Excitatory cholinergic responses in mouse primary bronchial smooth muscle require both Ca2+ entry via l-type Ca2+ channels and store operated Ca2+ entry via Orai channels.

Malfunctions in airway smooth muscle Ca2+-signalling leads to airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Ca2+-release from intracellular stores is important in mediating agonist-induced contractions, but the role of influx via l-type Ca2+ channels is controversial. We re-examined roles of the sarcoplasmic reticulum Ca2+ store, refilling of this store via store-operated Ca2+ entry (SOCE) and l-type Ca2+ channel pathways on carbachol (CCh, 0.1-10 µM)-induced contractions of mouse bronchial rings and intracellular Ca2+ signals of mouse bronchial myocytes. In tension experiments, the ryanodine receptor (RyR) blocker dantrolene (100 µM) reduced CCh-responses at all concentrations, with greater effects on sustained rather than initial components of contraction. 2-Aminoethoxydiphenyl borate (2-APB, 100 µM), in the presence of dantrolene, abolished CCh-responses, suggesting the sarcoplasmic reticulum Ca2+ store is essential for contraction. The SOCE blocker GSK-7975A (10 µM) reduced CCh-contractions, with greater effects at higher (e.g. 3 and 10 µM) CCh concentrations. Nifedipine (1 µM), abolished remaining contractions in GSK-7975A (10 µM). A similar pattern was observed on intracellular Ca2+-responses to 0.3 µM CCh, where GSK-7975A (10 µM) substantially reduced Ca2+ transients induced by CCh, and nifedipine (1 µM) abolished remaining responses. When nifedipine (1 µM) was applied alone it had less effect, reducing tension responses at all CCh concentrations by 25% - 50%, with greater effects at lower (e.g. 0.1 and 0.3 µM) CCh concentrations. When nifedipine (1 µM) was examined on the intracellular Ca2+-response to 0.3 µM CCh, it only modestly reduced Ca2+ signals, while GSK-7975A (10 µM) abolished remaining responses. In conclusion, Ca2+-influx from both SOCE and l-type Ca2+ channels contribute to excitatory cholinergic responses in mouse bronchi. The contribution of l-type Ca2+ channels was especially pronounced at lower doses of CCh, or when SOCE was blocked. This suggests l-type Ca2+ channels might be a potential target for bronchoconstriction under certain circumstances.

Effect of a novel BKCa opener on BKCa currents and contractility of the rabbit corpus cavernosum.

Large-conductance Ca(2+)-activated K(+) (BKCa) channels are thought to play a key role in the regulation of corpus cavernosum smooth muscle (CCSM) excitability. Few BKCa channel openers have been accepted for clinical development. The effect of the novel BKCa channel opener GoSlo-SR5-130 on electrical activity in isolated rabbit CCSM cells and mechanical activity in strips of rabbit CCSM was examined. Single-channel currents were observed in inside-out patches. These channels were sensitive to Ca(2+), blocked by penitrem A, and had a conductance of 291 ± 20 pS (n = 7). In the presence of GoSlo-SR5-130, the number of open BKCa channels increased. Using voltage-ramp protocols, GoSlo-SR5-130 caused currents to activate at more negative potentials in a concentration-dependent manner, shifting the half-maximal activation voltage potential to the left on the voltage axis. Therefore, BKCa channels were open within the physiological range of membrane potentials in the presence of GoSlo-SR5-130. GoSlo-SR5-130 also resulted in an increase in the activity of spontaneous transient outward currents in myocytes isolated from CCSM, and this effect was reversed by iberiotoxin. In current-clamp mode, GoSlo-SR5-130 hyperpolarized the cell membrane. Isometric tension recording of strips of rabbit corpus cavernosum showed that GoSlo-SR5-130 inhibited spontaneous contractions in a concentration-dependent manner. This effect was reversed in the presence of iberiotoxin, suggesting that GoSlo-SR5-130 exerts its effect through BKCa channels. These findings suggest that GoSlo-SR5-130 is an effective tool for the study of BKCa channels and that these channels can modulate CCSM activity and are possible targets for the treatment of erectile dysfunction.

Effects of the novel BK (KCa 1.1) channel opener GoSlo-SR-5-130 are dependent on the presence of BKß subunits.

