TY - JOUR
T1 - Functional and molecular identification of a novel chloride conductance in canine colonic smooth muscle
AU - Dick, Gregory M.
AU - Bradley, Karri K.
AU - Horowitz, Burton
AU - Hume, Joseph R.
AU - Sanders, Kenton M.
PY - 1998/10
Y1 - 1998/10
N2 - Swelling-activated or volume-sensitive Cl- currents are found in numerous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Recently, the molecular entity responsible for swelling-activated Cl- current in cardiac myocytes has been identified as ClC-3. The goal of our study was to determine whether such a channel exists in smooth muscle cells of the canine colon using both molecular biological and electrophysiological techniques and, if present, to characterize its functional and molecular properties. We hypothesized that ClC-3 is present in colonic smooth muscle and is regulated in a manner similar to the molecular entity cloned from heart. Indeed, the ClC-3 gene was expressed in colonic myocytes, as demonstrated by reverse transcriptase polymerase chain reaction performed on isolated cells. The current activated by decreasing extracellular osmolarity from 300 to 250 mosM was outwardly rectifying and dependent on the Cl- gradient. Current magnitude increased and reversed at more negative potentials when Cl- was replaced by I- or Br-. Tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]- l,2-diphenyl-1-butene; 10 μM) and DIDS (100 μM) inhibited the current, whereas 25 μM niflumic acid, 10 μM nicardipine, and Ca2+ removal had no effect. Current was inhibited by I mM extracellular ATP in a voltage- dependent manner. Cl- current was also regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM) decreased Cl- current magnitude, while chelerythrine chloride (30 μM) activated it under isotonic conditions. Our findings indicate that a current activated by hypotonic solution is present in colonic myocytes and is likely mediated by ClC-3. Furthermore, we suggest that the ClC-3 may be an important mechanism controlling depolarization and contraction of colonic smooth muscle under conditions that impose physical stress on the cells.
AB - Swelling-activated or volume-sensitive Cl- currents are found in numerous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Recently, the molecular entity responsible for swelling-activated Cl- current in cardiac myocytes has been identified as ClC-3. The goal of our study was to determine whether such a channel exists in smooth muscle cells of the canine colon using both molecular biological and electrophysiological techniques and, if present, to characterize its functional and molecular properties. We hypothesized that ClC-3 is present in colonic smooth muscle and is regulated in a manner similar to the molecular entity cloned from heart. Indeed, the ClC-3 gene was expressed in colonic myocytes, as demonstrated by reverse transcriptase polymerase chain reaction performed on isolated cells. The current activated by decreasing extracellular osmolarity from 300 to 250 mosM was outwardly rectifying and dependent on the Cl- gradient. Current magnitude increased and reversed at more negative potentials when Cl- was replaced by I- or Br-. Tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]- l,2-diphenyl-1-butene; 10 μM) and DIDS (100 μM) inhibited the current, whereas 25 μM niflumic acid, 10 μM nicardipine, and Ca2+ removal had no effect. Current was inhibited by I mM extracellular ATP in a voltage- dependent manner. Cl- current was also regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM) decreased Cl- current magnitude, while chelerythrine chloride (30 μM) activated it under isotonic conditions. Our findings indicate that a current activated by hypotonic solution is present in colonic myocytes and is likely mediated by ClC-3. Furthermore, we suggest that the ClC-3 may be an important mechanism controlling depolarization and contraction of colonic smooth muscle under conditions that impose physical stress on the cells.
KW - Adenosine 5'-triphosphate
KW - Chloride channels
KW - Gastrointestinal motility
KW - Myogenic response
KW - Protein kinase C
UR - http://www.scopus.com/inward/record.url?scp=0031751195&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.1998.275.4.c940
DO - 10.1152/ajpcell.1998.275.4.c940
M3 - Article
C2 - 9755047
AN - SCOPUS:0031751195
SN - 0363-6143
VL - 275
SP - C940-C950
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 4 44-4
ER -