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The function and molecular expression of ATP-sensitive potassium (KATP) channels in murine colonic smooth muscle was investigated by intracellular electrical recording from intact muscles, patch-clamp techniques on isolated smooth muscle myocytes, and reverse transcription polymerase chain reaction (RT-PCR) on isolated cells. Lemakalim (1 microM) caused hyperpolarization of intact muscles (17. 2 +/- 3 mV). The hyperpolarization was blocked by glibenclamide (1-10 microM). Addition of glibenclamide (10 microM) alone resulted in membrane depolarization (9.3 +/- 1.7 mV). Lemakalim induced an outward current of 15 +/- 3 pA in isolated myocytes bathed in 5 mM external K+ solution. Application of lemakalim to cells in symmetrical K+ solutions (140/140 mM) resulted in a 97 +/- 5 pA inward current. Both currents were blocked by glibenclamide (1 microM). Pinacidil (1 microM) also activated an inwardly rectifying current that was insensitive to 4-aminopyridine and barium. In single-channel studies, lemakalim (1 microM) and diazoxide (300 microM) increased the open probability of a 27-pS K+ channel. Openings of these channels decreased with time after patch excision. Application of ADP (1 mM) or ATP (0.1 mM) to the inner surface of the patches reactivated channel openings. The conductance and characteristics of the channels activated by lemakalim were consistent with the properties of KATP. RT-PCR demonstrated the presence of Kir 6.2 and SUR2B transcripts in colonic smooth muscle cells; transcripts for Kir 6.1, SUR1, and SUR2A were not detected. These molecular studies are the first to identify the molecular components of KATP in colonic smooth muscle cells. Together with the electrophysiological experiments, we conclude that KATP channels are expressed in murine colonic smooth muscle cells and suggest that these channels may be involved in dual regulation of resting membrane potential, excitability, and contractility.

The role of intracellular guanosine 3′,5′-cyclic monophosphate concentration ([cGMP]i) in nitric oxide (NO)-mediated relaxations in the uterus has become controversial. We found the NO donor S-nitroso-l-cysteine (CysNO) to potently (IC50 = 30 nM) inhibit spontaneous contractions in the nonpregnant human myometrium. CysNO treatment increased [cGMP]i significantly ( P < 0.001), and this increase was blocked by the guanylyl cyclase inhibitors methylene blue (10 μM) or LY-83583 (1 μM); however, pretreatment with these guanylyl cyclase inhibitors failed to block CysNO-mediated relaxations. Intracellular cAMP concentrations were not altered by treatment of tissues with 10 μM CysNO. Incubation with the cGMP analogs 8-bromo-cGMP or β-phenyl-1, N 2-etheno-cGMP did not significantly affect spontaneous contractility. Pretreatment of tissues with charybdotoxin [a calcium-dependent potassium channel (BK) blocker] completely reversed CysNO-induced relaxations. We conclude that NO is a potent inhibitor of spontaneous contractile activity in the nonpregnant human uterus and that, although guanylyl cyclase and BK activities are increased by NO, increases in [cGMP]i are not required for NO-induced relaxations in this tissue.

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]-1,2-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 1 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.

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