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Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant [Na.sup.+] current ([Na.sub.v]1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type [Ca.sup.2+] currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type [Ca.sup.2+] channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type [Ca.sup.2+] channels over ICC and smooth muscle [Na.sup.+] channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type [Ca.sup.2+] and [Na.sup.+] currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type [Ca.sup.2+] channel [[alpha].sub.1] 3.3b + [[beta].sub.2], the human intestinal L-type [Ca.sup.2+] channel subunits [[alpha].sub.1c] + [[beta].sub.2], or [Na.sub.v]1.5. Mibefradil significantly reduced expressed T-type [Ca.sup.2+] current at concentrations [greater than or equal to] 0.1 [micro]M ([IC.sub.50] = 0.29 [micro]M), L-type [Ca.sup.2+] current at > 1 [micro]M ([IC.sub.50] = 2.7 [micro]M), and [Na.sup.+] current at [greater than or equal to] 0.3 [micro]M ([IC.sub.50] = 0.98 [micro]M). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant [Na.sup.+] channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed. ion channel; gastrointestinal tract; patch clamp

Interstitial cells of Cajal (ICC) generate the electrical slow wave required for normal gastrointestinal motility. The ionic conductances expressed in human intestinal ICC are unknown. The aim of this study was to determine expression of a [Na.sup.+] current in human intestinal ICC and to determine the effects of the [Na.sup.+] current on the slow wave. Visually identified, freshly dissociated, single ICC were verified by the presence of c-kit mRNA by using single-cell RT-PCR. Standard whole cell currents were recorded from patch-clamped ICC held at -100 mV between pulse protocols. A [Na.sup.+] current was identified in human intestinal ICC. The current activated at -55 mV and peaked at -30 mV. Extracellular N-methyl-D-glucamine abolished and QX-314 (500 [micro]M) blocked the [Na.sup.+] current, but nifedipine and [Ni.sup.2+] did not. The [Na.sup.+] current was activated by shear stress. Single-cell RT-PCR detected mRNA for the [Na.sup.+] [alpha]-subunit SCN5A in single human intestinal ICC. Lidocaine (200 [micro]m) and QX-314 (500 [micro]M) decreased slow wave frequency, and stretch increased slow wave frequency. A mechanosensitive [Na.sup.+] channel current is present in human intestinal ICC and appears to play a role in the control of intestinal motor function. pacemaker; small intestine; patch clamp; smooth muscle

A [Na.sup.+] current is present in human jejunal circular smooth muscle cells. The aim of the present study was to determine the role of the cytoskeleton in the regulation of the [Na.sup.+] current. Whole cell currents were recorded by using standard patch-clamp techniques with [Cs.sup.+] in the pipette to block [K.sup.+] currents. Cytochalasin D and gelsolin were used to disrupt the actin cytoskeleton and phalloidin to stabilize it. Colchicine was used to disassemble the microtubule cytoskeleton (and intermediate filaments) and paclitaxel to stabilize it. Acrylamide was used to disrupt the intermediate filament cytoskeleton. Perfusion of the recording chamber at 10 ml/ min increased peak [Na.sup.+] current recorded from jejunal smooth muscle cells by 27 [+ or -] 3%. Cytochalasin D and gelsolin abolished the perfusion-induced increase in [Na.sup.+] current, whereas incubation with phalloidin, colchicine, paclitaxel, or acrylamide had no effect. In conclusion, the [Na.sup.+] current expressed in human jejunal circular smooth muscle cells appears to be regulated by the cytoskeleton. An intact actin cytoskeleton is required for perfusion-induced activation of the [Na.sup.+] current. small intestine; patch clamp; actin; microtubules

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the [[alpha].sub.1C] L-type calcium channel subunit ([Ca.sub.v]1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a [[beta].sub.2] calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac [[alpha].sub.1C] splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the [[alpha].sub.1C] COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming [[alpha].sub.1C]-subunit. smooth muscle; mechanogated; voltage gated

