ channel blockers on Ca²⁺ homeostasis and their interactions with dantrolene in human skeletal muscle.

Methods

Human skeletal muscle samples were obtained by biopsy and divided into two groups according to the results of the Ca²⁺-induced Ca²⁺ release rate test. Differentiated myotubes were labeled with Fura-2, and changes in the 340/380-nm ratio were used to calculate changes in Ca²⁺ concentration following nifedipine treatment in the absence or presence of dantrolene.

Results

Nifedipine induced a transient increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. The half-maximal concentration (EC₅₀) for nifedipine was 0.718?±?0.329?μM in the accelerated group and 1.389?±?0.482?μM in the nonaccelerated group (P?=?0.009). The addition of 50?μM dantrolene attenuated by 15.4% the increase in [Ca²⁺]_i caused by the 0.5?μM nifedipine.

Conclusion

Ca²⁺ channel blockers led to increased [Ca²⁺]_i in human skeletal muscle cells. The increase is thus scarcely affected by dantrolene treatment. Data provide a greater physiologic basis for avoiding the use of Ca²⁺ channel blockers during MH crisis.     

Voltage-Gated calcium channels and nonvoltage-gated calcium uptake pathways in the rat incisor odontoblast plasma membrane

Odontoblasts participate actively in the transport and accumulation of Ca²⁺ ions to the mineralization front during dentinogenesis. These cells are known to carry membrane-bound ATP-driven pumps and Na⁺/Ca²⁺ antiports for Ca²⁺ extrusion, but little is known about Ca²⁺ influx mechanisms into these cells. It has been shown that the administration of Ca²⁺ channel blockers in vivo strongly impairs Ca²⁺ uptake in the mineral phase during dentinogenesis in the rat; the present in vitro study is aimed at further elucidating odontoblast Ca²⁺ uptake mechanisms. Dissected rat incisor odontoblasts exhibited a pronounced fluorescence when incubated with a fluorescently-labeled (STBodipy) dihydropyridine, which is specific for voltage-gated Ca²⁺ channels of the L-type, and this binding was competitively abolished by nifedipine. As assayed by fluorescence spectrometry, odontoblast Ca²⁺ uptake was enhanced by the agonistic dihydropyridine BAYK-8644 (5 μM) as well as by plasma membrane depolarization in a high K⁺ (120 mM) medium. The Ca²⁺ uptake after depolarization was impaired by nifedipine (5 μM). When treated with the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), a nonvoltage-gated uptake of ⁴⁵Ca²⁺ was identified. This uptake was not influenced by nifedipine (20 μM) but was impaired by lanthanum ions (200 μM). A nonvoltage-gated uptake of Mn²⁺ into CPA-treated cells could be traced using the fura-2 quenching technique. This CPA-induced Ca²⁺ flux was not caused by an alteration of the plasma membrane potential, as assayed with di-8-ANEPPS. The results demonstrate that Ca²⁺ flux into dentinogenically active odontoblasts occurs through voltage-gated Ca²⁺ channels of the L-type and by nonvoltage-gated, agonist-sensitive Ca²⁺ uptake pathways. Received: 6 November 1995 / Accepted: 21 February 1996     

Enhanced Glucose Tolerance by SK4 Channel Inhibition in Pancreatic ��-Cells

OBJECTIVE

Ca²⁺-regulated K⁺ channels are involved in numerous Ca²⁺-dependent signaling pathways. In this study, we investigated whether the Ca²⁺-activated K⁺ channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic β-cell function.

RESEARCH DESIGN AND METHODS

Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca²⁺ concentration ([Ca²⁺]_c) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis.

RESULTS

SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO β-cells stimulated with 15 mmol/l glucose had an increased Ca²⁺ action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca²⁺]_c. In addition, glucose responsiveness of membrane potential, [Ca²⁺]_c, and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K⁺ currents and concomitant membrane hyperpolarization could be evoked in WT β-cells by the SK4 channel opener DCEBIO (100 μmol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 μmol/l) partly inhibited K_Ca currents and induced electrical activity at a threshold glucose concentration. In stimulated WT β-cells, TRAM-34 further increased [Ca²⁺]_c and broadened action potentials similar to those seen in SK4-KO β-cells. SK4 channels were found to substantially contribute to K_slow (slowly activating K⁺ current).

