Effect of ß1 /ß2 -adrenoceptor blockade on ß3 -adrenoceptor activity in the rat cremaster muscle artery.

BACKGROUND AND PURPOSE: The physiological role of vascular ß3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac ß3 -adrenoceptors are functionally effective after down-regulation of ß1 /ß2 -adrenoceptors. The functional interaction between the ß3 -adrenoceptor and other ß-adrenoceptor subtypes in rat striated muscle arteries was investigated. EXPERIMENTAL APPROACH: Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. ß-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of ß3 -adrenoceptor agonists and antagonists and other ß-adrenoceptor ligands were measured using pressure myography. KEY RESULTS: All three ß-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The ß3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the ß3 -adrenoceptor agonist SR 58611A and the nonselective ß-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of ß1/2 -adrenoceptor antagonists nadolol (10 µM), atenolol (1 µM) and ICI 118,551 (0.1 µM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The ß3 -adrenoceptor antagonist L 748,337 (1 µM) inhibited vasodilation caused by ß3 -adrenoceptor agonists (in the presence of ß1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation. CONCLUSION AND IMPLICATIONS: All three ß-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but ß3 -adrenoceptor mediated vasodilation was only evident after blockade of ß1/2 -adrenoceptors. This suggests constitutive ß1/2 -adrenoceptor activity inhibits ß3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.

TRPV3 expression and vasodilator function in isolated uterine radial arteries from non-pregnant and pregnant rats.

This study investigated the expression and function of transient receptor potential vanilloid type-3 ion channels (TRPV3) in uterine radial arteries isolated from non-pregnant and twenty-day pregnant rats. Immunohistochemistry (IHC) suggested TRPV3 is primarily localized to the smooth muscle in arteries from both non-pregnant and pregnant rats. IHC using C' targeted antibody, and qPCR of TRPV3 mRNA, suggested pregnancy increased arterial TRPV3 expression. The TRPV3 activator carvacrol caused endothelium-independent dilation of phenylephrine-constricted radial arteries, with no difference between vessels from non-pregnant and pregnant animals. Carvacrol-induced dilation was reduced by the TRPV3-blockers isopentenyl pyrophosphate and ruthenium red, but not by the TRPA1 or TRPV4 inhibitors HC-030031 or HC-067047, respectively. In radial arteries from non-pregnant rats only, inhibition of NOS and sGC, or PKG, enhanced carvacrol-mediated vasodilation. Carvacrol-induced dilation of arteries from both non-pregnant and pregnant rats was prevented by the IKCa blocker TRAM-34. TRPV3 caused an endothelium-independent, IKCa-mediated dilation of the uterine radial artery. NO-PKG-mediated modulation of TRPV3 activity is lost in pregnancy, but this did not alter the response to carvacrol.

Electrical slow waves in the mouse oviduct are dependent upon a calcium activated chloride conductance encoded by Tmem16a.

Myosalpinx contractions are critical for oocyte transport along the oviduct. A specialized population of pacemaker cells-oviduct interstitial cells of Cajal-generate slow waves, the electrical events underlying myosalpinx contractions. The ionic basis of oviduct pacemaker activity is unknown. We examined the role of a new class of Ca(2+)-activated Cl(-) channels (CaCCs)-anoctamin 1, encoded by Tmem16a-in oviduct slow wave generation. RT-PCR revealed the transcriptional expression of Tmem16a-encoded CaCCs in the myosalpinx. Intracellular microelectrode recordings were performed in the presence of two pharmacologically distinct Cl(-) channel antagonists, anthracene-9-carboxylic acid and niflumic acid. Both of these inhibitors caused membrane hyperpolarization, reduced the duration of slow waves, and ultimately inhibited pacemaker activity. Niflumic acid also inhibited propagating calcium waves within the myosalpinx. Slow waves were present at birth in wild-type and heterozygous oviducts but failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16a(tm1Bdh/tm1Bdh)). These data suggest that Tmem16a-encoded CaCCs contribute to membrane potential and are responsible for the upstroke and plateau phases of oviduct slow waves.

Electrical slow waves in the mouse oviduct are dependent on extracellular and intracellular calcium sources.