BACKGROUND AND PURPOSE: GoSlo-SR compounds are efficacious BK (KCa 1.1) channel openers, but little is known about their mechanism of action or effect on bladder contractility. We examined the effects of two closely related compounds on BK currents and bladder contractions. EXPERIMENTAL APPROACH: A combination of electrophysiology, molecular biology and synthetic chemistry was used to examine the effects of two novel channel agonists on BK channels from bladder smooth muscle cells and in HEK cells expressing BKα alone or in combination with either ß1 or ß4 subunits. KEY RESULTS: GoSlo-SR-5-6 shifted the voltage required for half maximal activation (V1/2 ) of BK channels approximately -100 mV, irrespective of the presence of regulatory ß subunits. The deaminated derivative, GoSlo-SR-5-130, also shifted the activation V1/2 in smooth muscle cells by approximately -100 mV; however, this was reduced by ∼80% in HEK cells expressing only BKα subunits. When ß1 or ß4 subunits were co-expressed with BKα, efficacy was restored. GoSlo-SR-5-130 caused a concentration-dependent reduction in spontaneous bladder contraction amplitude and this was abolished by iberiotoxin, consistent with an effect on BK channels. CONCLUSIONS AND IMPLICATIONS: GoSlo-SR-5-130 required ß1 or ß4 subunits to mediate its full effects, whereas GoSlo-SR-5-6 worked equally well in the absence or presence of ß subunits. GoSlo-SR-5-130 inhibited spontaneous bladder contractions by activating BK channels. The novel BK channel opener, GoSlo-SR-5-130, is approximately fivefold more efficacious on BK channels with regulatory ß subunits and may be a useful scaffold in the development of drugs to treat diseases such as overactive bladder.

The cardiac sodium current Na(v)1.5 is functionally expressed in rabbit bronchial smooth muscle cells.

A collagenase-proteinase mixture was used to isolate airway smooth muscle cells (ASMC) from rabbit bronchi, and membrane currents were recorded using the whole cell patch-clamp technique. Stepping from -100 mV to a test potential of -40 mV evoked a fast voltage-dependent Na(+) current, sometimes with an amplitude of several nanoamperes. The current disappeared within 15 min of exposure to papain + DTT (n = 6). Comparison of the current in ASMC with current mediated by NaV1.5 α-subunits expressed in human embryonic kidney cells revealed similar voltage dependences of activation (V1/2 = -42 mV for NaV1.5) and sensitivities to TTX (IC50 = 1.1 and 1.2 µM for ASMC and NaV1.5, respectively). The current in ASMC was also blocked by lidocaine (IC50 = 160 µM). Although veratridine, an agonist of voltage-gated Na(+) channels, reduced the peak current by 33%, it slowed inactivation, resulting in a fourfold increase in sustained current (measured at 25 ms after onset). In current-clamp mode, veratridine prolonged evoked action potentials from 37 ± 9 to 1,053 ± 410 ms (n = 8). Primers for NaV1.2-1.9 were used to amplify mRNA from groups of ∼20 isolated ASMC and from whole bronchial tissue by RT-PCR. Transcripts for NaV1.2, NaV1.3, and NaV1.5-1.9 were detected in whole tissue, but only NaV1.2 and NaV1.5 were detected in single cells. We conclude that freshly dispersed rabbit ASMC express a fast voltage-gated Na(+) current that is mediated mainly by the NaV1.5 subtype.

Investigation of L-type Ca(2+) current in the aganglionic bowel segment in Hirschsprung's disease.