CONCLUSIONS

SK4 channels are involved in β-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated β-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.SK4 channels are Ca²⁺-activated K⁺ channels of intermediate conductance (synonymous with IK1 and KCa3.1) encoded by the KCNN4 gene. They are primarily expressed in cells of the hematopoietic system, where they represent the Gardos channel (1). Channel activation requires Ca²⁺ increase and determines the cell volume of T-cells and erythrocytes by elevating K⁺ efflux. In organs regulating salt and fluid transport (e.g., colon, salivary glands, and lung), SK4 current provides the driving force for secondary electrogenic ion transport (2–4). SK4 channels are suggested to be involved in mast cell stimulation (5), and channel upregulation is important for lymphocyte activation and cell proliferation (6,7). For enteric neurons, SK4 channels seem to mediate the late after-hyperpolarization (8). In 1997, SK4 channels were cloned from human pancreatic tissue (9). A detailed investigation of mRNA and protein expression of K_Ca channels of intermediate (SK4) and small conductance (SK1–3) was performed by Tamarina et al. (10) showing mRNA expression of these channels in murine islets.In the past, ATP-sensitive K⁺ (K_ATP) channels were considered to be essential for glucose homeostasis. Consequently, K_ATP channel inhibitors are important drugs to augment insulin secretion in type 2 diabetic subjects. However, with the generation of two K_ATP channel-deficient mouse models (SUR1 and Kir6.2 knockout), it was shown that K_ATP channels are not indispensable for glycemic control (11–14). Neither SUR1 nor Kir6.2 knockout mice show severe hypoglycemia or any symptoms of insulin hypersecretion. Several reports provide evidence that efficient blood glucose regulation and even glucose-dependent insulin secretion (15–17) is possible despite K_ATP channel ablation. In the search for compensatory mechanisms, modulation of insulin release by other K⁺ channels gains particular interest.Besides K_Ca channels, pancreatic β-cells express K⁺ channels exclusively regulated by voltage (K_v channels) (10,18,19). Several studies indicate that K_v channel activation plays a role in action potential (AP) repolarization (20–22). Blocking these channels broadens APs and increases insulin secretion (23–25). Recently, it was shown that K_v2.1 ablation drastically reduces K_v currents of isolated β-cells (26). Interestingly, this coincides with improved glucose tolerance pointing to a specific role for K_v2.1 in the regulation of insulin secretion.For decades, it was discussed whether K_Ca channels participate in the regulation of β-cell activity (27). An early report (28) described K_Ca currents that were periodically activated by inositol-trisphosphate–dependent Ca²⁺ mobilization. The existence of large conductance K_Ca channels (BK channels) in pancreatic β-cells and insulin-secreting cell lines has been verified by several groups (29–31). However, since blockage of BK channels does not alter membrane potential oscillations (31,32), these channels are not considered to play a major role in glucose-stimulated insulin release. In 1999, a K⁺ current activating with increasing Ca²⁺ influx during burst phases of glucose-stimulated β-cells was detected (33). The current, termed K_slow because of its delayed and slow onset, strongly depends on [Ca²⁺]_c. Further analysis suggested that ∼50% could be ascribed to K_ATP current (34). However, the remaining sulfonylurea-insensitive component of K_slow does not resemble the characteristics of any known K_Ca channel (33), and its precise nature remains to be identified. It has been suggested that K_Ca channels of small conductance (SK1–3) play a functional role in β-cells (10,35), but at present, there is only limited information about their contribution to glucose handling of the whole organism.Because up to now nothing is known about the significance of SK4 channels in pancreatic β-cells, this study was performed to elucidate whether SK4 channels are suitable candidates for modulation of β-cell function. We demonstrate that SK4 channels are expressed in murine islets and investigated the influence of constitutive SK4 channel knockout (SK4-KO) and of pharmacological SK4 channel inhibition on glucose homeostasis, insulin sensitivity, and the stimulus-secretion cascade of murine pancreatic β-cells.     

Effects of Schisandra chinensis extract on the contractility of corpus cavernosal smooth muscle (CSM) and Ca2+ homeostasis in CSM cells

What's known on the subject? and What does the study add? Schisandra chinensis extract (SCE) has been known to have relaxative effects on penile smooth muscle. A recent study showed that SCE could enhance slidenafil citrate‐induced relaxation of penile corpus cavernosum. The current study investigated the mechanism of action of SCE and its constituents on corporal smooth muscle cells. And this study shows that SCE induced relaxation of CSM primarily through an endothelium independent pathway and the relaxation effects of SCE on corporal smooth muscle are, in part, due to the activation of K⁺ channels and inhibition of TRPC6 channels, resulting in decreased [Ca²⁺].