Spontaneous contractions of the myosalpinx are critical for oocyte transport along the oviduct. Slow waves, the electrical events that underlie myosalpinx contractions, are generated by a specialized network of pacemaker cells called oviduct interstitial cells of Cajal (ICC-OVI). The ionic basis of oviduct pacemaker activity is unknown. Intracellular recordings and Ca(2+) imaging were performed to examine the role of extracellular and intracellular Ca(2+) sources in slow wave generation. RT-PCR was performed to determine the transcriptional expression of Ca(2+) channels. Molecular studies revealed most isoforms of L- and T-type calcium channels (Cav1.2,1.3,1.4,3.1,3.2,3.3) were expressed in myosalpinx. Reduction of extracellular Ca(2+) concentration ([Ca(2+)](o)) resulted in the abolition of slow waves and myosalpinx contractions without significantly affecting resting membrane potential (RMP). Spontaneous Ca(2+) waves spread through ICC-OVI cells at a similar frequency to slow waves and were inhibited by reduced [Ca(2+)](o). Nifedipine depolarized RMP and inhibited slow waves; however, pacemaker activity returned when the membrane was repolarized with reduced extracellular K(+) concentration ([K(+)](o)). Ni(2+) also depolarized RMP but failed to block slow waves. The importance of ryanodine and inositol 1,4,5 trisphosphate-sensitive stores were examined using ryanodine, tetracaine, caffeine, and 2-aminoethyl diphenylborinate. Results suggest that although both stores are involved in regulation of slow wave frequency, neither are exclusively essential. The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump inhibitor cyclopiazonic acid inhibited pacemaker activity and Ca(2+) waves suggesting that a functional SERCA pump is necessary for pacemaker activity. In conclusion, results from this study suggest that slow wave generation in the oviduct is voltage dependent, occurs in a membrane potential window, and is dependent on extracellular calcium and functional SERCA pumps.

Increased complexity of Tmem16a/Anoctamin 1 transcript alternative splicing.

BACKGROUND: TMEM16A (Anoctamin 1; ANO1) is an eight transmembrane protein that functions as a calcium-activated chloride channel. TMEM16A in human exhibits alternatively spliced exons (6b, 13 and 15), which confer important roles in the regulation of channel function. Mouse Tmem16a is reported to consist of 25 exons that code for a 956 amino acid protein. In this study our aim was to provide details of mouse Tmem16a genomic structure and to investigate if Tmem16a transcript undergoes alternative splicing to generate channel diversity. RESULTS: We identified Tmem16a transcript variants consisting of alternative exons 6b, 10, 13, 14, 15 and 18. Our findings indicate that many of these exons are expressed in various combinations and that these splicing events are mostly conserved between mouse and human. In addition, we confirmed the expression of these exon variants in other mouse tissues. Additional splicing events were identified including a novel conserved exon 13b, tandem splice sites of exon 1 and 21 and two intron retention events. CONCLUSION: Our results suggest that Tmem16a gene is significantly more complex than previously described. The complexity is especially evident in the region spanning exons 6 through 16 where a number of the alternative splicing events are thought to affect calcium sensitivity, voltage dependence and the kinetics of activation and deactivation of this calcium-activated chloride channel. The identification of multiple Tmem16a splice variants suggests that alternative splicing is an exquisite mechanism that operates to diversify TMEM16A channel function in both physiological and pathophysiological conditions.

