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B Horowitz

Publications and source records attributed to B Horowitz.

At least 19 recordsLinked to original sources
PubMed2005

Differential expression of P2X-purinoceptor subtypes in circular and longitudinal muscle of canine colon.

Adenosine triphosphate (ATP) mediates excitatory junction potentials through P2X receptors in many smooth muscles. However, relatively little is known about postjunctional intestinal P2X receptors. We examined the effect of exogenous ATP on circular and longitudinal myocytes of canine colon using the patch clamp technique at 32 degrees C. In both cell types, ATP induced inward currents (I(ATP)) at -70 mV in a concentration-dependent manner. The potency profile of ATP analogues in circular myocytes was: ATP approximately 2-methylthio-ATP > alpha,beta-methylene ATP, and that in longitudinal myocytes was: alpha,beta-methylene ATP approximately ATP > 2-methylthio-ATP. Pretreatment of circular myocytes with alpha,beta-methylene ATP inhibited the response to subsequent ATP, suggesting receptor desensitization. I-V relationships of I(ATP) were linear with inward rectification and E(rev) of -13 mV. I(ATP) at -70 mV was carried predominantly by Na+ as determined by shifts in E(rev) when extracellular Na+ was lowered. In RT-PCR, circular myocytes expressed mRNAs encoding P2X2, 3 and 4, while longitudinal myocytes expressed mRNAs for P2X3 and 5. P2X7 was absent in both cells. Fragments of each subtype were cloned and sequenced. We failed to clone P2X1 and P2X6 genes. Overall, different P2X receptor subtypes are expressed in circular and longitudinal canine colonic myocytes. Their activation produces non-selective cation currents that can depolarize and excite muscles of both layers.

Adenosine Triphosphate↗
PubMed2004

Constitutive and functional expression of cyclooxygenase 2 in the murine proximal colon.

Recent reports suggest that cyclo-oxygenase (COX)-2, an inducible COX isoform may be constitutively expressed in gastrointestinal tissues. This study has evaluated the expression and function of COX-2 in the tunica muscularis of the murine proximal colon. Cyclo-oxygenase-2-like (COX-2-LI) immunoreactivity was found in a subpopulation of neurones in the myenteric and submucosal ganglia and in interstitial cells of Cajal within the muscle layers (IC-IM). Reverse transcriptase polymerase chain reaction (RT-PCR) verified expression of COX-2 in colonic muscles, and quantitative PCR demonstrated that COX-1 transcriptional expression was greater than COX-2. To test the functional significance of COX-2 expression, the effects of a COX-2 inhibitor were compared with the effects of indomethacin (COX-1/COX-2 inhibitor) on circular muscle contractions. The experiments indicate that indomethacin and the specific COX-2 inhibitor, GR253035X, increased the amplitude of phasic contractions, suggesting production of inhibitory prostaglandins tonically dampen contractile activity. The effects of indomethacin were reduced when tested on phasic contractions of muscles from COX-2 knockout mice. GR253035X did not affect contractions in muscles of COX-2 knockout animals. These studies demonstrate constitutive expression of COX-2 in the tunica muscularis of the proximal colon. The COX-2 appears to contribute a significant amount of the prostaglandins that affect the contractile behaviour of colonic muscles.

Animals↗
PubMed2004

WHO Expert Committee on Biological Standardization.

This report presents the recommendations of a WHO Expert Committee commissioned to coordinate activities leading to the adoption of international requirements for the production and control of vaccines and other biologicals and the establishment of international biological reference materials. The report starts with a discussion of general issues brought to the Committee's attention and provides information on the status and development of reference materials for various antibodies, antigens, blood products and related substances, cytokines, growth factors, and endocrinological substances. The second part of the report, of particular relevance to manufacturers and national regulatory authorities, contains recommendations for the production and quality control of meningococcal group C conjugate vaccines, guidelines for regulatory expectations for clinical evaluation of vaccines, guidelines for the production and quality control of inactivated oral cholera vaccines and guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products.

Advisory Committees↗
PubMed2003

Expression and function of KCNH2 (HERG) in the human jejunum.

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERG(USO)) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3-S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10(-6) M), atropine (10(-6) M). and l-nitroarginine (10(-4) M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10(-8) M) and MK-499 (3 x 10(-8) M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10(-6) M) produced a 10-20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10(-6) M) did not affect [(14)C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.