OBJECTIVE

• ? To evaluate the relaxant effects of Schisandra chinensis extract (SCE) on corporal tissue in the penis and to investigate the mechanism of action of SCE and its constituents on corporal smooth muscle (CSM) cells.

MATERIALS AND METHODS

• ? The fruit of SC was collected and extracted with ethanol. Six SC lignans (schisandrol A, schisandrol B, schisandrin A, schisandrin B, gomisin N, and schisandrin C) were isolated and purified, and the chemical structures were confirmed by ¹H‐nuclear magnetic resonance (NMR) and ¹³C‐NMR data.
• ? Isolated rabbit CSM strips were mounted in an organ‐bath system, and the effects of SCE were evaluated.
• ? To estimate the intracellular Ca²⁺ level ([Ca²⁺]_i), we used a Fura‐2 fluorescent technique, and a conventional whole‐cell patch‐clamp technique was used to measure the calcium‐sensitive K⁺ channels (K_Ca), inward rectifier K⁺ channels (K_IR), and canonical transient receptor potential cation channel 6 (TRPC6) currents.

RESULTS

• ? SCE induced concentration‐dependent relaxation in contracted CSM tissue, and the removal of the endothelium did not significantly affect their relaxation potencies.
• ? In CSM cells, extracellular application of SCE significantly increased whole‐cell K_Ca currents (117.4%) and K_IR currents (110.0%). These effects were completely abolished by charybdotoxin or BaCl₂.
• ? In contrast, carbachol‐induced TRPC6 channel activity was significantly inhibited (87.3%) by SCE in green fluorescent protein‐TRPC6 pcDNA transfected HEK 293 cells. [Ca²⁺]_i measurements showed that SCE effectively reduced basal [Ca²⁺]_i in both cell lines (CSM cells and A7r5 cells) and the [Arg8]‐vasopressin (AVP)‐induced [Ca²⁺]_i increase in A7r5 cells.
• ? Among the six SC lignans, schisandrin A and schisandrin B most effectively attenuated the AVP‐induced [Ca²⁺]_i increase.

CONCLUSIONS

• ? SCE induced relaxation of CSM that occurred primarily via an endothelium‐independent pathway.
• ? The relaxation effects of SCE on CSM were, in part, due to the activation of K⁺ channels and inhibition of TRPC6 channels, resulting in decreased [Ca²⁺]_i.

     

Glutamate-induced Calcium Transients in Rat Neurons of the Dorsal Motor Nucleus of the Vagus

The dorsal motor nucleus of the vagus (DMNV) integrates peripheral and central signals and sends efferent output to the gastrointestinal system. Glutamate, the major excitatory neurotransmitter of the central nervous system, causes increases in intracellular calcium in DMNV neurons. The mechanisms by which glutamate activates calcium signaling in the DMNV were examined. DMNV neurons were isolated from neonatal rat brainstem using microdissection and enzymatic digestion. Exposure to glutamate caused intracellular Ca²⁺ increments in greater than 80% of cells. Removal of extracellular Ca²⁺ abolished intracellular Ca²⁺ transients. Kynurenic acid, a nonspecific glutamate receptor antagonist, abolished intracellular Ca²⁺ transients. Exposure to glutamate while blocking AMPA receptors with GYKI 52466 abolished the Ca²⁺ response. Exposure to (S)AMPA, an AMPA receptor agonist, caused intracellular Ca²⁺ increments in 97% of cells. Activation and antagonism of NMDA and kainate receptors produced no changes compared to control experiments. NiCl, a nonspecific Ca²⁺ channel blocker, abolished intracellular Ca²⁺ transients. Blocking T-type Ca²⁺ channels with mibefradil abolished the Ca²⁺ response in 76% of cells. Blockade of L-type and N-type Ca²⁺ channels did not affect the Ca²⁺ response. Glutamate mediates intracellular Ca²⁺ currents in DMNV neurons via the AMPA receptor and T-type Ca²⁺ channels, allowing influx of extracellular Ca²⁺.     

Induced Expression of STIM1 Sensitizes Intestinal Epithelial Cells to Apoptosis by Modulating Store-Operated Ca2+ Influx

Introduction

Apoptosis plays a critical role in the maintenance of gut mucosal epithelial homeostasis and is tightly regulated by numerous factors including intracellular Ca²⁺. Canonical transient receptor potential channel-1 (TRPC1) is expressed in intestinal epithelial cells (IECs) and functions as a store-operated Ca²⁺ channel. We have recently demonstrated that increased TRPC1 activity sensitizes IECs to apoptosis, but the upstream signaling initiating TRPC1 activation remains elusive. The novel protein, stromal interaction molecule 1 (STIM1), is shown to act as a store Ca²⁺ sensor, and it can rapidly translocate to the plasma membrane where it directly interacts with TRPC1. The current study determined whether STIM1 plays an important role in the regulation of IEC apoptosis by activating TRPC1 channel activity.

