Biomedical subjects

B Horowitz

Publications and source records attributed to B Horowitz.

At least 37 records · Page 2Linked to original sources

PubMed2000

Blocking of cloned and native delayed rectifier K channels from visceral smooth muscles by phencyclidine.

We investigated the effect of phencyclidine (PCP) on three native delayed rectifier K+ currents and three channels cloned from canine and human circular colonic myocytes using voltage-clamp techniques. Native delayed rectifier K+ current in canine circular colon is composed of at least three components: (i) a rapidly activating, 4-aminopyridine-sensitive component (termed IdK(f)); (ii) a slowly activating, tetraethylammonium (TEA)-sensitive component (IdK(s)); and (iii) a rapidly activating, TEA-sensitive component, which has a steady-state inactivation curve shifted towards more negative potentials (IdK(n)). PCP blocked all three components with EC50 values of 45, 27 and 59 micromol L-1, respectively. Blocking was neither use-dependent nor voltage-dependent. Delayed rectifier K+ channels cloned from canine (Kv1.2, Kv1.5) and from human (Kv2.2) colon were expressed in Xenopus oocytes. PCP blocked all three currents with similar potency. In contrast, PCP (up to 10-4 mol L-1) did not reduce the magnitude of Ca2+-dependent outward current of large conductance Ca2+-activated K+ channels (BK channels).

Animals↗

PubMed2000

Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal.

Located within the tunica muscularis of the gastrointestinal (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(2) and M(3), neurokinin receptors NK(1) and NK(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.

Animals↗

PubMed2000

Kir3.1/3.2 encodes an I(KACh)-like current in gastrointestinal myocytes.

Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K(+) channel subfamily encode G protein-gated inwardly rectifying K(+) channels (I(KACh)) in other tissues, including the heart and brain. In the GI tract, I(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 microM) reversibly activated a Ba(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(KACh) current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.

Acetylcholine↗

PubMed2000

Molecular distribution of volume-regulated chloride channels (ClC-2 and ClC-3) in cardiac tissues.

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 < 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.

Actins↗

PubMed2000

Anion transport in heart.

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.

Animals↗

PubMed2000

Efforts in minimizing risk of viral transmission through viral inactivation.

The viral safety of blood and blood products has improved substantially over the last decade on account of the development of new viral screening and virucidal procedures. For nearly 15 years, virally inactivated blood derivatives, prepared by using advanced virucidal procedures, have amassed an extraordinary safety record with respect to hepatitis B and C and HIV. This record of safety has spawned the development of newer virucidal procedures designed to eliminate nonenveloped viruses from blood derivatives and viruses and other pathogens from blood components, including cellular components. Ongoing tests that include clinical studies will demonstrate how close we are to achieving a blood supply that is free of viruses, bacteria, and parasites.

Blood Substitutes↗

PubMed2000

Photochemical decontamination of red blood cell concentrates with the silicon phthalocyanine PC 4 and red light.

Various approaches are being developed for virus inactivation of red blood cell concentrates (RBCC) in order to increase the safety of the blood supply. We have been studying the silicon phthalocyanine Pc 4 for this purpose, a photosensitizer activated with red light. Pc 4 targets the envelope of pathogenic viruses such as HIV. To protect RBC during the process two main approaches are used: (i) inclusion of quenchers of reactive oxygen species produced during the treatment. Tocopherol succinate was found to be most effective for this purpose; (ii) formulation of Pc 4, a lipophilic compound, in liposomes that reduce its binding to RBC but not to viruses. As a light source we used a light emitting diode array emitting at 670-680 nm. An efficient mixing device ensures homogenous light exposure during treatment of intact RBCC. Treatment of 50 ml RBCC with 5 microM Pc 4 and 18 J/cm(2) light results in the inactivation of > or = 5.5 log(10) HIV, > or = 6.3 log(10), VSV and > or = 5 log(10) of PRV and BVDV. The relative sensitivities of these viruses based on the slope of virus kill versus light dose are 1.0, 1.25, 1.5 and 1.9 for HIV, VSV, PRV and BVDV, respectively. To achieve the same level of virus inactivation in 350 ml RBCC, the light dose needed is 40 J/cm(2). HIV actively replicating in CEM cells is as sensitive as cell-free and HIV in latently infected cells is 3-4 times more sensitive. Parasites that can be transmitted by blood transfusion (P. falciparum and T. cruzi) are even more sensitive than viruses. Following treatment, RBCC can be stored for 28 days at 4 degrees C with haemolysis below 1%. Previous studies under less favourable conditions showed that baboon RBC circulated with an acceptable 24 hr recovery and half-life. Genetic toxicological studies of Pc 4 with or without light exposure (mutagenicity in bacteria, mammalian cells in vitro and clastogenicity in vivo) were negative. We conclude that a process using Pc 4 and red light can potentially reduce the risk of transmitting pathogens in RBCC.

