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ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance [Ca.sup.2+]-activated [K.sup.+] channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current ([I.sub.VNSCC]) and a relatively high-threshold voltage-activated (L-type) [Ca.sup.2+] current ([I.sub.L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect [I.sub.VNSCC] or [I.sub.L] in dialyzed cells. ATP (1 mM) increased [I.sub.VNSCC] and decreased 1L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on [I.sub.VNSCC]. ADP decreased [I.sub.L] but had no effect on [I.sub.VNSCC]. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2, 4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on [I.sub.VNSCC]. ATP stimulation of [I.sub.VNSCC] was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that [P2Y.sub.4] is expressed by routine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates [I.sub.VNSCC] and depresses [I.sub.L] via binding of [P2Y.sub.4] receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system

The voltage-dependent [K.sup.+] channel 4.3 ([K.sub.v] 4.3) is one of the major molecular correlates encoding a class of rapidly inactivating [K.sup.+] currents, including the transient outward current in the heart ([I.sub.to]) and A currents ([I.sub.A]) in neuronal and smooth muscle preparations. Recent studies have shown that [I.sub.to] in human atrial myocytes and [I.sub.A] in murine colonic myocytes are modulated by [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII): however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate [K.sub.v]4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged [K.sub.v]4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at $550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of [K.sub.v]4.3 currents by a direct effect at $550A, located at the COOH-terminal region of the channel. transient outward current: potassium channel; inactivation: phosphorylation

The serum- and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase (PK) transcriptionally regulated by corticoids, serum, and cell volume. SGK regulates cell volume of various cells by effects on [Na.sup.+] and [K.sup.+] transport through membrane channels. We hypothesized a role for SGK in the activation of volume-sensitive osmolyte and anion channels (VSOACs) in cultured canine pulmonary artery smooth muscle cells (PASMCs). Intracellular dialysis through the patch electrode of recombinant active SGK, but not kinase-dead [DELTA]60-SGK-K127M, heat-inactivated SGK, or active Akt1, partially activated VSOACs under isotonic conditions. Dialysis of active SGK before cell exposure to hypotonic medium significantly accelerated the activation kinetics and increased the maximal density of VSOAC current. Exposure of PASMCs to hypotonic medium (230 mosM) activated phosphatidylinositol 3-kinases (PI3Ks) and their downstream targets Akt/PKB and SGK but not PKC-[epsilon]. Inhibition of PI3Ks with wortmannin reduced the activation rate and maximal amplitude of VSOACs. Immunoprecipirated C1C-3 channels were phosphorylated by PKC-[epsilon] but not by SGK in vitro, suggesting that SGK may activate VSOACs indirectly. These data indicate that the PI3K-SGK cascade is activated on hypotonic swelling of PASMCs and, in turn, affects downstream signaling molecules linked to activation of VSOACs. hypotonic cell swelling; [Cl.sup.-] conductance; phosphatidylinositol 3-kinase; Akt/protein kinase B; protein kinase C-[epsilon]; pulmonary artery smooth muscle cells; volume-sensitive osmolyte and anion channels; serum- and glucocorticoid-inducible kinase