Methods

Studies were conducted in IEC-6 cells (derived from rat intestinal crypts) and stable TRPC1-transfected IECs (IEC-TRPC1). Apoptosis was induced by tumor necrosis factor-?? (TNF-??)/cycloheximide (CHX), and intracellular free Ca²⁺ concentration ([Ca²⁺]_cyt) was measured by fluorescence digital imaging analysis. Functions of STIM1 were investigated by specific siRNA (siSTIM1) and ectopic overexpression of the constitutively active STIM1 EF-hand mutants.

Results

Stable STIM1-transfected IEC-6 cells (IEC-STIM1) showed increased STIM1 protein expression (~5 fold) and displayed a sustained increase in Ca²⁺ influx after Ca²⁺ store depletion (~2 fold). Susceptibility of IEC-STIM1 cells to TNF-??/CHX-induced apoptosis increased significantly as measured by changes in morphological features, DNA fragmentation, and caspase-3 activity. Apoptotic cells were increased from ~20% in parental IEC-6 cells to ~40% in stable IEC-STIM1 cells 4 h after exposure to TNF-??/CHX (p?2+ influx after store depletion in cells overexpressing TRPC1. Levels of Ca²⁺ influx due to store depletion were decreased by ~70% in STIM1-silenced populations. Similarly, exposure of IEC-STIM1 cells to Ca²⁺-free medium also blocked increased sensitivity to apoptosis.

Conclusions

These results indicate that (1) STIM1 plays an important role in the regulation of IEC apoptosis by altering TRPC1 activity and (2) ectopic STIM1 expression sensitizes IECs to apoptosis through induction in TRPC1-mediated Ca²⁺ influx.     

Store–Operated Ca2+ Channels in Mesangial Cells Inhibit Matrix Protein Expression

Accumulation of extracellular matrix derived from glomerular mesangial cells is an early feature of diabetic nephropathy. Ca²⁺ signals mediated by store–operated Ca²⁺ channels regulate protein production in a variety of cell types. The aim of this study was to determine the effect of store–operated Ca²⁺ channels in mesangial cells on extracellular matrix protein expression. In cultured human mesangial cells, activation of store–operated Ca²⁺ channels by thapsigargin significantly decreased fibronectin protein expression and collagen IV mRNA expression in a dose-dependent manner. Conversely, inhibition of the channels by 2-aminoethyl diphenylborinate significantly increased the expression of fibronectin and collagen IV. Similarly, overexpression of stromal interacting molecule 1 reduced, but knockdown of calcium release–activated calcium channel protein 1 (Orai1) increased fibronectin protein expression. Furthermore, 2-aminoethyl diphenylborinate significantly augmented angiotensin II–induced fibronectin protein expression, whereas thapsigargin abrogated high glucose– and TGF-β1–stimulated matrix protein expression. In vivo knockdown of Orai1 in mesangial cells of mice using a targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV, and mice showed significant mesangial expansion compared with controls. Similarly, in vivo knockdown of stromal interacting molecule 1 in mesangial cells by recombinant adeno–associated virus–encoded shRNA markedly increased collagen IV protein expression in renal cortex and caused mesangial expansion in rats. These results suggest that store–operated Ca²⁺ channels in mesangial cells negatively regulate extracellular matrix protein expression in the kidney, which may serve as an endogenous renoprotective mechanism in diabetes.     

Ca2+ channel modulation alters halothane-induced depression of ventricular myocytes

Purpose

This study examined the direct myocardial depressant effect of halothane and determined whether an L-type Ca²⁺ channel agonist and antagonists altered the myocardial depression induced by halothane in cultured rat ventricular myocytes.

Methods

Ventricular myocytes were obtained from neonatal rats by enzymatic digestion with collagenase and then cultured for 6 to 7 days. The myocytes were stabilized in a serum-free medium, and the spontaneous beating rate and amplitude were measured. To assess the halothane-induced conformational changes in L-type Ca²⁺ channel, receptor binding study was performed using a dihydropyridine derivative, [³H] PN 200-110, in cardiac membrane preparation.

