Membrane currents in cultured human intestinal smooth muscle cells

Abstract

Using whole‐cell patch‐clamp recording techniques, we have examined voltage‐gated ion currents in a cultured human intestinal smooth muscle cell line (HISM). Experiments were performed at room temperature on cells after passages 16 and 17. Two major components of the whole‐cell current were a tetraethylammonium‐sensitive (IC50= 9 mm), iberiotoxin‐resistant, delayed rectifier K+ current and a Na+ current inhibited by tetrodotoxin (IC50= 100 nm). No measurable inward current via voltage‐gated Ca2+ channels could be detected in these cells even with 10 mm Ca2+ or Ba2+ in the external solution. No current attributable to calcium‐activated K+ channels was found and no cationic current in response to muscarinic receptor activation was present. In divalent cation‐free external solution two additional currents were activated: an inwardly rectifying hyperpolarization‐activated current, IHA, and a depolarization‐activated current, IDA. I HA and IDA could be carried by several monovalent cations; the sizes of currents in descending order were: K+ > Cs+ > Na+ for IHA and Na+ > K+ >> Cs+ for IDA. IHA was activated and deactivated instantaneously and showed no inactivation whereas IDA was activated, inactivated and deactivated within tens of milliseconds. These currents were inhibited by external calcium with an IC50 of 0.3 μm for IDA and an IC50 of 20 μm for IHA. Cyclopiazonic acid (CPA) induced an outward, but not an inward current. SK&F 96365, a blocker of store‐operated Ca2+ channels, suppressed IDA with a half‐maximal inhibitory concentration of 9 μm but was ineffective in inhibiting IHA at concentrations up to 100 μm. Gd3+ and La3+ strongly suppressed IDA at 1 and 10 μm, respectively and were less effective in blocking IHA (complete inhibition required a concentration of 100 μm for both). Carbachol at 10–100 μm evoked about a 3‐fold increase in IHA amplitude and completely abolished IDA. We conclude that IHA and IDA are Ca2+‐blockable cationic currents with different ion selectivity profiles that are carried by different channels. IDA shows novel voltage‐dependent properties for a cationic current.

Characterization of a voltage-dependent Na+current in human esophageal smooth muscle

“…The TTX sensitivity of the current (IC 50 ϳ20 nM), combined with its insensitivity to Cd 2ϩ , indicates that cultured HESMCs express channels similar to those found in skeletal muscle and neurons, but not those found in cardiac muscle (2). This finding is consistent with the characteristics of some Na ϩ channels found in the smooth muscle of humans (28,35,38) and animals (6,25,30). In contrast, a recent report of voltage-dependent Na ϩ channels in human jejunal SMCs found them to be relatively resistant to blockade by TTX (20).…”

“…In the gastrointestinal tract, Na ϩ current has been observed in the ileum (30), large intestine (35), and fundus (25). Zholos et al (38) recently provided electrophysiological evidence of a TTX-sensitive Na ϩ current in human intestinal smooth muscle. The observed differences in activation and inactivation ranges of these currents have led to the suggestion that considerable heterogeneity may exist among smooth muscle Na ϩ channels (7).…”

Targeting the T-type calcium channel Cav3.2 in GABAergic arcuate nucleus neurons to treat obesity

“…The regulatory effects of Cav3.2 on energy expenditure may also work through peripheral mechanisms. The Cav3.2 encoding gene expresses in cells from many peripheral tissues, including the adipocytes from WAT and BAT [ 79 ], myocytes from skeletal muscle [ 80 ] and smooth muscle [ 81 ], sensory dorsal root ganglion (DRG) neurons, and spinal dorsal horn (SDH) neurons, but not sympathetic neurons [ 82 ]. Cav3.2 has been shown to play essential roles in the physiological functions of excitable tissues, i.e., neurons and muscle.…”

Muscarinic receptor‐activated cationic channels in murine ileal myocytes

“…The effect of well known nonselective cation channel blockers such as quinine, SK&F 96365 and La 3+ were also tested in order to compare murine intestinal myocytes with other cell types (Chen et al ., 1993; Boulay et al ., 1997; Wayman et al ., 1997; Lin et al ., 1998; Zholos et al ., 2000a). We have used nearly saturating blocking concentrations of 20 μ M of quinine, 30 μ M of SK&F 96365 and 2 m M of La 3+ .…”