Carbon monoxide (CO) is a gaseous vasodilator produced by many cell types, including endothelial and smooth muscle cells. The goal of the present study was to investigate signaling mechanisms responsible for CO activation of large-conductance [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channels in newborn porcine cerebral arteriole smooth muscle cells. In intact cells at 0 mV, CO (3 [micro]M) or CO released from dimanganese decacarbonyl (10 [micro]M), a novel light-activated CO donor, increased [K.sub.Ca] channel activity 4.9- or 3.5-fold, respectively. [K.sub.Ca] channel activation by CO was not blocked by 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (25 [micro]M), a soluble guanylyl cyclase inhibitor. In inside-out patches at 0 mV, CO shifted the [Ca.sup.2+] concentration-response curve for [K.sub.Ca] channels leftward and decreased the apparent dissociation constant for [Ca.sup.2+] from 31 to 24 [micro]M. Western blotting data suggested that the low [Ca.sup.2+] sensitivity of newborn [K.sub.Ca] channels may be due to a reduced [beta]-subunit-to-[alpha]-subunit ratio. CO activation of [K.sub.Ca] channels was [Ca.sup.2+] dependent. CO increased open probability 3.7-fold with 10 [micro]M free [Ca.sup.2+] at the cytosolic membrane surface but only 1.1-fold with 300 nM [Ca.sup.2+]. CO left shifted the current-voltage relationship of cslo-[alpha] currents expressed in HEK-293 cells, increasing currents 2.2-fold at +50 mV. In summary, data suggest that in newborn arteriole smooth muscle cells, CO activates low-affinity [K.sub.Ca] channels via a direct effect on the [alpha]-subunit that increases apparent [Ca.sup.2+] sensitivity. The optimal tuning by CO of the micromolar [Ca.sup.2+] sensitivity of [K.sub.Ca] channels will lead to preferential activation by signaling modalities, such as [Ca.sup.2+] sparks, which elevate the subsarcolemmal [Ca.sup.2+] concentration within this range. patch-clamp electrophysiology; cerebrovascular circulation

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000-40,000 cells and ~60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched ~6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested ~2,000-4,000 cells from single gastric corpus-antrum muscles and detected an ~30-fold increase in c-kit mRNA, no enrichment of mast cells, and an ~4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses. mouse; c-kit; macrophage; dendritic cell; mast cell

Baker, Salah A., Violeta Mutafova-Yamboliva, Kevin Monaghan, Burton Horowitz, Kenton M. Sanders, and Sang Don Koh. Mechanism of active repolarization of inhibitory junction potential in murine colon. Am J Physiol Gastrointest Liver Physiol 285: G813-G821, 2003; 10.1152/ajpgi. 00115.2003.--Enteric inhibitory responses in gastrointestinal (GI) smooth muscles involve membrane hyperpolarization that transiently reduce the excitability of GI muscles. We examined the possibility that an active repolarization mechanism participates in the restoration of resting membrane potential after fast inhibitory junction potentials (IJPs) in the murine colon. Previously, we showed these cells express a voltage-dependent nonselective cation conductance (NSCC) that might participate in active repolarization of IJPs. Colonic smooth muscle cells were impaled with microelectrodes and voltage responses to nerve-evoked IJPs, and locally applied ATP were recorded. [Ba.sup.2.sup.+] (500 [micro]M), a blocker of the NSCC, slowed the rate of repolarization of IJPs. We also tested the effects of [Ba.sup.2.sup.+], [Ni.sup.2.sup.+], and mibefradil, all blockers of the NSCC, on responses to locally applied ATP. Spritzes of ATP caused transient hyperpolarization, and the durations of these responses were significantly increased by the blockers of the NSCC. We considered whether NSCC blockers might affect ATP metabolism and found that [Ni.sup.2.sup.+] decreased ATP breakdown in colonic muscles. Mibefradil had no effect on ATP metabolism. Because both [Ni.sup.2.sup.+] and mibefradil had similar effects on prolonging responses to ATP, it appears that restoration of resting membrane potential after ATP spritzes is not primarily due to ATP metabolism. Neurally, released enteric inhibitory transmitter and locally applied ATP resulted in transient hyperpolarizations of murine colonic muscles. Recovery of membrane potential after these responses appears to involve an active repolarization mechanism due to activation of the voltage-dependent NSCC expressed by these cells. gastrointestinal motility; adenosine 5'-triphosphate; voltagedependent nonselective cation current