Results

Halothane (1%, 2%, 3%, 4%) decreased the beating rate and amplitude in a concentration-dependent manner (P < 0.05). The myocardial depressant effects of halothane were potentiated by nifedipine or verapamil (P < 0.05). Bay K 8644, an L-type Ca²⁺ channel agonist, completely prevented the halothane-induced depression in amplitude (P < 0.05), but affected the beating rate less. Adding halothane (2%) decreased (P < 0.05) the maximum binding site density for [³H] PN 200-110 (from 198.6 ± 23.7 fmol·mg^?1 protein to 115.3 ± 21.6 fmol·mg^?1 protein) but did not affect binding affinity (from 0.461 ± 0.077 nM to 0.307 ± 0.055 nM).

Conclusion

The reduction of Ca²⁺ current via sarcolemmal L-type Ca²⁺ channel, probably due to conformational changes in dihydropyridine binding sites, plays an important role in halothane-induced myocardial depression in living heart cells.     

Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function. INTRODUCTION: During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. MATERIALS AND METHODS: Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. RESULTS: mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. CONCLUSION: These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.     

Mechanisms of control of the free Ca2+ concentration in the endoplasmic reticulum of mouse pancreatic β-cells: interplay with cell metabolism and [Ca2+]c and role of SERCA2b and SERCA3

OBJECTIVE

Sarco-endoplasmic reticulum Ca²⁺-ATPase 2b (SERCA2b) and SERCA3 pump Ca²⁺ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca²⁺ concentration ([Ca²⁺]_ER) and the role of SERCA3 in the control of insulin secretion and ER stress.

RESEARCH DESIGN AND METHODS

β-Cell [Ca²⁺]_ER of SERCA3^+/+ and SERCA3^−/− mice was monitored with an adenovirus encoding the low Ca²⁺-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and [Ca²⁺]_ER were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied.

RESULTS

Glucose elicited synchronized [Ca²⁺]_ER and [Ca²⁺]_c oscillations. [Ca²⁺]_ER oscillations were smaller in SERCA3^−/− than in SERCA3^+/+ β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca²⁺]_ER rise in SERCA3^+/+ and SERCA3^−/− β-cells. In a Ca²⁺-free medium, glucose moderately raised [Ca²⁺]_ER from a highly buffered cytosolic Ca²⁺ pool. Increasing [Ca²⁺]_c with high [K] elicited a [Ca²⁺]_ER rise that was larger but more transient in SERCA3^+/+ than SERCA3^−/− β-cells because of the activation of a Ca²⁺ release from the ER in SERCA3^+/+ β-cells. Glucose-induced insulin release was larger in SERCA3^−/− than SERCA3^+/+ islets. SERCA3 ablation did not induce ER stress.

CONCLUSIONS

[Ca²⁺]_c and [Ca²⁺]_ER oscillate in phase in response to glucose. Upon [Ca²⁺]_c increase, Ca²⁺ is taken up by SERCA2b and SERCA3. Strong Ca²⁺ influx triggers a Ca²⁺ release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca²⁺ uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.Pancreatic β-cells stimulated by glucose display oscillations of the free cytosolic Ca²⁺ concentration ([Ca²⁺]_c) resulting from intermittent Ca²⁺ influx (1,2). Their endoplasmic reticulum (ER) takes up cytosolic Ca²⁺ by two sarco-endoplasmic reticulum Ca²⁺-ATPases (SERCAs): SERCA2b, ubiquitously expressed, and SERCA3, expressed only in islet β-cells (3,4). The role played by the ER in the [Ca²⁺]_c response to glucose is unclear. In particular, it has been suggested that Ca²⁺ influx through voltage-dependent Ca²⁺ channels facilitates the uptake of Ca²⁺ by the ER (5–10) or, on the contrary, triggers a release of Ca²⁺ from the ER (11–14), which might contribute to glucose-induced [Ca²⁺]_c oscillations (11,14) or to a sustained and pronounced [Ca²⁺]_c rise (12,13).The method of choice to monitor the free ER Ca²⁺ concentration ([Ca²⁺]_ER) in living cells uses genetically encoded, Ca²⁺-sensitive probes targeted to the organelle (15,16). One of them, D1ER, a ratiometric Ca²⁺ indicator, has been used in several cell types (17,18). However, the D1 Ca²⁺ sensor has a relatively high affinity for Ca²⁺ (60 µmol/L) (19). To yield a more suitable probe to monitor higher [Ca²⁺]_ER, we replaced D1 by D4 that has a lower affinity for Ca²⁺ (195 µmol/L) (20), and expressed it under the control of the insulin promoter in clusters of β-cells. In most experiments, [Ca²⁺]_ER (D4ER) and [Ca²⁺]_c (FuraPE3) were simultaneously recorded to evaluate the interplay between both parameters. Because SERCA2b and SERCA3 have been suggested to play distinct roles (4,5), we evaluated their respective roles on [Ca²⁺]_c and [Ca²⁺]_ER by using β-cells from wild-type (SERCA3^+/+, expressing SERCA2b and SERCA3) and SERCA3 knockout mice (SERCA3^−/−, expressing SERCA2b only) (21). We also assessed the role of SERCA3 in glucose tolerance, insulin secretion, and ER stress, as it was found that missense mutations of the human SERCA3 gene are associated with type 2 diabetes (22), SERCA3 expression is reduced in diabetic rat models (23), and SERCA3 is involved in ER stress (24).     