Acetylcholine↗
PubMed2002

Modulation of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton.

The regulation of cardiac delayed rectifier potassium (Kv) currents by cAMP-dependent protein kinase (PKA) contributes to the control of blood pressure and heart rate. We investigated the modulation by PKA and protein phosphatases of cloned Kv1.5 channels expressed in Xenopus laevis oocytes. Exposure of oocytes to activators of PKA (100 nM forskolin, 1 mM 8-bromo-cAMP, or 1 mM 3-isobutyl-1-methylxanthine) had no effect on the amplitude of Kv1.5 currents. Inhibition of PKA by injection of protein kinase A inhibitor peptide or exposure to myristoylated protein kinase A inhibitor peptide (M-PKI; 100 nM) reduced currents mediated by Kv1.5. M-PKI also reduced the amplitude of currents mediated by mutated Kv1.5 channels in which the COOH terminal PKA phosphorylation sites and PSD-95, Disc-large, and ZO-1-binding domain were removed. The reduction of Kv1.5 currents by M-PKI was attenuated by inhibition of actin polymerization by 1 microM cytochalasins B and D, but was not affected by 10 microM phalloidin (stabilizes actin filaments) or 50 microM colchicine (disrupts microtubules). Treatment of oocytes with antisense oligonucleotides against alpha-actinin-2 abolished the reduction in Kv1.5 current by M-PKI. These observations suggest that Kv1.5 currents are activated by endogenous PKA in "resting" oocytes and that inhibition of PKA activity reveals the action of endogenous phosphatases. Indeed, injection of alkaline phosphatase reduced currents mediated by Kv1.5. Further preincubation of oocytes with 1 mM sodium orthovanadate (a protein tyrosine phosphatase inhibitor) abolished the reduction in Kv1.5 currents by M-PKI. We conclude that currents encoded by Kv1.5 are regulated by PKA and protein tyrosine phosphatase and that this regulation requires an intact actin cytoskeleton and alpha-actinin-2.

Actinin↗
PubMed2002

Novel human and mouse genes encoding an acid phosphatase family member and its downregulation in W/W(V) mouse jejunum.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) are pacemakers and mediators of motor neurotransmission in gastrointestinal smooth muscles. ICC require cellular signalling via Kit, a receptor tyrosine kinase, for development and maintenance of phenotype. Much of the evidence demonstrating the functions of ICC comes from studies of W/W(V) mice, which have reduced Kit function and reductions in specific populations of ICC. The aim of the present study was to differentially examine gene expression in the small intestines of wild-type and W/W(V) mutant mice. METHODS AND RESULTS: RNA from the jejunums of wild-type and W/W(V) mutants was analysed using a differential gene display method. Eighteen queries were identified as novel genes that were differentially displayed in wild-type and W/W(V) mice. One candidate gene, encoding a novel acid phosphatase-like protein, was significantly suppressed in fed and starved W/W(V) mice. The full length clone of the murine gene and its human counterpart were designated acid phosphatase-like protein 1 (ACPL1). Human ACPL1 cDNA encodes a protein of 428 amino acids with homology to human prostatic acid phosphatase protein. This gene is located at 1q21. ACPL1 was abundantly expressed in the human small intestine and colon. Gene products were found to be cytoplasmic in transfected COS-7 cells. Reverse transcription-polymerase chain reaction analysis revealed expression of ACPL1 mRNA within single isolated ICCs. CONCLUSIONS: Gene analysis showed that ACPL1 was differentially expressed in the small intestines of normal and W/W(V) mice. ICC within the small intestine expressed mRNA for ACPL1. Specific downregulation of ACPL1 in the jejunums of W/W(V) mice and high expression in human intestinal tissue suggest that the ACPL1 gene could be associated with ICC function in mice and humans.

Acid Phosphatase↗
PubMed2001

Molecular composition of 4-aminopyridine-sensitive voltage-gated K(+) channels of vascular smooth muscle.