Muscarinic activation of Ca2+-activated Cl- current in interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) provide pacemaker activity and functional bridges between enteric motor nerve terminals and gastrointestinal smooth muscle cells. The ionic conductance(s) in ICC that are activated by excitatory neural inputs are unknown. Transgenic mice (Kit(copGFP/+)) with constitutive expression of a bright green fluorescent protein were used to investigate cellular responses of ICC to cholinergic stimulation. ICC displayed spontaneous transient inward currents (STICs) under voltage clamp that corresponded to spontaneous transient depolarizations (STDs) under current clamp. STICs reversed at 0 mV when E(Cl) = 0 mV and at -40 mV when E(Cl) was -40 mV, suggesting the STICs were due to a chloride conductance. Carbachol (CCh, 100 nm and 1 µm) induced a sustained inward current (depolarization in current clamp) and increased the amplitude and frequency of STICs and STDs. CCh responses were blocked by atropine (10 µm) or 4-DAMP (100 nm), an M(3) receptor antagonist. STDs were blocked by niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (both 100 µm), and CCh had no effect in the presence of these drugs. The responses of intact circular muscles to CCh and stimulation of intrinsic excitatory nerves by electrical field stimulation (EFS) were also compared. CCh (1 µm) caused atropine-sensitive depolarization and increased the maximum depolarization of slow waves. Similar atropine-sensitive responses were elicited by stimulation of intrinsic excitatory neurons. Niflumic acid (100 µm) blocked responses to EFS but had minor effect on responses to exogenous CCh. These data suggest that different ionic conductances are responsible for electrical responses elicited by bath-applied CCh and cholinergic nerve stimulation.

Cardiac-specific, inducible ClC-3 gene deletion eliminates native volume-sensitive chloride channels and produces myocardial hypertrophy in adult mice.

Native volume-sensitive outwardly rectifying anion channels (VSOACs) play a significant role in cell volume homeostasis in mammalian cells. However, the molecular correlate of VSOACs has been elusive to identify. The short isoform of ClC-3 (sClC-3) is a member of the mammalian ClC gene family and has been proposed to be a molecular candidate for VSOACs in cardiac myocytes and vascular smooth muscle cells. To directly test this hypothesis, and assess the physiological role of ClC-3 in cardiac function, we generated a novel line of cardiac-specific inducible ClC-3 knock-out mice. These transgenic mice were maintained on a doxycycline diet to preserve ClC-3 expression; removal of doxycycline activates Cre recombinase to inactivate the Clcn3 gene. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the knock-out mice at both 1.5 and 3 weeks off doxycycline. In mice off doxycycline, time-dependent inactivation of ClC-3 gene expression was confirmed in atrial and ventricular cells by qRT-PCR and Western blot analysis. Electrophysiological examination of native VSOACs in isolated atrial and ventricular myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, VSOAC current densities were significantly reduced, compared to age-matched control mice maintained on doxycycline. These results indicate that ClC-3 is a key component of native VSOACs in mammalian heart and plays a significant cardioprotective role against cardiac hypertrophy and failure.

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.

Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles.

Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles, but the mechanism(s) of pacemaker activity are controversial. Several conductances, such as Ca(2+)-activated Cl() channels (CaCC) and non-selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. We investigated the expression and function of a new class of CaCC, anoctamin 1 (ANO1), encoded by Tmem16a, which was discovered to be highly expressed in ICC in a microarray screen. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non-human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4-diisothiocyano-2,2-stillbene-disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration-dependent manner. Unitary potentials, small stochastic membrane depolarizations thought to underlie slow waves, were insensitive to CaCC blockers. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16a(tm1Bdh)(/tm1Bdh)) and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. Loss of function of ANO1 did not inhibit the development of ICC networks that appeared structurally normal as indicated by Kit antibodies. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC.

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.

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.

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.

Functional and molecular identification of pH-sensitive K+ channels in murine urinary bladder smooth muscle.

OBJECTIVE: To examine the role of pH-sensitive K(+) channels in setting the resting membrane potential in murine bladder smooth muscle, as bladder contractility is influenced by the resting membrane potential, which is mainly regulated by background K(+) conductances. MATERIALS AND METHODS: Using conventional microelectrode recordings, isometric tension measurements, patch-clamp recordings, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry, we assessed bladder smooth muscle cells and tissues. RESULTS: Acidic pH (pH 6.5) depolarized the resting membrane potential of murine bladder smooth muscles and increased muscle tone and contractility. The pH-induced changes were not abolished by neuronal blockers or classical K(+)-channel antagonists. Lidocaine (1 mM) and bupivacaine (100 microm) mimicked the effects of acidifying the external solution, and in the presence of lidocaine no further increase in contractility was induced by reducing the pH to 6.5. Voltage-clamp experiments on freshly dispersed bladder myocytes showed that pH 6.5 decreased the outward current. Pre-treatment of bladder myocytes with the classical K(+) antagonists tetraethylammonium (10 mm), 4-aminopyridine (5 mM), glibenclamide (10 microm) or apamin (300 nM) did not inhibit the effects of low pH on outward current. However, treatment with lidocaine (1 mM) abolished the effects of acidic pH on outward current. RT-PCR showed the expression of the acid-sensitive K(+) channel (TASK)-1 and TASK-2 gene transcripts in murine bladder, and immunohistochemistry and Western blot analysis showed TASK-1 and TASK-2 channel expression and distribution in smooth muscle tissues and cells. CONCLUSION: TASK channels are expressed in bladder smooth muscle and contribute to the basal K(+) conductances responsible for resting membrane potential.