Characteristics of spontaneous calcium oscillations in renal tubular epithelial cells

Background

The kidney is a major organ involved in calcium (Ca²⁺) metabolism. Ca²⁺ is transported through renal tubular epithelial cells. The intracellular free calcium concentration ([Ca²⁺]_i) is tightly controlled at a low concentration, but transient increases and oscillations in [Ca²⁺]_i are induced by various conditions. In this study, we investigated the mechanisms underlying the spontaneous [Ca²⁺]_i oscillations observed in MDCK cells.

Methods

[Ca²⁺]_i was monitored in fura-2-loaded Madin-Darby canine kidney (MDCK) cells using a calcium imaging system. We investigated the mechanism by which [Ca²⁺]_i changed by applying drugs or by changing the extracellular Ca²⁺ concentration.

Results

Spontaneous [Ca²⁺]_i oscillations occurred in MDCK cells. The oscillations occurred irregularly and were not transmitted to neighboring cells. Spontaneous [Ca²⁺]_i oscillations in MDCK cells were initiated by Ca²⁺ release from ryanodine/IP₃-sensitive intracellular calcium stores, and their frequency was largely unaffected by the extracellular Ca²⁺ concentration. Moreover, the frequency of the oscillations was increased by extracellular nucleotide, but was decreased when the nucleotides were removed.

Conclusions

Our study suggested that [Ca²⁺]_i release from ryanodine/IP₃-sensitive intracellular calcium stores mediates spontaneous [Ca²⁺]_i oscillations in MDCK cells. Calcium oscillations may be associated with the function of the renal tubular epithelial cells.     

Signal transduction in cavernous smooth muscle

Summary Knowledge of intracellular signal propagation in smooth-muscle tone regulation is of major importance to the understanding of both the physiology of erection and the pathophysiology of erectile dysfunction and the development of new and selective pharmacological agents in the treatment of erectile dysfunction. Cavernous smooth-muscle tone depends heavily on the amount of intracellular free Ca²⁺. In the resting state the sarcoplasmic free Ca²⁺ amounts to about 120–270 nM, whereas in the extracellular fluid the Ca²⁺ level is in the range of 1.5–2 mM. Electromechanical and pharmacomechanical coupling induces an increase in the levels of free sarcoplasmic Ca²⁺ by a factor of 2–3 to 550–700 nM that triggers myosin phosphorylation and subsequent smooth muscle contraction. In this case, modulation of membrane-bound ion channels and regulation of the intracellular second-messenger system are attractive and feasible targets for pharmacological intervention. Besides the amount of free sarcoplasmic Ca²⁺ smooth muscle tone is also modulated by the regulation of Ca2+ sensitivity (Ca-sensitization) and Ca²⁺-independent contraction processes.     