Animals↗

PubMed1999

Purinoceptor-coupled Cl- channels in mouse heart: a novel, alternative pathway for CFTR regulation.

1. P2-purinoceptors couple extracellular ATP to the activation of a Cl- current (ICl,ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of ICl,ATP activation in mouse heart. 2. Extracellular adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS; 100 microM) activated ICl,ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPgammaS while a PKC activator, phorbol 12, 13-dibutyrate (PDBu) activated a current with identical properties to ICl,ATP. Maximal activation of ICl,ATP by ATPgammaS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl- channels. 3. Isoprenaline increased ICl,ATP pre-activated by ATPgammaS or PDBu, while isoprenaline or forskolin alone failed to activate any Cl- current in these myocytes. Adenosine 3',5'-cyclic monophosphothionate, a PKA inhibitor, prevented ATPgammaS or PDBu activation of ICl,ATP. Thus, ICl,ATP is regulated by dual intracellular phosphorylation pathways involving both PKA and PKC in a synergistic manner similar to cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. 4. Glibenclamide (50 microM) significantly blocked ICl,ATP activated by ATPgammaS or by the CFTR channel activator, levamisole. 5. The slope conductance of the unitary ICl,ATP in cell-attached patches was 11.8 +/- 0.3 pS, resembling the known properties of CFTR Cl- channels in cardiac myocytes. 6. The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. 7. We conclude that ICl,ATP in mouse heart is due to activation of CFTR Cl- channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.

Adenosine Triphosphate↗

PubMed1999

Inward rectifier potassium conductance regulates membrane potential of canine colonic smooth muscle.

1. The membrane potential of gastrointestinal smooth muscles determines the open probability of ion channels involved in rhythmic electrical activity. The role of Ba2+-sensitive K+ conductances in the maintenance of membrane potential was examined in canine proximal colon circular muscle. 2. Application of Ba2+ (1-100 microM) to strips of tunica muscularis produced depolarization of cells along the submucosal surface of the circular muscle layer. Significantly higher concentrations of Ba2+ were needed to depolarize preparations from which the submucosal and myenteric pacemaker regions were removed. 3. Elevation of extracellular [K+]o (from 5.9 to 12 mM) brought membrane potentials closer to EK (the Nernst potential for K+ ions), suggesting activation of a K+ conductance. This occurred at potentials much more negative than the activation range for delayed rectifier channels (Kv). 4. Forskolin (1 microM) caused hyperpolarization and a leftward shift in the dose-response relationship for Ba2+, suggesting that forskolin may activate a Ba2+-sensitive conductance. 5. Patch-clamp recordings from interstitial cells of Cajal (ICC) revealed the presence of a Ba2+-sensitive inward rectifier potassium conductance. Far less of this conductance was present in smooth muscle cells. 6. Kir2.1 was expressed in the circular muscle layer of the canine proximal colon, duodenum, jejunum and ileum. Kir2.1 mRNA was expressed in greater abundance along the submucosal surface of the circular muscle layer in the colon. 7. These results demonstrate that ICC express a Ba2+-sensitive conductance (possibly encoded by Kir2.1). This conductance contributes to the generation and maintenance of negative membrane potentials between slow waves.

Animals↗

PubMed1999

Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072.

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-alpha channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Ialpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-alpha by direct phosphorylation at serine 1072.

Base Sequence↗

PubMed1999

Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells.

1. The molecular nature of the strong inward rectifier K+ channel in vascular smooth muscle was explored by using isolated cell RT-PCR, cDNA cloning and expression techniques. 2. RT-PCR of RNA from single smooth muscle cells of rat cerebral (basilar), coronary and mesenteric arteries revealed transcripts for Kir2.1. Transcripts for Kir2.2 and Kir2.3 were not found. 3. Quantitative PCR analysis revealed significant differences in transcript levels of Kir2.1 between the different vascular preparations (n = 3; P < 0.05). A two-fold difference was detected between Kir2.1 mRNA and beta-actin mRNA in coronary arteries when compared with relative levels measured in mesenteric and basilar preparations. 4. Kir2.1 was cloned from rat mesenteric vascular smooth muscle cells and expressed in Xenopus oocytes. Currents were strongly inwardly rectifying and selective for K+. 5. The effect of extracellular Ba2+, Ca2+, Mg2+ and Cs2+ ions on cloned Kir2.1 channels expressed in Xenopus oocytes was examined. Ba2+ and Cs+ block were steeply voltage dependent, whereas block by external Ca2+ and Mg2+ exhibited little voltage dependence. The apparent half-block constants and voltage dependences for Ba2+, Cs+, Ca2+ and Mg2+ were very similar for inward rectifier K+ currents from native cells and cloned Kir2.1 channels expressed in oocytes. 6. Molecular studies demonstrate that Kir2.1 is the only member of the Kir2 channel subfamily present in vascular arterial smooth muscle cells. Expression of cloned Kir2.1 in Xenopus oocytes resulted in inward rectifier K+ currents that strongly resemble those that are observed in native vascular arterial smooth muscle cells. We conclude that Kir2.1 encodes for inward rectifier K+ channels in arterial smooth muscle.