Functional characterization of novel alternatively spliced ClC-2 chloride channel variants in the heart.

A novel volume-regulated hyperpolarization-activated chloride inward rectifier channel (Cl.ir) was identified in mammalian heart. To investigate whether ClC-2 is the gene encoding Cl.ir channels in heart, ClC-2 cDNAs cloned from rat (rClC-2) and guinea pig (gpClC-2) hearts were functionally characterized. When expressed in NIH/3T3 cells, full-length rClC-2 yielded inwardly rectifying whole-cell currents with very slow activation kinetics (time constants > 1.7 s) upon hyperpolarization under hypotonic condition. The single-channel rClC-2 currents had a unitary slope conductance of 3.9 +/- 0.2 picosiemens. A novel variant with an in-frame deletion at the beginning of exon 15 that leads to a deletion of 45 bp (corresponding to 15 amino acids in alpha-helices O and P, rClC-2(Delta509-523)) was identified in rat heart. The relative transcriptional expression levels of full-length rClC-2 and rClC-2(Delta509-523) in rat heart were 0.018 +/- 0.003 and 0.028 +/- 0.006 arbitrary units, respectively, relative to glyceraldehyde-3-phosphate dehydrogenase (n = 5, p = nonsignificant). A similar partial exon 15 skipping with a deletion of 105 bp (35 amino acids in alpha-helices O-Q, gpClC-2(Delta509-543)) was also identified in guinea pig heart. Expression of both rClC-2(Delta509-523) and gpClC-2(Delta509-543) resulted in functional channels with phenotypic activation kinetics and many properties identical to those of endogenous Cl.ir channels in native rat and guinea pig cardiac myocytes, respectively. Intracellular dialysis of anti-ClC-2 antibody inhibited expressed ClC-2 channels and endogenous Cl.ir currents in native rat and guinea pig cardiac myocytes. These results demonstrate that novel deletion variants of ClC-2 due to partial exon 15 skipping may be expressed normally in heart and contribute to the formation of endogenous Cl.ir channels in native cardiac cells.

Comparison of the properties of CLCA1 generated currents and I(Cl(Ca)) in murine portal vein smooth muscle cells.

Calcium-activated chloride currents (I(Cl(Ca))) have been recorded in various smooth muscle cells but, to date, there has been no information as to the molecular nature of the channel underlying this conductance. We have characterised native I(Cl(Ca)) in freshly dispersed smooth muscle cells isolated from murine portal vein using whole-cell voltage clamp. I(Cl(Ca)) exhibited time-dependent activation at depolarised potentials and rapid deactivation upon repolarisation. The reversal potential of I(Cl(Ca)) was close to the theoretical equilibrium potential (E(Cl)) and was shifted by replacement of external Cl- by SCN- or isethionate. Dithiothreitol (DTT, 1 mM), a blocker of CLCA1, had no effect on the I(Cl(Ca)) current in myocytes. RT-PCR demonstrated the expression of mCLCA1 transcripts, but not mCLCA3 transcripts, in various murine smooth muscle cells including portal vein, as well as cardiomyocytes, and the levels of mCLCA1 transcriptional expression were quantified by real time quantitative RT-PCR. Stable transfection of HEK293 cells with the cDNA encoding mCLCA1 cloned from murine portal vein smooth muscle yielded a current with notable differences in Ca2+ sensitivity, channel kinetics and modulation by DTT from the native I(Cl(Ca)). However, there was some similarity in the pore properties and these data suggest that mCLCA1 alone does not comprise the Cl- channel in portal vein smooth muscle cells.