The current study was undertaken to test the existence and possible role of ether-a-go-go-related gene 1 (ERG1) protein [K.sup.+] channels in gallbladder smooth muscle (GBSM). Transcripts encoding ERG1 were detected in human, mouse, and guinea pig GBSM, and ERG1 immunoreactivity was observed in GBSM cells. In intracellular voltage recordings, addition of E-4031 (100 nM-1 [micro]M) or cisapride (100 nM-2 [micro]M) caused concentration-dependent excitation of guinea pig GBSM that was not affected by 500 nM TTX + 5 [micro]M atropine, and E-4031 also depolarized the resting membrane potential. In muscle strip studies, E-4031 either induced phasic contractions or significantly increased the amplitude of phasic contractions in spontaneously active tissues (P = 0.001). E-4031 also potentiated bethanechol-induced contractions. In conclusion, ERG1 channels are expressed in the GBSM, where they play a role in excitation-contraction coupling probably by contributing to repolarization of the plateau phase of the action potential and to the resting membrane potential. biliary motility; rapidly activated delayed rectifier; E-4031; cisapride

Calmodulin (Cal) plays important roles for contractile activity in smooth muscles. Recently, two distinct [Ca.sup.2+]-binding protein superfamilies with sequence similarities to Cal have been identified in neuronal cells: neuronal [Ca.sup.2+]-binding proteins (NCBPs) and Cal-like [Ca.sup.2+]-binding proteins (CaBPs). Some NCBPs and CaBPs play significant roles for [Ca.sup.2+]-dependent cellular signaling in the nervous system. In gastrointestinal smooth muscles (GISMs), Cal functions as the regulator of contractile behavior and electrical rhythmicity. However, the molecular identification of NCBPs and CaBPs has not been elucidated in GISMs. Here, we have identified NCBPs and CaBPs expressed in GISMs and determined the expression levels of their transcripts by quantitative RT-PCR. Of 12 NCBPs, the transcripts for neuronal [Ca.sup.2+] sensor 1, neural visinin-like proteins 1, 2, and 3, and [K.sup.+] channel-interacting proteins 1 and 3 were detected in proximal colon, gastric fundus, gastric antrum, and jejunum. On the other hand, of seven CaBPs including alternatively spliced variants, only CaBP1L transcripts were detected in GISMs. EF-hand motif; quantitative PCR; ion channel regulation; rhythmicity; contractile activity

Interstitial cells of Cajal (ICC) are the pacemaker cells responsible for the generation and propagation of electrical slow waves in phasic muscles of the gastrointestinal (GI) tract. The pacemaker current that initiates each slow wave derives from a calcium-inhibited, voltage-independent, nonselective cation channel. This channel in ICC displays properties similar to that reported for the transient receptor potential (TRP) family of nonselective cation channels, particularly those seen for TRPC3 and TRPC4. We have identified transcripts for TRPC4 in individually isolated ICC and have cloned the two alternatively spliced forms of TRPC4, TRPC4[alpha] and TRPC4[beta], from GI muscles. TRPC4[beta] is missing an 84-amino acid segment from the carboxy terminus. Expression of either form using the whole cell patch-clamp technique led to calcium-inhibited, nonselective cation channels as determined by N-methyl-D-glucamine replacement experiments and BAPTA dialysis. Expression of TRPC4[beta] channels recorded at the whole cell level had characteristics similar to the nonselective cation current in ICC. The single-channel conductance of TRPC4[beta] was determined to be 17.5 pS. Application of calmidazolium to cells expressing TRPC4[beta] led to a significant increase in the inward current of these cells at both the whole cell and single-channel level, and currents were sensitive to block by 10 [micro]M lanthanum, niflumic acid, and DIDS. Comparison of the properties reported for the nonselective cation current in ICC and those identified here for TRPC4[beta] led us to conclude that a TRPC4-like current encodes the plasmalemmal pacemaker current in murine small intestine. cation channel; gastrointestinal; smooth muscle; calmodulin

