Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells.

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.

Serum response factor-dependent MicroRNAs regulate gastrointestinal smooth muscle cell phenotypes.

BACKGROUND & AIMS: Smooth muscle cells (SMCs) change phenotypes under various pathophysiological conditions. These changes are largely controlled by the serum response factor (SRF), a transcription factor that binds to CC (A/T)6 GG (CArG) boxes in SM contractile genes. MicroRNAs (miRNA) regulate transitions among SMC phenotypes. The SMC miRNA transcriptome (SMC miRNAome) and its regulation by SRF have not been determined. METHODS: We performed massively parallel sequencing to identify gastrointestinal (GI) SMC miRNA transcriptomes in mice and humans. SMC miRNA transcriptomes were mapped to identify all CArG boxes, which were confirmed by SRF knockdown and microarrays. Quantitative polymerase chain reaction was used to identify SMC-phenotypic miRNAs in differentiated and proliferating SMCs. Bioinformatics and target validation analysis showed regulation of SMC phenotype by SRF-dependent, SMC-phenotype miRNAs. RESULTS: We cloned and identified GI miRNA transcriptomes using genome-wide analyses of mouse and human cells. The SM miRNAome consisted of hundreds of unique miRNAs that were highly conserved among both species. We mapped miRNAs CArG boxes and found that many had an SRF-dependent signature in the SM miRNAome. The SM miRNAs CArG boxes had several distinct features. We also identified approximately 100 SMC-phenotypic miRNAs that were induced in differentiated or proliferative SMC phenotypes. We showed that SRF-dependent, SMC-phenotypic miRNAs bind and regulate Srf and its cofactors, myocadin (Myocd) and member of ETS oncogene family Elk1. CONCLUSIONS: The GI SMC phenotypes are controlled by SRF-dependent, SMC-phenotypic miRNAs that regulate expression of SRF, MYOCD, and ELK1.

Role of TREK-1 potassium channel in bladder overactivity after partial bladder outlet obstruction in mouse.

PURPOSE: Mouse models of partial bladder outlet obstruction cause bladder hypertrophy. Expression of a number of ion channels is altered in hypertrophic detrusor muscle, resulting in bladder dysfunction. We determined whether mechanosensitive TREK-1 channels are present in the murine bladder and whether their expression is altered in partial bladder outlet obstruction, resulting in abnormal filling responses. MATERIALS AND METHODS: Partial bladder outlet obstruction was surgically induced in CD-1 mice and the mice recovered for 14 days. Cystometry was done to evaluate bladder pressure responses during filling at 25 microl per minute in partial bladder outlet obstruction mice and sham operated controls. TREK-1 channel expression was determined at the mRNA and protein levels by quantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively, and localized in the bladder wall using immunohistochemistry. RESULTS: Obstructed bladders showed about a 2-fold increase in weight vs sham operated bladders. TREK-1 channel protein expression on Western blots from bladder smooth muscle strip homogenates was significantly decreased in obstructed mice. Immunohistochemistry revealed a significant decrease in TREK-1 channel immunoreactivity in detrusor smooth muscle in obstructed mice. On cystometry the TREK-1 channel blocker L-methioninol induced a significant increase in premature contractions during filling in sham operated mice. L-methioninol had no significant effect in obstructed mice, which showed an overactive detrusor phenotype. CONCLUSIONS: TREK-1 channel down-regulation in detrusor myocytes is associated with bladder overactivity in a murine model of partial bladder outlet obstruction.

Functional role of CLC-2 chloride inward rectifier channels in cardiac sinoatrial nodal pacemaker cells.