Voltage-gated K(+) channels (Kv) play a critical role in regulating arterial tone by modulating the membrane potential of vascular smooth muscle cells. Our previous work demonstrated that the dominant 4-aminopyridine (4-AP)-sensitive, delayed rectifier Kv current of rabbit portal vein (RPV) myocytes demonstrates similar 4-AP sensitivity and biophysical properties to Kv1alpha-containing channels. To identify the molecular constituents underlying the 4-AP-sensitive Kv current of vascular myocytes, we characterized the expression pattern of Kv1alpha subunits and their modulatory Kvbeta subunits in RPV. The mRNAs encoding pore-forming subunits Kv1.2, Kv1.4, and Kv1.5 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Kv1.1, Kv1.3, and Kv1.6 transcripts were undetectable. Kvbeta1.1, beta1.2, beta1.3, beta2.1, and beta2.2 messages were expressed, whereas Kvbeta3.1 and beta4 mRNAs were undetected by RT-PCR. Kv1.2, Kv1.4, Kv1.5, Kvbeta1.2, beta1.3, and beta2.1 proteins were detected in RPV by Western blotting and/or immunocytochemistry of freshly isolated myocytes. We provide the first evidence, from coimmunoprecipitation studies, for the formation of heteromultimeric Kv channel complexes composed of Kv1.2, Kv1.5, and Kvbeta1.2 subunits in vascular smooth muscle.

4-Aminopyridine↗
PubMed2001

TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. An essential role in smooth muscle inhibitory neurotransmission.

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.

Animals↗
PubMed2001

Tamoxifen activates smooth muscle BK channels through the regulatory beta 1 subunit.

Estrogen (17beta-estradiol; 17betaE) and xenoestrogens, estrogenic compounds that are not steroid hormones, have non-genomic actions at plasma membrane receptors unrelated to the nuclear estrogen receptor. The open probability (P(o)) of large conductance Ca(2+)/voltage-sensitive k(+)(BK) channels is increased by 17betaE through the regulatory beta1 subunit. The pharmacological nature of the putative membrane binding site is unclear. We probed the site by determining whether tamoxifen ((Z)-1-(p-dimethylaminoethoxy-phenyl)-1,2-diphenyl-1-butene; Tx), a chemotherapeutic xenoestrogen, increased P(o) in clinically relevant concentrations (0.1-10 microm). In whole cell patch clamp recordings on canine colonic myocytes, which express the beta1 subunit, Tx activated charybdotoxin-sensitive K(+) current. In single channel experiments, Tx increased the NP(o) (P(o) x number channels; N) and decreased the unitary conductance (gamma) of BK channels. Tx increased NP(o) (EC(50) = 0.65 microm) in excised membrane patches independent of Ca(2+) changes. The Tx mechanism of action requires the beta1 subunit, as Tx increased the NP(o) of Slo alpha expressed in human embryonic kidney cells only in the presence of the beta1 subunit. Tx decreased gamma of the alpha subunit expressed alone, without effect on NP(o). Our data indicate that Tx increases BK channel activity in therapeutic concentrations and reveal novel pharmacological properties attributable to the alpha and beta1 subunits. These data shed light on BK channel structure and function, non-genomic mechanisms of regulation, and physiologically and therapeutically relevant effects of xenoestrogens.

Animals↗
PubMed2001

Functional and molecular expression of a voltage-dependent K(+) channel (Kv1.1) in interstitial cells of Cajal.

1. Located within the gastrointestinal (GI) musculature are networks of cells known as interstitial cells of Cajal (ICC). ICC are associated with several functions including pacemaker activity that generates electrical slow waves and neurotransmission regulating GI motility. In this study we identified a voltage-dependent K(+) channel (Kv1.1) expressed in ICC and neurons but not in smooth muscle cells. 2. Transcriptional analyses demonstrated that Kv1.1 was expressed in whole tissue but not in isolated smooth muscle cells. Immunohistochemical co-localization of Kv1.1 with c-kit (a specific marker for ICC) and vimentin (a specific marker of neurons and ICC) indicated that Kv1.1-like immunoreactivity (Kv1.1-LI) was present in ICC and neurons of GI tissues of the dog, guinea-pig and mouse. Kv1.1-LI was not observed in smooth muscle cells of the circular and longitudinal muscle layers. 3. Kv1.1 was cloned from a canine colonic cDNA library and expressed in Xenopus oocytes. Pharmacological investigation of the electrophysiological properties of Kv1.1 demonstrated that the mamba snake toxin dendrotoxin-K (DTX-K) blocked the Kv1.1 outward current when expressed as a homotetrameric complex (EC(50) = 0.34 nM). Other Kv channels were insensitive to DTX-K. When Kv1.1 was expressed as a heterotetrameric complex with Kv1.5, block by DTX-K dominated, indicating that one or more subunits of Kv1.1 rendered the heterotetrameric channel sensitive to DTX-K. 4. In patch-clamp experiments on cultured murine fundus ICC, DTX-K blocked a component of the delayed rectifier outward current. The remaining, DTX-insensitive current (i.e. current in the presence of 10(-8) M DTX-K) was outwardly rectifying, rapidly activating, non-inactivating during 500 ms step depolarizations, and could be blocked by both tetraethylammonium (TEA) and 4-aminopyridine (4-AP). 5. In conclusion, Kv1.1 is expressed by ICC of several species. DTX-K is a specific blocker of Kv1.1 and heterotetrameric channels containing Kv1.1. This information is useful as a means of identifying ICC and in studies of the role of delayed rectifier K(+) currents in ICC functions.