Ion channels encoded by ether-a-go-go-related genes (ERG) have been implicated in repolarization of the cardiac action potential and also as components of the resting membrane conductance in various cells. The aim of the present study was to determine whether ERG channels were expressed in smooth muscle cells isolated from portal vein. RT-PCR demonstrated the expression of murine ERG (mERG), and real-time quantitative PCR showed that the mERG1b isoform predominated over the mERG1a, mERG2, and mERG3 in portal vein. Single myocytes from portal vein displayed membrane staining with an ERG1-specific antibody. Whole cell voltage-clamp experiments were performed to determine whether portal vein myocytes expressed functional ERG channels. Large inward currents with distinctive kinetics were elicited that were inhibited rapidly by E-4031 (mean amplitude of the E-4031-sensitive current at -120 mV was -205 [+ or -] 24 pA; n = 14). Deactivation of the E-4031-sensitive current was voltage dependent (mean time constants at -80 and -120 mV were 103 [+ or -] 9 and 33 [+ or -] 2 ms, respectively; n = 13). Because of the rapid kinetics of mERG currents at more negative potentials, there was a substantial noninactivating 'window' current that reached a maximum of -66 [+ or -] 10 pA at -70 mV. Complete portal veins exhibited spontaneous contractile activity in isometric tension experiments, and this activity was modified significantly by E-4031. These data show that ERG channels are expressed in murine portal vein myocytes that may contribute to the resting membrane conductance. vascular smooth muscle; ether-a-go-go-related genes; potassium channel

Electrical rhythmicity in gastrointestinal muscles and the enteric nervous system is discussed relative to its cellular/molecular basis and ionic species involved. The channels involved, when assignment of molecular entities to underlying ionic events has been achieved, might be targets for therapeutic agents. BLocking drugs can more easily be tested in expression systems than in native cells. Smooth muscle cells and interstitial cells of Cajal are involved in the body, which complicates study with different types of ionic conductance, and the relationship between the two is the focus here.

Studies were performed to investigate the existence of swelling-activated or volume-sensitive Cl- currents, also identified as the CIC-3 channel in cardiac myocytes, in the smooth muscle cells of the canine colon. The morphologic and molecular properties of the CIC-3 channel were described. The findings confirmed CIC-3 gene expression in the tonic and phasic smooth muscles of the dog and mouse gastro-intestinal (GI) tracts. They also suggest the involvement of CIC-3 channels in altering extracellular osmolarity and radical stretch in the GI smooth muscle cells.

Research was conducted to examine the activation and expression of p38 mitogen-activated protein kinases (MAPK) in smooth muscle through G protein-linked, seven-transmembrane-spanning receptors in mammalian smooth muscle. The p38 MAPK expressed in canine smooth muscles was cloned and sequenced to study the role of p38 MAPK in smooth muscle. Results indicate that elements of the stress-response pathway can be linked to transcriptional control and to cytoskeletal and contractile protein phosphorylation in mammalian smooth muscle.

The molecular identification of a component of delayed rectifier current, Kv2.2, in gastrointestinal smooth muscles is described. RNA isolated from dispersed and selected smooth muscle cells was analyzed using RT-PCR while RNA prepared from tissues were analyzed through northern hybridization technique. Results indicate that Kv2.2 is expressed in smooth muscle cells located in all regions of the canine gastrointestinal (GI) tract and in various vascular tissues. It is concluded that Kv2.2 may contribute to a delayed rectifier current in native GI smooth muscles.

Research suggests that the putative chloride channel protein CIC-3 is responsible for encoding volume-regulated chloride current in mammalian cells. The functional expression of NIH/3T3 cells is achieved through cloning techniques. Further research is required to ascertain the molecular relationship between cell volume and channel activation.

The characteristics and requirements of canine proximal colon organ cultures were analyzed to determine the effectivity of canine blood serum in the preservation of the physiological properties of the cultured organ. Canine serum maintained the cell/tissue morphology, ionic mechanisms for membrane potential and spontaneous electrical rhythmicity in canine proximal colon organ cultures for six days. Furthermore, canine serum maintained the normal gradient in resting membrane potential across the circular smooth muscles of canine proximal colon.