A novel Cl(-) inward rectifier channel (Cl,ir) encoded by ClC-2, a member of the ClC voltage-gated Cl(-) channel gene superfamily, has been recently discovered in cardiac myocytes of several species. However, the physiological role of Cl,ir channels in the heart remains unknown. In this study we tested the hypothesis that Cl,ir channels may play an important role in cardiac pacemaker activity. In isolated guinea-pig sinoatrial node (SAN) cells, Cl,ir current was activated by hyperpolarization and hypotonic cell swelling. RT-PCR and immunohistological analyses confirmed the molecular expression of ClC-2 in guinea-pig SAN cells. Hypotonic stress increased the diastolic depolarization slope and decreased the maximum diastolic potential, action potential amplitude, APD(50), APD(90), and the cycle-length of the SAN cells. These effects were largely reversed by intracellular dialysis of anti-ClC-2 antibody, which significantly inhibited Cl,ir current but not other pacemaker currents, including the hyperpolarization-activated non-selective cationic "funny" current (I(f)), the L-type Ca(2+) currents (I(Ca,L)), the slowly-activating delayed rectifier I(Ks) and the volume-regulated outwardly-rectifying Cl(-) current (I(Cl,vol)). Telemetry electrocardiograph studies in conscious ClC-2 knockout (Clcn2(-/-)) mice revealed a decreased chronotropic response to acute exercise stress when compared to their age-matched Clcn2(+/+) and Clcn2(+/-) littermates. Targeted inactivation of ClC-2 does not alter intrinsic heart rate but prevented the positive chronotropic effect of acute exercise stress through a sympathetic regulation of ClC-2 channels. These results provide compelling evidence that ClC-2-encoded endogenous Cl,ir channels may play an important role in the regulation of cardiac pacemaker activity, which may become more prominent under stressed or pathological conditions.

Functional properties of murine bestrophin 1 channel.

Bestrophins form Ca2+-activated Cl- channels when they are expressed heterologously. Here we report the functional characterization of murine bestrophin 1 (mBest1). We isolated mBest1 transcript from mouse heart and analyzed the biophysical properties and expression of this channel protein using a tetracycline inducible system. mBest1 expression is localized at the membrane of transfected HEK cells, in agreement with its role as a channel. Whole-cell patch clamp experiments revealed a calcium sensitive, time independent chloride current. mBest1 current displayed slight voltage dependence, exhibited an anion permeability sequence of SCN- > I- > Cl- and was sensitive to DIDS and niflumic acid. Anion replacement studies were also performed on mBest2 and mBest3 and differences were observed in their relative permeability and slope conductance to SCN-. Our study provides the first characterization of the biophysical properties of mBest1 and a framework for the elucidation of the physiological role of bestrophins.

Cardiac-specific overexpression of the human short CLC-3 chloride channel isoform in mice.

1. ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2. Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3. Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4. The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I(-) > Cl(-) > Asp(-)) and inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5. Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6. In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.

Effects of aging on Ca2+ signaling in murine mesenteric arterial smooth muscle cells.

Pathophysiological changes in arterial smooth muscle structure and function occur with aging and there are a number of reports illustrating reductions in vascular responsiveness with aging. While much is known about arterial remodeling and functional adaptations with aging, very little is known about the biophysical adaptations in individual arterial myocytes. Cytosolic Ca2+ signaling, involving activation of L-type Ca2+ channels on the plasma membrane as well as InsP3 and ryanodine receptors on the sarcoplasmic reticulum, is integral to vascular tone and reactivity. Thus, we tested the hypothesis that aging results in reductions in the functional expression of L-type channels and temporal aspects of ryanodine receptor and InsP3 receptor Ca2+ signaling, in mesenteric arterial smooth muscle cells isolated from 6 and 30 months old C57Bl/6 mice. Comparisons of L-type current activity were made using dialyzed, whole-cell voltage-clamp techniques and Ba2+ as charge carrier. Ca2+ signaling was measured using fura-2 fluorescence microscopy techniques. Cell morphological changes were also investigated using electrophysiological and immunocytochemical approaches. The amplitudes of L-type Ca2+ currents were increased in older mice, but this was associated with membrane surface area increases of approximately 50%, due to increases in cell length not cell width. Consequently, L-type Ca2+ current densities were preserved with age, indicating functional channel expression was unchanged. In contrast, aging was associated with decrements in Ca2+ signaling in response to either ryanodine receptor stimulation by caffeine or InsP3 receptor activation with phenylephrine. These changes with aging may be related to the previously reported depression in myogenic reactivity.

Low osmolarity transforms ventricular fibrillation from complex to highly organized, with a dominant high-frequency source.