Animals↗
PubMed2001

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody.

Intracellular dialysis of NIH/3T3 cells with a commercially available anti-ClC-3 polyclonal antibody (Ab) for approximately 30 min completely inhibited expressed guinea-pig ClC-3 currents (IgpClC-3), while intracellular dialysis with antigen-preabsorbed anti-ClC-3 Ab failed to affect IgpClC-3. Anti-ClC-3 Ab was used as a selective probe to examine the relationship between endogenous ClC-3 expression and native volume-sensitive outwardly rectifying anion channels (VSOACs) in guinea-pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti-ClC-3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen-preabsorbed anti-ClC-3 Ab. It is suggested that endogenous ClC-3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti-ClC-3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC-3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

3T3 Cells↗
PubMed2001

Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

8,11,14-Eicosatrienoic Acid↗
PubMed2001

Differential expression and alternative splicing of TRP channel genes in smooth muscles.

Nonselective cation channels (NSCC) are targets of excitatory agonists in smooth muscle, representing the nonselective cation current I(cat). Na(+) influx through NSCC causes depolarizations and activates voltage-dependent Ca(2+) channels, resulting in contraction. The molecular identity of I(cat) in smooth muscle has not been elucidated; however, products of the transient receptor potential (TRP) genes have characteristics similar to native I(cat). We have determined the levels of TRP transcriptional expression in several murine and canine gastrointestinal and vascular smooth muscles and have analyzed the alternative processing of these transcripts. Of the seven TRP gene family members, transcripts for TRP4, TRP6, and TRP7 were detected in all murine and canine smooth muscle cell preparations. TRP3 was detected only in canine renal artery smooth muscle cells. The full-length cDNAs for TRP4, TRP6, and TRP7, as well as one splice variant of TRP4 and two splice variants of TRP7, were cloned from murine colonic smooth muscle. Quantitative RT-PCR determined the relative amounts of TRP4, TRP6, and TRP7 transcripts, as well as that of the splice variants, in several murine smooth muscles. TRP4 is the most highly expressed, while TRP6 and TRP7 are expressed at a lower level in the same tissues. Splice variants for TRP7, deleted for exons encoding amino acids including transmembrane segment S1, predominated in murine smooth muscles, while the full-length form of the transcript was expressed in canine smooth muscles.

Alternative Splicing↗
PubMed2001

Molecular properties of small-conductance Ca2+-activated K+ channels expressed in murine colonic smooth muscle.

Small-conductance Ca2+-activated K+ (SK) channels are important participants in inhibitory neurotransmission in gastrointestinal smooth muscles. Three isoforms of an SK channel family were cloned from murine proximal colon smooth muscle. The transcripts encoding these subunits (SK1, SK2, and SK3) were detected in murine proximal colon and other peripheral tissues. The mRNA of each subunit was expressed at different levels in murine and canine colonic smooth muscles. The mRNA quantitative ratio of SK transcriptional expression in murine proximal colon is SK2 > SK3 > SK1; transcriptional expression of SK isoforms in canine proximal colon is minimal. SK3 immunohistochemical localization in murine small intestine (jejunum) and proximal colon showed immunoreactivity in circular and longitudinal muscularis. In transversely sectioned muscularis, staining was localized at the cell membrane in smooth muscle cells. Immunoreactivity was more intense in myenteric ganglia between longitudinal and circular muscularis and neuronal processes in circular and longitudinal muscularis. Transient expression of mSK1, mSK2, and mSK3 in COS cells resulted in Ca2+-activated voltage-independent channels. mSK1 is less sensitive to apamin compared with SK2 and showed intracellular Ca2+ sensitivity (10(-8) to 10(-6) M) in asymmetrical K+ (5/140 mM K+) gradients. Our results suggest that SK channel expression varies in colonic myocytes from different species and may contribute differentially to inhibitory junction potentials.