BACKGROUND: Studies on animal models of Hirschsprung's disease (HD) suggest that L-type Ca(2+) channels are down-regulated in the aganglionic bowel segment, however, this has yet to be confirmed in HD patients. The objective of this study was to test the hypothesis that L-type Ca(2+) current density is decreased in smooth muscle cells (SMC) obtained from the aganglionic bowel segment of patients with HD in comparison with those from the ganglionic segment. METHODS: Smooth muscle cells were freshly isolated from colon samples obtained from HD patients undergoing pull-through surgery. L-type Ca(2+) currents were recorded using the perforated patch configuration of the whole cell voltage clamp technique and the expression levels of CACNA1C transcripts (which encode L-type Ca(2+) channels) in the ganglionic and aganglionic bowel segments were compared using real-time quantitative PCR. KEY RESULTS: All SMC displayed robust currents that had activation/inactivation kinetics typical of L-type Ca(2+) current, were inhibited by nifedipine and enhanced by the L-type Ca(2+) channel agonists FPL 64176 and Bay K 8644. Moreover, FPL 64176 activated currents were also inhibited by nifedipine. However, there was no significant difference in L-type Ca(2+) current density, CACNA1C subunit expression or sensitivity to the pharmacological agents noted above, between SMC isolated from the ganglionic and aganglionic regions of the HD colon. CONCLUSIONS & INFERENCES: In contrast to studies on genetic animal models of HD, L-type Ca(2+) currents are not down-regulated in the aganglionic bowel segment of HD patients and are therefore unlikely to account for the impaired colonic peristalsis observed in these patients.

Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra.

We have characterized the native voltage-dependent K(+) (K(v)) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K(v)2.1 and K(v)2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEK(Kv2.1) and HEK(Kv2.2)). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch-clamp technique with K(+)-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca(2+)-activated K(+) (BK) currents and depolarized to +40 mV for 500 ms to evoke K(v) currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3-5) but were blocked by stromatoxin-1 (ScTx, IC(50) ∼130 nM), consistent with the idea that the currents were carried through K(v)2 channels. RNA was detected for K(v)2.1, K(v)2.2, and the silent subunit K(v)9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K(v)2 subtypes and K(v)9.3 in isolated RUSMC. HEK(Kv2.1) and HEK(Kv2.2) currents were blocked in a concentration-dependent manner by ScTx, with estimated IC(50) values of ∼150 nM (K(v)2.1, n = 5) and 70 nM (K(v)2.2, n = 6). The mean half-maximal voltage (V(1/2)) of inactivation of the USMC K(v) current was -56 ± 3 mV (n = 9). This was similar to the HEK(Kv2.1) current (-55 ± 3 mV, n = 13) but significantly different from the HEK(Kv2.2) currents (-30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K(v)2.1 channels contribute significantly to the K(v) current in RUSMC.

Cajal beyond the gut: interstitial cells in the urinary system--towards general regulatory mechanisms of smooth muscle contractility?

Interstitial cells of Cajal (ICC), similar to GI pacemakers have been identified throughout the urinary system. Although each part of the system serves a different function, ranging from peristalsis of the ureters, storage of urine by the bladder, and a sphincteric action by the urethra, they share a common mechanism in being able to generate phasic myogenic contractions. Even the urethra, often considered to be a 'tonic' smooth muscle, achieves an apparently sustained contraction by averaging numerous small asynchronous 'phasic' contractions. This activity can occur in the absence of any neural input, implying the presence of an intrinsic pacemaker. Intracellular microelectrode recordings from urethral muscle strips reveal electrical slow waves similar to those of the GI tract. To study this further, we isolated single cells from rabbit urethra and found not only smooth muscle cells (SMC), but a second cell type comprising -10% of the total. The latter cells were branched and non-contractile and closely resembled intestinal ICC. Electrophyiological studies revealed that, while the isolated smooth muscle cells were electrically quiescent, the 'ICC' fired electrical slow waves similar to those observed in the whole tissue. The basis of this difference was the presence of a large pacemaker current involving the activation of calcium-activated Cl channels by spontaneous intracellular Ca2+ waves. These, in turn, have been shown to be modulated by neurotransmitters such as nitric oxide, noradrenaline and ATP, thus providing a possible mechanism whereby neural regulation of the urethra, as well as spontaneous tone, may be mediated via ICC.

Ionic currents in intimal cultured synoviocytes from the rabbit.