Complementary deoxyribonucleic acids clones (cDNAs) that encode the alpha- and beta-subunits of the slo gene were expressed in the gastrointestinal tract to determine the properties of Ca2+-activated K+ channels. The canine smooth muscle forms of the beta- and alpha-subunits of large conductance BK channels are expressed in the messenger ribonucleic acid level in the GI muscles. Heterogeneous transcript sizes were detected in Northern blot analysis and a degree of diversity was detected in the carboxy-terminal end of the splice variant at site 5.

A study of the charybdotoxin (CTX) sensitivity of two cloned smooth muscle rectifier K+ channels expressed in oocytes confirms the formation of heterotetramers by these channels. However, the sensitivity of these heterotetrameric channels to CTX is nullified in the presence of CTX-insensitive subunit. Variation in the amino acid near the pore region of the two K+ channel clones inhibits CTX access to its binding site, thereby determining CTX sensitivity of the channel.

Northern blot analysis of a newly expression-cloned and characterized Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle exhibits amino acid sequence similarity with other K+ channels of the Kv1.5 family in the core region between transmembrane segments S1 and S6. The cKv1.5 in oocytes exhibits a preference for K+ and is activated by depolarization to membrane potentials positive to -40 mV in a voltage-dependent fashion. The channel is characterized by a linear current-voltage curve with a slope condutance of 9.8 plus or minus 1.1 pS. cKv1.5 expression in smooth muscle of the gastrointestinal tract and in several vascular smooth muscles is detected by Northern blot analysis. cKv1.5 denotes a delayed rectifier current component in both vascular and visceral smooth muscles.

cDNA cloning and functional expression of a delayed rectifier K+ channel of the Kv1.2 class from canine colonic circular smooth muscle were used to explore the characteristics of K+ currents of gastrointestinal smooth muscles. A K+ current that activated in a voltage-dependent fashion upon depolarization was established by the functional expression of a cDNA (CSMK1) in Xenopus oocytes. The K+ channel examined may be expressed only in gastrointestinal smooth muscles because CSMK1 expression is absent in portal vein, renal artery and uterus.

The molecular and structural identity of cystic fibrosis transmembrane conductance regulator (CFTR), present in the Cl- channels of the heart, is analyzed, and its homology with the CFTR expressed in epithelial tissues is tested. The CFTR transcript is demonstrated to be alternately cleaved in the heart, leading to the cardiac expression of a specific isoform of CFTR. CFTR expression in cardiac tissues is exceptional, as demonstrated by the unique presence of a adenosine 3',5'-cyclic monophosphate-dependent Cl- conductance in native cells.

Canine colonic smooth muscle shows an inward calcium current, controlled by various kinds of calcium channels, whose properties are determined. The circular layer of the canine proximal colon yields freshly dispersed smooth muscle cells which are employed in the experiment. Eighty millimols of Ba2+ is used as the charge carrier to calculate the single-channel currents. Voltage steps are depolarized to reveal small- and large-conductance single-channel currents. Ten to the power of minus 6 mols of nifedipine is incorporated into the solution to hinder both the currents of the single-channel.

Small-conductance [Ca.sup.2+]-activated [K.sup.+] (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 [is greater than] SK3 [is greater than] 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 [Ca.sup.2+]-activated voltage-independent channels, mSK1 is less sensitive to apamin compared with SK2 and showed intracellular [Ca.sup.2+] sensitivity ([10.sup.-8] to [10.sup.-6] M) in asymmetrical [K.sup.+] (5/140 mM [K.sup.+]) gradients. Our results suggest that SK channel expression varies in colonic myocytes from different species and may contribute differentially to inhibitory junction potentials. gastroenteric motility; apamin; small-conductance calcium-activated potassium channel isoform