Background & Aims: Interstitial cells of Cajal (ICC) are required for normal gastrointestinal motility. Loss of ICC is associated with several motility disorders. The mechanisms modulating ICC survival and proliferation are poorly understood. This study aimed to establish whether 5-hydroxytryptamine (5-HT) plays a role in regulating ICC proliferation. Methods: Expression of 5-HT receptor mRNA was investigated in muscle strips, in purified populations of ICC, and in identified single cells. The effect of 5-HT2B receptor ligands on ICC numbers was studied in primary cell cultures. Proliferation of ICC was determined by counting Ki67-positive cells in culture. Results: Of the 5-HT receptors known to be involved in proliferation, 5-HT2B receptor mRNA was detected by reverse transcriptase–polymerase chain reaction (RT-PCR) in jejunal muscle, whereas 5-HT1A, 5-HT1D, and 5-HT2C receptor mRNAs were not. 5-HT2B receptor mRNA was found in single ICC and cells purified by flow cytometry. Exogenous 5-HT (1 μmol/L) increased (66% ± 9%, P < .005) ICC numbers in culture. The 5-HT2 receptor antagonist, ritanserin, and the 5-HT2B receptor antagonist, SB204741, inhibited the effect of 5-HT. The 5-HT2B receptor agonist BW 723C86 induced a concentration-dependent increase in ICC number (50% ± 6% at 50 nM, P < .04) and increased ICC proliferation (25% ± 3% vs 19 ± 1% in controls, P < .03). Conclusions: These studies establish that 5-HT2B receptors are expressed on ICC. Exogenous 5-HT regulates ICC numbers through 5-HT2B receptors in part by increasing ICC proliferation. The 5-HT2B receptor may serve as a novel pathway to regulate ICC numbers. [Copyright &y& Elsevier]

T-type Ca(2+) currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca(2+) channel alpha subunit responsible for these currents has not been determined. We established that the alpha subunit of the alpha(1H) Ca(2+) channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and Cd(2+) resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the alpha(1H) Ca(2+) channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the alpha(1H) Ca(2+) channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the alpha(1H) Ca(2+) channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the alpha(1H) Ca(2+) channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.

SCN5A encodes the alpha subunit of the cardiac muscle and intestinal smooth muscle mechanosensitive Na(+) channel. Mechanosensitivity in the intestine requires an intact cytoskeleton. We report, using laser capture microdissection, single cell PCR, and immunohistochemistry, that syntrophins, scaffolding proteins, were expressed in human intestinal smooth muscle cells. The distribution of syntrophin gamma 2 was similar to that of SCN5A. Yeast two-hybrid and glutathione S-transferase pull-down experiments show that SCN5A and syntrophin gamma 2 co-express and that the PDZ domain of syntrophin gamma 2 directly interacts with the C terminus of SCN5A. In native cells, disruption of the C terminus-syntrophin gamma 2 PDZ domain interaction using peptides directed against either region result in loss of mechanosensitivity. Co-transfection of syntrophin gamma 2 with SCN5A in HEK293 cells markedly shifts the activation kinetics of SCN5A and reduces the availability of Na(+) current. We propose that syntrophin gamma 2 is an essential Na(+) channel-interacting protein required for the full expression of the Na(+) current and that the SCN5A-syntrophin gamma 2 interaction determines mechanosensitivity and current availability.

K(+) currents are known to regulate the excitability of corpus cavernosum myocytes and therefore to play a role in the control of penile erection and detumescence. We used electrophysiology and molecular cloning techniques to identify ion channel proteins that contribute to K(+) currents in rabbit cavernosal myocytes. Currents were recorded from freshly isolated myocytes using whole-cell patch clamp techniques. Cavernosal myocytes expressed a delayed rectifier voltage-gated K(+) current that appeared to contribute to the resting membrane potential. This voltage-gated K(+) (K(v)) current was inhibited by the nonselective compounds 4-aminopyridine (1-10 mM), (+)-fenfluramine (10 micro M-1 mM), and Grammostola spatulata venom (1:100) in a dose-dependent and reversible fashion. Hanatoxin-1 (1 micro M), a selective Kv2 channel inhibitor, partially inhibited the current, but alpha-dendrotoxin (200 nM), a Kv1 channel blocker, had no effect. The nucleotide sequence of K(+) channel subunits was determined by polymerase chain reaction-based cloning techniques using RNA derived from cavernosal muscle strips and single identified myocytes. Molecular cloning techniques identified the full-length sequence of the rabbit ortholog of the Kv2.2 alpha subunit. This sequence contains 911 amino acid residues and is 92% identical to the recently revised human Kv2.2 sequence. Identified cavernosal myocytes of the type used in physiological recordings expressed Kv2.2 messenger RNA. We conclude that Kv2.2 alpha subunits contribute to whole-cell currents in rabbit canvernosal myocytes. Further, K(v) currents play a role in regulating membrane potential and hence excitability in rabbit cavernosal myocytes.