Hyaluronan, a joint lubricant and regulator of synovial fluid content, is secreted by fibroblast-like synoviocytes lining the joint cavity, and secretion is greatly stimulated by Ca(2+)-dependent protein kinase C. This study aimed to define synoviocyte membrane currents and channels that may influence synoviocyte Ca(2+) dynamics. Resting membrane potential ranged from -30 mV to -66 mV (mean -45 ± 8.60 mV, n = 40). Input resistance ranged from 0.54 GΩ to 2.6 GΩ (mean 1.28 ± 0.57 GΩ; ν = 33). Cell capacitance averaged 97.97 ± 5.93 pF. Voltage clamp using C(s+) pipette solution yielded a transient inward current that disappeared in Ca(2+)-free solutions and was blocked by 1 µM nifedipine, indicating an L-type calcium current. The current was increased fourfold by the calcium channel activator FPL 64176 (300 nM). Using K(+) pipette solution, depolarizing steps positive to -40 mV evoked an outward current that showed kinetics and voltage dependence of activation and inactivation typical of the delayed rectifier potassium current. This was blocked by the nonspecific delayed rectifier blocker 4-aminopyridine. The synoviocytes expressed mRNA for four Kv1 subtypes (Kv1.1, Kv1.4, Kv1.5, and Kv1.6). Correolide (1 µM), margatoxin (100 nM), and α-dendrotoxin block these Kv1 subtypes, and all of these drugs significantly reduced synoviocyte outward current. The current was blocked most effectively by 50 nM κ-dendrotoxin, which is specific for channels containing a Kv1.1 subunit, indicating that Kv1.1 is critical, either as a homomultimeric channel or as a component of a heteromultimeric Kv1 channel. When 50 nM κ-dendrotoxin was added to current-clamped synoviocytes, the cells depolarized by >20 mV and this was accompanied by an increase in intracellular calcium concentration. Similarly, depolarization of the cells with high external potassium solution caused an increase in intracellular calcium, and this effect was greatly reduced by 1 µM nifedipine. In conclusion, fibroblast-like synoviocytes cultured from the inner synovium of the rabbit exhibit voltage-dependent inward and outward currents, including Ca(2+) currents. They thus express ion channels regulating membrane Ca(2+) permeability and electrochemical gradient. Since Ca(2+)-dependent kinases are major regulators of synovial hyaluronan secretion, the synoviocyte ion channels are likely to be important in the regulation of hyaluronan secretion.

The PI-PLC inhibitor U-73122 is a potent inhibitor of the SERCA pump in smooth muscle.

In this issue MacMillan and McCarron in 2010 demonstrated that the phospholipase C (PLC) inhibitor U-73122 can potently inhibit Ca(2+) release from isolated smooth muscle cells independent of its effect on PLC. Their data suggest that the PLC inhibitor can block the sarcoplasmic/endoplasmic reticulum calcium ATPase pump in smooth muscle and cast doubt on the reliability of U-73122 as the main pharmacological tool to assess the role of the phosphotidyl inositol-PLC pathway in cellular signalling.

Spontaneous electrical activity in sheep mesenteric lymphatics.

BACKGROUND: It has recently become apparent that the lymph pump is an electrical entity that rivals the heart in complexity. Many interesting currents have been demonstrated by voltage clamping isolated lymphatic smooth muscle cells, but until now the role of these currents in the intact syncitium has not been studied. METHODS AND RESULTS: Intracellular microelectrode recordings were made from smooth muscle of sheep mesenteric lymphatics to investigate the electrophysiological basis of lymphatic pumping. Approximately 50% of the vessels exhibited spontaneous electrical activity, varying from regular oscillations in membrane potential to spike complexes. Spike complexes generally consisted of one or more action potentials superimposed on a slower depolarization or 'plateau' phase and were often preceded by a slow diastolic depolarization or 'pre-potential'. Norepinephrine (5 microM) induced depolarizing events in quiescent preparations. Both agonist-induced oscillations and spike complexes were attenuated or completely abolished by 2-aminoethoxydiphenyl borate (2-APB); 10-100 microM). Cesium (1 mM) reduced the frequency of spontaneous firing by approximately 30% by flattening the pre-potential phase. In addition to having a negative inotropic effect, 10 mM Cs(+) also caused gradual membrane depolarization and prolonged the plateau. 1 microM nifedipine abolished spontaneous events while tetrodotoxin (TTX; 0.5-1 muM) decreased the amplitude and maximum dV/dt of the spike upstroke or stopped activity completely. Spontaneously active segments of lymphatic vessel were inhibited by the chloride channel blocker, anthracene-9-carboxylic acid (9-AC; 250 microM - 1 mM) suggesting that I(Cl(Ca)) plays a significant role in the generation of spontaneous activity in this tissue. Penitrem-A (0.1 microM) did not affect resting membrane potential but increased action potential amplitude and prolonged the plateau, suggesting that calcium-activated potassium current does not make a significant contribution to resting membrane conductance but is important in membrane repolarization following calcium influx during the action potential. In contrast 4-aminopyridine (4-AP; 5 microM) caused significant membrane depolarization, suggesting the existence of an active 4-AP-sensitive current at rest. CONCLUSIONS: These results demonstrate that the currents found in isolated voltage-clamped cells from sheep mesenteric lymphatics do play a significant role in the shaping of spontaneous electrical activity of the intact syncitium.