Parathyroid Hormone Activates TRPV5 via PKA-Dependent Phosphorylation

Low extracellular calcium (Ca²⁺) promotes release of parathyroid hormone (PTH), which acts on multiple organs to maintain overall Ca²⁺ balance. In the distal part of the nephron, PTH stimulates active Ca²⁺ reabsorption via the adenylyl cyclase–cAMP–protein kinase A (PKA) pathway, but the molecular target of this pathway is unknown. The transient receptor potential vanilloid 5 (TRPV5) channel constitutes the luminal gate for Ca²⁺ entry in the distal convoluted tubule and has several putative PKA phosphorylation sites. Here, we investigated the effect of PTH-induced cAMP signaling on TRPV5 activity. Using fluorescence resonance energy transfer, we studied cAMP and Ca²⁺ dynamics during PTH stimulation of HEK293 cells that coexpressed the PTH receptor and TRPV5. PTH increased cAMP levels, followed by a rise in TRPV5-mediated Ca²⁺ influx. PTH (1 to 31) and forskolin, which activate the cAMP pathway, mimicked the stimulation of TRPV5 activity. Remarkably, TRPV5 activation was limited to conditions of strong intracellular Ca²⁺ buffering. Cell surface biotinylation studies demonstrated that forskolin did not affect TRPV5 expression on the cell surface, suggesting that it alters the single-channel activity of a fixed number of TRPV5 channels. Application of the PKA catalytic subunit, which phosphorylated TRPV5, directly increased TRPV5 channel open probability. Alanine substitution of threonine-709 abolished both in vitro phosphorylation and PTH-mediated stimulation of TRPV5. In summary, PTH activates the cAMP-PKA signaling cascade, which rapidly phosphorylates threonine-709 of TRPV5, increasing the channel''s open probability and promoting Ca²⁺ reabsorption in the distal nephron.Calcium (Ca²⁺) is essential for numerous physiologic functions and many cellular processes, including signal transduction, cell motility, and morphology.^1,2 The body maintains extracellular Ca²⁺ levels within a narrow range. Slight perturbations in Ca²⁺ homeostasis are detected by the Ca²⁺-sensing receptor, located in the parathyroid glands.³ Low extracellular Ca²⁺ levels inhibit Ca²⁺-sensing receptor activity, causing the release of parathyroid hormone (PTH) into the circulation.^3,4 In the kidney, PTH reduces Ca²⁺ excretion by stimulating Ca²⁺ reabsorption from the distal part of the nephron.⁵ Secreted PTH consists of 84 amino acids. Upon binding to the PTH receptor (PTH1R), it is able to activate both protein kinase C (PKC), via phospholipase C (PLC), and adenylyl cyclase-cAMP, depending on the cell type.^6,7 Activation of the latter pathway requires the first two residues within the amino-terminus of PTH. Consequently, PTH (3 to 34) does not affect basal cAMP levels in contrast to PTH (1 to 34).^8–10 Use of full-length PTH and these fragments has implicated both pathways in PTH-induced stimulation of renal Ca²⁺ reabsorption.^5,7,11,12The transient receptor potential vanilloid 5 (TRPV5) Ca²⁺ channel constitutes the apical entry gate for active (transcellular) Ca²⁺ reabsorption from the distal part of the nephron.^13,14 It is, therefore, a likely target of PTH. Support for this notion is provided by the observation that long-term exposure to PTH enhances TRPV5 activity via two distinct mechanisms. PTH increases TRPV5 expression¹⁵ and causes accumulation of the channel at the plasma membrane. The latter effect involves PLC signaling and a subsequent PKC-dependent phosphorylation.¹⁶ In contrast, the molecular target for the adenylyl cyclase–cAMP–protein kinase A (PKA) pathway activated by PTH has not been identified. Using ex vivo models of active Ca²⁺ reabsorption, PTH-induced cAMP signaling was demonstrated to be essential for rapid stimulation of luminal Ca²⁺ uptake.^5,11,17,18 Interestingly, primary cultures of isolated rabbit connecting tubules (CNT) and cortical collecting duct cells, which express endogenous TRPV5, displayed increased transcellular Ca²⁺ transport after exposure to forskolin or cAMP-elevating hormones, including PTH.^12,19,20 Thus, despite the large number of studies investigating the role of PTH in renal Ca²⁺ handling, the molecular mechanism connecting the cAMP pathway with subsequent stimulation of active Ca²⁺ reabsorption has not yet been determined. Because TRPV5 has several putative PKA phosphorylation sites and is fundamental to renal Ca²⁺ handling, this channel is a likely target for PTH stimulation via the cAMP-signaling pathway.The aim of this study, therefore, was to elucidate the potential role of TRPV5 in PTH-induced stimulation of transcellular Ca²⁺ reabsorption via cAMP-PKA signaling. First, we confirmed that PTH increases ⁴⁵Ca²⁺ influx in HEK293 cells coexpressing TRPV5 and PTH1R. Next, after exposure to PTH we monitored intracellular Ca²⁺, phosphatidylinositol-4,5-bisphosphate (PIP₂) and cAMP levels in this model system by dynamic fluorescence resonance energy transfer (FRET) assays. Furthermore, we investigated the consequence of forskolin-induced cAMP elevation on TRPV5 function. Finally, we determined TRPV5 cell surface abundance and single channel activity after activation of the cAMP-PKA signaling cascade.     