Animals↗

PubMed1999

Contribution of delayed rectifier potassium currents to the electrical activity of murine colonic smooth muscle.

1. We used intracellular microelectrodes to record the membrane potential (Vm) of intact murine colonic smooth muscle. Electrical activity consisted of spike complexes separated by quiescent periods (Vm approximately -60 mV). The spike complexes consisted of about a dozen action potentials of approximately 30 mV amplitude. Tetraethylammonium (TEA, 1-10 mM) had little effect on the quiescent periods but increased the amplitude of the action potential spikes. 4-Aminopyridine (4-AP, >= 5 mM) caused continuous spiking. 2. Voltage clamp of isolated myocytes identified delayed rectifier K+ currents that activated rapidly (time to half-maximum current, 11.5 ms at 0 mV) and inactivated in two phases (tauf = 96 ms, taus = 1.5 s at 0 mV). The half-activation voltage of the permeability was -27 mV, with significant activation at -50 mV. 3. TEA (10 mM) reduced the outward current at potentials positive to 0 mV. 4-AP (5 mM) reduced the early current but increased outward current at later times (100-500 ms) consistent with block of resting channels relieved by depolarization. 4-AP inhibited outward current at potentials negative to -20 mV, potentials where TEA had no effect. 4. Qualitative PCR amplification of mRNA identified transcripts encoding delayed rectifier K+ channel subunits Kv1.6, Kv4.1, Kv4.2, Kv4.3 and the Kvbeta1.1 subunit in murine colon myocytes. mRNA encoding Kv 1.4 was not detected. 5. We find that TEA-sensitive delayed rectifier currents are important determinants of action potential amplitude but not rhythmicity. Delayed rectifier currents sensitive to 4-AP are important determinants of rhythmicity but not action potential amplitude.

4-Aminopyridine↗

PubMed1999

Angiotensin II type 1 receptor-mediated inhibition of K+ channel subunit kv2.2 in brain stem and hypothalamic neurons.

Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function.

Angiotensin II↗

PubMed1999

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C.

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.

Animals↗

PubMed1999

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.

BACKGROUND & AIMS: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS: Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS: ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.

Animals↗

PubMed1999

A serine residue in ClC-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume.

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

Animals↗

PubMed1999

Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscles.

Regulation of gastrointestinal (GI) motility is intimately coordinated with the modulation of ionic conductance expressed in GI smooth muscle and nonmuscle cells. Interstitial cells of Cajal (ICC) act as pacemaker cells and possess unique ionic conductances that trigger slow wave activity in these cells. The slow wave mechanism is an exclusive feature of ICC: Smooth muscle cells may lack the basic ionic mechanisms necessary to generate or regenerate slow waves. The molecular identification of the components for these conductances provides the foundation for a complete understanding of the ionic basis for GI motility. In addition, this information will provide a basis for the identification or development of therapeutics that might act on these channels. It is much easier to study these conductances and develop blocking drugs in expression systems than in native GI muscle cells. This review focuses on the relationship between ionic currents in native GI smooth muscle cells and ICC and their molecular counterparts.

Animals↗

PubMed1999

Molecular diversity of K(V) alpha- and beta-subunit expression in canine gastrointestinal smooth muscles.

Voltage-activated K(+) (K(V)) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the K(V) currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different K(V) channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of K(V)1.4, K(V)1.5, K(V)1.6, K(V)2.2, and K(V)4.3 transcripts in all regions of the GI tract examined. Transcripts encoding K(V)1.2, K(V)beta1.1, and K(V)beta1.2 subunits were differentially expressed. K(V)1.1, K(V)1.3, K(V)2.1, K(V)3.1, K(V)3.2, K(V)3.4, K(V)4.1, K(V)4.2, and K(V)beta2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these K(V) channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that K(V)1.2, K(V)1.4, K(V)1.5, and K(V)2.2 are expressed at the protein level in GI tissues and smooth muscle cells. K(V)2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of K(V) channel subunits.

Animals↗