Ca2+ signalling in urethral interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) in the urethra have been proposed as specialized pacemakers that are involved in the generation of urethral tone and therefore the maintenance of urinary continence. Recent studies on freshly dispersed ICC from the urethra of rabbits have demonstrated that pacemaker activity in urethra ICC is characterized by spontaneous transient depolarizations (STDs) under current clamp and spontaneous transient inward currents (STICs) under voltage clamp. When these events were simultaneously recorded with changes in intracellular Ca(2+) (using a Nipkow spinning disk confocal microscope) they were found to be associated with global Ca(2+) oscillations. In this short review we will consider some of these recent findings regarding the contribution of intracellular Ca(2+) stores and Ca(2+) influx to the generation of pacemaker activity in urethral ICC with particular emphasis on the contribution of reverse Na(+)/Ca(2+) exchange (NCX).

Interstitial cells of Cajal in the urethra.

The smooth muscle layer of the urethra generates spontaneous myogenic tone that is thought to make a major contribution to urinary continence. The mechanisms underlying generation of tone remain unclear, however recent studies from our laboratory highlighted a role for a specialised population of pacemaker cells which we originally referred to as interstitial cells (IC) and now term ICC. Urethra ICC possess an electrical pacemaker mechanism characterised by rhythmic activation of Ca(2+)-activated Cl(-) channels leading to spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarisations (STDs) under current clamp conditions. Both STICS and STDs are now known to be associated with spontaneous Ca(2+) oscillations that result from a complex interplay between release of Ca(2+) from intracellular stores and Ca(2+) influx across the plasma membrane. In this review we will consider some of the precise mechanisms involved in the generation of pacemaker activity and discuss how these are modulated by excitatory and inhibitory neurotransmitters.

Contribution of reverse Na+-Ca2+ exchange to spontaneous activity in interstitial cells of Cajal in the rabbit urethra.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+ oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+ concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+-Ca2+ exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+ were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+ concentration [Na+]o from 130 to 13 mM, the frequency of global Ca2+ oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB-R7943 and SEA0400 decreased the frequency of Ca2+ oscillations and STICs. Application of caffeine (10 mM) and noradrenaline (10 microM) induced transient Ca2+-activated chloride currents (I(ClCa)) at -60 mV due to release of Ca2+ from ryanodine- and inositol trisphosphate (IP3)-sensitive Ca2+ stores, respectively, but these responses were not blocked by KB-R7943 or SEA0400 suggesting that neither drug blocked Ca2+-activated chloride channels or Ca2+ release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration-dependent fashion. Finally, single cell RT-PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+-Ca2+ exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+ entry via reverse NCX.

Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves.

In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch-clamp technique. The nitric oxide donor, DEA-NO, the soluble guanylyl cyclase activator YC-1 and the membrane-permeant analogue of cGMP, 8-Br-cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current-clamp and voltage-clamp conditions, respectively. Caffeine-evoked Cl- currents were unaltered in the presence of SP-8-Br-PET-cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl- channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline-evoked Cl- currents were attenuated by SP-8-Br-PET-cGMPs, suggesting that activation of cGMP-dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R). When urethral interstitial cells (ICC) were loaded with Fluo4-AM (2 microm), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA-NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2-APB (100 microm). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.

Calcium oscillations in interstitial cells of the rabbit urethra.

Measurements were made (using fast confocal microscopy) of intracellular Ca2+ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca2+ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with D-myo-inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release using 100 microm 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca2+, and frequency was directly proportional to the external Ca2+ concentration. Frequency of Ca2+ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca2+ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca2+ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca2+ concentration and that conversion of the primary oscillation to a propagated Ca2+ wave depends upon IP3-induced Ca2+ release.