Differential expression and alternative splicing of TRP channel genes in smooth muscles. Am J Physiol Cell Physiol 280: C1184-C1192, 2001.--Nonselective cation channels (NSCC) are targets of excitatory agonists in smooth muscle, representing the nonselective cation current [I.sub.cat]. [Na.sup.+] influx through NSCC causes depolarizations and activates voltage-dependent [Ca.sup.2+] channels, resulting in contraction. The molecular identity of [I.sub.cat] in smooth muscle has not been elucidated; however, products of the transient receptor potential (TRP) genes have characteristics similar to native [I.sub.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. calcium channels; gastrointestinal; vascular; ribonucleic acid expression

Britton, Fiona C., William J. Hatton, Charles F. Rossow, Dayue Duan, Joseph R. Hume, and Burton Horowitz. Molecular distribution of volume-regulated chloride channels (ClC-2 and ClC-3) in cardiac tissues. Am J Physioi Heart Circ Physiol 279: H2225-H2233, 2000.--The molecular identification of cardiac chloride channels has provided probes to investigate their distribution and abundance in heart. In this study, the molecular expression and distribution of volume-regulated chloride channels ClC-2 and ClC-3 in cardiac tissues were analyzed and quantified. Total RNA was isolated from atria and ventricles of several species (dog, guinea pig, and rat) and subjected to a quantitative RT-PCR strategy. ClC-2 and ClC-3 mRNA expression were calculated relative to [Beta]-actin expression within these same tissues. The transcriptional levels of ClC-3 mRNA were between 1.8 and 10.2% of [Beta]-actin expression in atria and between 3.4 and 8.6% of [Beta]-actin in ventricles (n = 3 for each tissue). The levels of ClC-2 in both atria and ventricles were significantly less than those measured for ClC-3 (n = 3; P [is less than] 0.05). ClC-2 mRNA levels were between 0.04-0.08% and 0.03-0.18% of [Beta]-actin expression in atria and ventricles, respectively (n = 3 for each tissue). Immunoblots of atrial and ventricular wall protein extracts demonstrated ClC-2 and ClC-3-specific immunoreactivity at 97 and 85 kDa, respectively. Immunohistochemical localization in guinea pig cardiac muscle demonstrates a ubiquitous distribution of ClC-2 and ClC-3 channels in the atrial and ventricular wall. Confocal analysis detected colocalization of ClC-2 and ClC-3 in sarcolemmal membranes and distinct ClC-3 immunoreactivity in cytoplasmic regions. The molecular expression of ClC-2 and ClC-3 in cardiac tissue is consistent with the proposed role of these chloride channels in the regulation of cardiac cell volume and the modulation of cardiac electrical activity. heart; anion channel; immunohistochemistry; cDNA

Epperson, Anne, William J. Hatton, Brid Callaghan, Philip Doherty, Rebecca L. Walker, Kenton M. Sanders, Sean M. Ward, and Burton Horowitz. Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal. Am J Physiol Cell Physiol 279: C529-C539, 2000.--Located within the tunica muscularis of the gastro-intestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types [M.sub.2] and [M.sdub.3], neurokinin receptors [NK.sub.1] and [NK.sub.3], and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form. gastrointestinal; smooth muscle; c-kit; development

Bradley, Karri K., William J. Hatton, Helen S. Mason, Rebecca L. Walker, Elaine R. M. Flynn, James L. Kenyon, and Burton Horowitz. Kir3.1/3.2 encodes an [I.sub.KACh]-like current in gastrointestinal myocytes. Am. J. Physiol. Gastrointest. Liver Physiol. 278: G289-G296, 2000.--Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this [K.sup.+] channel subfamily encode G protein-gated inwardly rectifying K channels ([I.sub.KACh]) in other tissues, including the heart and brain. In the GI tract, [I.sub.KACh] could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to [Beta]-actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 [micro] M) reversibly activated a [Ba.sup.2+]-sensitive and inwardly rectifying K+ current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an [I.sub.KACh] current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers. colon; motility; smooth muscle; potassium; ion channel

HUME, JOSEPH R. I. INTRODUCTION Anion channels in the heart have been the subject of electrophysiological examination for nearly four decades dating back to the original work in 1961 of [...]