BACKGROUND: An osmotic challenge activates volume-regulated chloride currents (I(Cl,vol)), resulting in depolarization of the resting membrane potential and shortening of action potential duration (APD). I(Cl,vol) is activated in ischemia/reperfusion, but the effects of osmotic challenges and I(Cl,vol) on ventricular fibrillation (VF) are unknown. OBJECTIVES: The purpose of this study was to investigate the influence of hypo-osmotic and hypotonic stress and I(Cl,vol) activation on VF dynamics. METHODS: Guinea pig hearts were isolated, stained with di-4 ANEPPS to optically map action potentials (APs) from epicardium using a photodiode array, and perfused with iso-osmotic (low NaCl Ringer plus 45 mM mannitol) or hypo-osmotic (low NaCl Ringer) solution. RESULTS: Hypo-osmotic solution shortened APDs (143 +/- 5 ms --> 115 +/- 10 ms) and increased APD gradients between right and left ventricles (21 +/- 7 ms --> 41 +/- 10 ms, n = 4). In VF induced by burst stimulation, switching to hypo-osmotic solution increased VF frequencies (15.3 +/- 1.2 Hz to 28.9 +/- 3.6 Hz, n = 11), transforming complex fast Fourier transformation spectra to a single dominant high frequency on the left but not the right ventricle. Perfusion with the I(Cl,vol) blocker indanyloxyacetic acid-94 (10 muM) reversed organized VF to complex VF with lower (13.5 +/- 3.7 Hz in left ventricle) frequencies (n = 8), indicating that I(Cl,vol) underlies the changes in VF dynamics. Consistent with this interpretation, the levels of ClC-3 channel protein were 27% greater on left than right ventricles (n = 10), and computer simulations showed that insertion of I(Cl,vol) transformed complex VF to a stable spiral. CONCLUSION: Activation of I(Cl,vol) by decreasing osmolarity (45 mOsm) has a major impact on VF dynamics by transforming random multiple wavelets to a highly organized VF with a single dominant frequency.

Targeted inactivation of cystic fibrosis transmembrane conductance regulator chloride channel gene prevents ischemic preconditioning in isolated mouse heart.

BACKGROUND: Recent evidence suggests that chloride channels may be involved in ischemic preconditioning (IPC). In this study, we tested whether the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels, which are expressed in the heart and activated by protein kinase A and protein kinase C, are important for IPC in isolated heart preparations from wild-type (WT) and CFTR knockout (CFTR-/-) mice. METHODS AND RESULTS: Hearts were isolated from age-matched WT or CFTR-/- (B6.129P2-Cftr(tm1Unc) and STOCKCftr(tm1Unc)-TgN 1Jaw) mice and perfused in the Langendorff or working-heart mode. All hearts were allowed to stabilize for 10 minutes before they were subjected to 30 or 45 minutes of global ischemia followed by 40 minutes of reperfusion (control group) or 3 cycles of 5 minutes of ischemia and reperfusion (IPC group) before 30 or 45 minutes of global ischemia and 40 minutes of reperfusion. Hemodynamic indices were recorded to evaluate cardiac functions. Release of creatine phosphate kinase (CPK) in the samples of coronary effluent and infarct size of the ventricles were used to estimate myocardial tissue injury. In WT adult hearts, IPC protected cardiac function during reperfusion and significantly decreased ischemia-induced CPK release and infarct size. A selective CFTR channel blocker, gemfibrozil, abrogated the protective effect of IPC. Furthermore, targeted inactivation of the CFTR gene in 2 different strains of CFTR-/- mice also prevented IPC's protection of cardiac function and myocardial injury against sustained ischemia. CONCLUSIONS: CFTR Cl- channels may serve as novel and crucial mediators in mouse heart IPC.

ClC-3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery.