Electrophysiological properties of the bladder

The electrophysiological properties of detrusor smooth muscle are described, in particular with regard to their influence on the contractile properties of the tissue. The Ca²⁺ and K⁺ channel activities are most important in generating action potentials, but the role of several other ionic currents is described, including Cl^–, Ca²⁺-activated, stretch-activated and ligand-gated channels. The variable appearance and functions of different ionic currents in disease states is discussed, as well as the question of whether electrical activity can transmit between adjacent smooth muscle cells. In addition, the precise role that electrophysiological phenomena play in the regulation of the contractile state of the smooth muscle cells, as well as the generation of bladder electromyograms, is discussed.     

Cellular and molecular mechanisms regulating vascular tone. Part 2: regulatory mechanisms modulating Ca<Superscript>2+</Superscript> mobilization and/or myofilament Ca<Superscript>2+</Superscript> sensitivity in vascular smooth muscle cells

Understanding the physiological mechanisms regulating vascular tone would lead to better circulatory management during general anesthesia. This two-part review provides an overview of current knowledge about the cellular and molecular mechanisms regulating the contractile state of vascular smooth muscle cells (i.e., vascular tone). The first part reviews basic mechanisms controlling the cytosolic Ca²⁺ concentration in vascular smooth muscle cells, and the Ca²⁺-dependent regulation of vascular tone. This second part reviews the regulatory mechanisms modulating Ca²⁺ mobilization and/or myofilament Ca²⁺ sensitivity in vascular smooth muscle cells—including Rho/Rho kinase, protein kinase C, arachidonic acid, Ca²⁺/calmodulin-dependent protein kinase II, caldesmon, calponin, mitogen-activated protein kinases, tyrosine kinases, cyclic nucleotides, Cl⁻ channels, and K⁺ channels.     

Membrane Potential-Dependent Inactivation of Voltage-Gated Ion Channels in ��-Cells Inhibits Glucagon Secretion From Human Islets

OBJECTIVE

To document the properties of the voltage-gated ion channels in human pancreatic α-cells and their role in glucagon release.

RESEARCH DESIGN AND METHODS

Glucagon release was measured from intact islets. [Ca²⁺]_i was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated α-cells identified by immunocytochemistry.

RESULTS

Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl₂. Glucose remained inhibitory in the presence of ZnCl₂ and after blockade of type-2 somatostatin receptors. Human α-cells are electrically active at 1 mmol/l glucose. Inhibition of K_ATP-channels with tolbutamide depolarized α-cells by 10 mV and reduced the action potential amplitude. Human α-cells contain heteropodatoxin-sensitive A-type K⁺-channels, stromatoxin-sensitive delayed rectifying K⁺-channels, tetrodotoxin-sensitive Na⁺-currents, and low-threshold T-type, isradipine-sensitive L-type, and ω-agatoxin-sensitive P/Q-type Ca²⁺-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40–70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, ω-agatoxin, and isradipine. The [Ca²⁺]_i oscillations depend principally on Ca²⁺-influx via L-type Ca²⁺-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below −20 mV and peaked at 0 mV. Blocking P/Q-type Ca²⁺-currents abolished depolarization-evoked exocytosis.

CONCLUSIONS

Human α-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.Glucagon is the principal hyperglycemic hormone (1,2). It is secreted from the pancreatic α-cells in response to a fall in plasma glucose levels, β-adrenergic stimulation, lipids, and amino acids (3–5). Glucagon secretion from α-cells is regulated by paracrine (3), neuronal (6), and intrinsic mechanisms (7). Diabetes involves both impaired insulin and glucagon secretion (8). Thus, hyperglucagonemia is thought to contribute to elevated blood glucose levels, and the impaired glucagon response to hypoglycemia represents a limiting factor for insulin treatment in both type 1 and type 2 diabetes (9,10).Ion channels and electrical activity play a key role in the regulation of glucagon secretion. The properties of rodent α-cells have been characterized in some detail (5,11–13). Rodent α-cells are electrically excitable and electrically active in the absence of glucose. Action potential firing depends on the opening of voltage-activated L- and N-type Ca²⁺-channels, tetrodotoxin (TTX)-sensitive Na⁺-channels, and A-type K⁺-channels (14).The α-cells make up ∼35% of the cell population in human islets (15,16). Here, we have characterized the electrophysiological properties of isolated human α-cells and correlated our findings to changes in glucagon secretion from intact human islets. Our data indicate that glucagon secretion depends on a complex interplay among a number of depolarizing and repolarizing membrane currents.