Amino Acid Sequence↗
PubMed2000

Intracellular cyclic AMP inhibits native and recombinant volume-regulated chloride channels from mammalian heart.

1. ClC-3 encodes a volume-regulated Cl- channel (ICl,vol) in heart. We studied the regulation of native and recombinant cardiac ICl,vol by intracellular cyclic AMP (cAMPi). 2. Symmetrical high Cl- concentrations were used to effectively separate outwardly rectifying ICl,vol from other non-rectifying Cl- currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- currents (ICl,CFTR and ICl,Ca, respectively), which are concomitantly expressed in cardiac myocytes. 3. 8-Bromo-cyclic AMP (8-Br-cAMP) significantly inhibited ICl,vol in most guinea-pig atrial myocytes. In approximately 30 % of the atrial myocytes examined, 8-Br-cAMP increased macroscopic Cl- currents. However, the 8-Br-cAMP-stimulated difference currents exhibited a linear current-voltage (I-V ) relation, consistent with activation of ICl,CFTR, not ICl,vol. 4. In canine atrial myocytes, isoprenaline (1 microM) consistently reduced ICl,vol in Ca2+-free hypotonic bath solutions with strong intracellular Ca2+ (Ca2+i) buffering. In Ca2+-containing hypotonic bath solutions with weak Ca2+i buffering, however, isoprenaline increased net macroscopic Cl- currents. Isoprenaline-stimulated difference currents were not outwardly rectifying, consistent with activation of ICl,Ca, not ICl, vol. 5. In NIH/3T3 cells transfected with gpClC-3 (the gene encoding ICl,vol), 8-Br-cAMP consistently inhibited ICl,ClC-3. These effects were prevented by a protein kinase A (PKA) inhibitor, KT5720, or by mutation of a single consensus protein kinase C (PKC) phosphorylation site (S51A) on the N-terminus of ClC-3, which also mediates PKC inhibition of ICl,ClC-3. 6. We conclude that cAMPi causes inhibition of ICl,vol in mammalian heart due to cross-phosphorylation of the same PKC consensus site on ClC-3 by PKA. Our results suggest that contamination of macroscopic ICl,vol by ICl,CFTR and/or ICl,Ca may account for some of the inconsistent and controversial effects of cAMPi on ICl,vol previously reported in native cardiac myocytes.

3T3 Cells↗
PubMed2000

A novel anionic inward rectifier in native cardiac myocytes.

Although the cationic inward rectifiers (Kir and hyperpolarization-activated I(f) channels) have been well characterized in cardiac myocytes, the expression and physiological role of anionic inward rectifiers in heart are unknown. In the present study, we report the functional and molecular identification of a novel chloride (Cl(-)) inward rectifier (Cl.ir) in mammalian heart. Under conditions in which cationic inward rectifier channels were blocked, membrane hyperpolarization (-40 to -140 mV) activated an inwardly rectifying whole-cell current in mouse atrial and ventricular myocytes. Under isotonic conditions, the current activated slowly with a biexponential time course (time constants averaging 179.7+/-23.4 [mean+/-SEM] and 2073.6+/-287.6 ms at -120 mV). Hypotonic cell swelling accelerated the activation and increased the current amplitude whereas hypertonic cell shrinkage inhibited the current. The inwardly rectifying current was carried by Cl(-) (I(Cl.ir)) and had an anion permeability sequence of Cl(-)>I(-)>>aspartate. I(Cl.ir) was blocked by 9-anthracene-carboxylic acid and cadmium but not by stilbene disulfonates and tamoxifen. A similar I(Cl.ir) was also observed in guinea pig cardiac myocytes. The properties of I(Cl.ir) are consistent with currents generated by expression of ClC-2 Cl(-) channels. Reverse transcription polymerase chain reaction and Northern blot analysis confirmed transcriptional expression of ClC-2 in both atrial and ventricular tissues and isolated myocytes of mouse and guinea pig hearts. These results indicate that a novel I(Cl.ir) is present in mammalian heart and support a potentially important role of ClC-2 channels in the regulation of cardiac electrical activity and cell volume under physiological and pathological conditions.

Animals↗