Cl- channels have been implicated in essential cellular functions including volume regulation, progression of cell cycle, cell proliferation and contraction, but the physiological functions of the ClC-3 channel are controversial. We tested the hypothesis that the ClC-3 gene (ClCn-3) is upregulated in hypertensive pulmonary arteries of monocrotaline-treated rats, and upregulated ClC-3 channel aids viability of pulmonary artery smooth muscle cells (PASMCs). Experimental pulmonary hypertension was induced in rats by a single subcutaneous administration of monocrotaline (60 mg kg(-1)). Injected animals developed characteristic features of pulmonary hypertension including medial hypertrophy of pulmonary arteries and right ventricular hypertrophy. Reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry and Western immunoblot analysis indicated that histopathological alterations were associated with upregulation of the ClC-3 mRNA and protein expression in both smooth muscle cells of hypertensive pulmonary arteries and in cardiac myocytes. RT-PCR analysis of mRNA, extracted from canine cultured PASMCs, indicated that incubation with the inflammatory mediators endothelin-1 (ET-1), platelet-derived growth factor (PDGF), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF alpha), but not transforming growth factor beta (TGFbeta), upregulated ClC-3 mRNA. Adenovirus-mediated delivery and overexpression of ClC-3 in canine PASMCs improved cell viability against increasing concentrations of hydrogen peroxide (H2O2, range 50-250 microM). In conclusion, upregulation of ClC-3 in rat hypertensive lung and heart is a novel observation. Our functional data suggest that upregulation of ClC-3 is an adaptive response of inflamed pulmonary artery, which enhances the viability of PASMCs against reactive oxygen species.

Differential tissue shrinkage and compression in the z-axis: implications for optical disector counting in vibratome-, plastic- and cryosections.

The optical disector is among the most efficient cell counting methods, but its accuracy depends on an undistorted particle distribution in the z-axis of tissue sections. Because the optical disector samples particle densities exclusively in the center of sections, it is essential for unbiased estimates of particle numbers that differential shrinkage or compression (and resulting differences in particle densities along the z-axis) are known and corrected. Here we examined, quantified, and compared differential shrinkage and compression of vibratome-, celloidin- and cryosections. Vibratome sections showed a significant z-axis distortion, while celloidin- and cryosections were minimally distorted. Results were directly compared with previous data obtained from paraffin and methacrylate sections. We conclude that z-axis distortion varies significantly between embedding and sectioning methods, and that vibratome-, methacrylate- and paraffin sections can result in grossly biased estimates. We describe a simple method for assessing differential z-axis shrinkage or compression, as well as simple strategies to minimize the bias of the optical disector. Minimal bias can be achieved by either adjusting the placement and extent of counting boxes and guard spaces for sampling, or by applying a correction factor in cases when guard spaces are deemed essential for particle recognition.

MicroRNAs dynamically remodel gastrointestinal smooth muscle cells.

Smooth muscle cells (SMCs) express a unique set of microRNAs (miRNAs) which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI) SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM) layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF), and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract.

Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart.

Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in the mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated by using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is present at the membrane. Using the patch-clamp technique, we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a K(D) for Ca(2+) of approximately 175 nM. The calcium-activated chloride current was found to be time and voltage independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN(-)>I(-)>Cl(-), and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a site-specific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a pore-forming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization, and functional properties of mBest3 cloned from heart.

Cell culture alters Ca2+ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells.

Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores activates capacitative Ca(2+) entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca(2+) stores and alters Ca(2+) entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura 2-loaded cells. Mn(2+) quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15-18 mmHg. In acutely isolated PASMCs, depletion of IP(3)-sensitive Ca(2+) stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular Ca(2+) transients but abolished 5-HT-induced Ca(2+) transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca(2+) transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [Ca(2+)](i). The transient rise in [Ca(2+)](i) was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [Ca(2+)](i) was inhibited by KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca(2+)](i) was inhibited by SKF-96365, Ni(2+), La(3+), and Gd(3+). In addition, store depletion or hypoxia increased the rate of Mn(2+) quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP(3) and ryanodine receptors into a common intracellular Ca(2+) compartment, and depletion of this store or hypoxia activates voltage-operated Ca(2+) entry, reverse mode NCX, and CCE.

Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells.

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes. These new antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90-92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozygous Clcn3+/+ mice but not from homozygous Clcn3-/- mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 antibodies specifically targeting the ClC-3 carboxy (C670-687 antibody) and amino terminus (A1-14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670-687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.

Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC.

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl(-) currents (I(Cl.vol)) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 microM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 microM) or Ro-31-8425 (0.1 microM), inhibitors of both conventional and novel PKC isozymes, significantly activated I(Cl.vol) and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 microM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with epsilon V1-2 (10 microM), a translocation inhibitory peptide derived from the V1 region of epsilon PKC, activated I(Cl.vol) under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with beta C2-2 (10 microM), a translocation inhibitory peptide derived from the C2 region of beta PKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of epsilon PKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound epsilon PKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.

Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytes.

A rapidly inactivating K(+) current (A-type current; I(A)) present in murine colonic myocytes is important in maintaining physiological patterns of slow wave electrical activity. The kinetic profile of colonic I(A) resembles that of Kv4-derived currents. We examined the contribution of Kv4 alpha-subunits to I(A) in the murine colon using pharmacological, molecular and immunohistochemical approaches. The divalent cation Cd(2+) decreased peak I(A) and shifted the voltage dependence of activation and inactivation to more depolarized potentials. Similar results were observed with La(3+). Colonic I(A) was sensitive to low micromolar concentrations of flecainide (IC(50) = 11 microM). Quantitative PCR indicated that in colonic and jejunal tissue, Kv4.3 transcripts demonstrate greater relative abundance than transcripts encoding Kv4.1 or Kv4.2. Antibodies revealed greater Kv4.3-like immunoreactivity than Kv4.2-like immunoreactivity in colonic myocytes. Kv4-like immunoreactivity was less evident in jejunal myocytes. To address this finding, we examined the expression of K(+) channel-interacting proteins (KChIPs), which act as positive modulators of Kv4-mediated currents. Qualitative PCR identified transcripts encoding the four known members of the KChIP family in isolated colonic and jejunal myocytes. However, the relative abundance of KChIP transcript was 2.6-fold greater in colon tissue than in jejunum, as assessed by quantitative PCR, with KChIP1 showing predominance. This observation is in accordance with the amplitude of the A-type current present in these two tissues, where colonic myocytes possess densities twice that of jejunal myocytes. From this we conclude that Kv4.3, in association with KChIP1, is the major molecular determinant of I(A) in murine colonic myocytes.

Characterization of the A-type potassium current in murine gastric antrum.

A-type currents are rapidly inactivating potassium currents that operate at subthreshold potentials. A-type currents have not been reported to occur in the phasic muscles of the stomach. We used conventional voltage-clamp techniques to identify and characterize A-type currents in myocytes isolated from the murine antrum. A-type currents were robust in these cells, with peak current densities averaging 30 pA pF(-1) at 0 mV. These currents underwent rapid inactivation with a time constant of 83 ms at 0 mV. Recovery from inactivation at -80 mV was rapid, with a time constant of 252 ms. The A-type current was blocked by 4-aminopyridine (4-AP) and was inhibited by flecainide, with an IC(50) of 35 microM. The voltage for half-activation was -26 mV, while the voltage of half-inactivation was -65 mV. There was significant activation and incomplete inactivation at potentials positive to -60 mV, which is suggestive of sustained current availability in this voltage range. Under current-clamp conditions, exposure to 4-AP or flecainide depolarized the membrane potential by 7-10 mV. In intact antral tissue preparations, flecainide depolarized the membrane potential between slow waves by 5 mV; changes in slow waves were not evident. The effect of flecainide was not abolished by inhibiting enteric neurotransmission or by blocking delayed rectifier and ATP-sensitive K(+) currents. Transcripts encoding Kv4 channels were detected in isolated antral myocytes by RT-PCR. Immunocytochemistry revealed intense Kv4.2- and Kv4.3-like immunoreactivity in antral myocytes. These data suggest that the A-type current in murine antral smooth muscle cells is likely to be due to Kv4 channels. This current contributes to the maintenance of negative resting membrane potentials.

Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3-/- mice.

ClC-3, a member of the large superfamily of ClC voltage-dependent Cl(-) channels, has been proposed as a molecular candidate responsible for volume-sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC-3 disrupted (Clcn3(-/-)) mice casts considerable doubt on this proposed role for ClC-3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3(+/+) and Clcn3(-/-) mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3(-/-) mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock-out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3(+/+) and Clcn3(-/-) mice are remarkably similar in terms of activation and inactivation kinetics, steady-state current densities, rectification, anion selectivity (I(-) > Cl(-)>> Asp(-)) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3(-/-) mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti-ClC-3 antibodies, and differential sensitivities to [ATP](i) and free [Mg(2+)](i). These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3(-/-) mice compared to Clcn3(+/+) mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC-1, ClC-2, and ClC-3, and protein expression profiles obtained using two-dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3(-/-) mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.