Characterization of T-type calcium current and its contribution to electrical activity in rabbit urethra.

Rabbit urethral smooth muscle cells were studied at 37 degrees C by using the amphotericin B perforated-patch configuration of the patch-clamp technique, using Cs(+)-rich pipette solutions. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca(2+) currents, were recorded. Fitting steady-state inactivation curves for the L current with a Boltzmann equation yielded a V(1/2) of -41 +/- 3 mV. In contrast, the T current inactivated with a V(1/2) of -76 +/- 2 mV. The L currents were reduced by nifedipine (IC(50) = 225 +/- 84 nM), Ni(2+) (IC(50) = 324 +/- 74 microM), and mibefradil (IC(50) = 2.6 +/- 1.1 microM) but were enhanced when external Ca(2+) was substituted with Ba(2+). The T current was little affected by nifedipine at concentrations <300 nM but was increased in amplitude when external Ca(2+) was substituted with Ba(2+). Both Ni(2+) and mibefradil reduced the T current with an IC(50) = 7 +/- 1 microM and approximately 40 nM, respectively. Spontaneous electrical activity recorded with intracellular electrodes from strips of rabbit urethra consisted of complexes comprising a series of spikes superimposed on a slow spontaneous depolarization (SD). Inhibition of T current reduced the frequency of these SDs but had no effect on either the number of spikes per complex or the amplitude of the spikes. In contrast, application of nifedipine failed to significantly alter the frequency of the SD but reduced the number and amplitude of the spikes in each complex.

Modulation of spontaneous Ca2+-activated Cl- currents in the rabbit corpus cavernosum by the nitric oxide-cGMP pathway.

The whole-cell perforated patch clamp technique was used to study membrane currents in isolated rabbit corpus cavernosum smooth muscle cells. Depolarization from -80 mV to the range -40 to -10 mV evoked a nifedipine-sensitive Ca(2+) current that was followed by a slower inward current that activated over several hundred milliseconds. The slow current reversed near the Cl(-) equilibrium potential (E(Cl)) and was reduced by anthracene-9-carboxylic acid (A9C; 1 mm) and niflumic acid (100 microm), suggesting that it was a Ca(2+)-activated Cl(-) current. When held constantly at -60 mV, over 70% of cells fired spontaneous transient inward currents (STICs), the amplitudes of which were reduced by A9C and niflumic acid. STICs reversed near E(Cl) in a symmetrical Cl(-) gradient and when [Cl(-)](o) was substituted with glutamate or I(-), the reversal potential shifted to more positive or more negative values, respectively, confirming that STICs were mediated by Cl(-) channels. STICS were also blocked by cyclopiazonic acid, 2-aminoethoxydiphenyl borate (2-APB) and 2-nitro-4-carboxyl-N,N-diphenylcarbamate (NCDC), suggesting that they depended on IP(3)-mediated Ca(2+)-release from the sarcoplasmic reticulum. Modulation by the NO-cGMP pathway was investigated by applying nitrosocysteine, 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1), and 8-bromo cGMP, all three of which abolished STIC activity. YC-1 also reduced noradrenaline-evoked inward currents, but had no effect on similar currents evoked by caffeine, suggesting that cGMP selectively inhibited IP(3)-mediated Ca(2+) release. We propose that Ca(2+)-activated Cl(-) currents underlie detumescent tone in the corpus cavernosum, and that modulation of this mechanism by the NO-cGMP pathway is important during penile erection.

T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra.

The purpose of the present study was to characterise Ca2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37 degrees C using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs+-rich pipette solutions to block K+ currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V1/2 of -45+/-5 mV. In contrast, the T current inactivated with a V1/2 of -80+/-3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50=159+/-54 nM) and Ni2+ (ED50=65+/-16 microM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by TTX, reduction in external Na+, application of nifedipine at concentrations below 300 nM or substitution of external Ca2+ with Ba2+, but was reduced by Ni2+ with an ED50 of 6+/-1 microM. When cells were stepped from -100 to -30 mV in Ca2+-free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg2+ with an ED50 of 32+/-16 microM. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents.

Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells.

Interstitial cells were isolated from strips of rabbit urethra for study using the amphotericin B perforated-patch technique. Depolarizing steps to -30 mV or greater activated a Ca2+ current (ICa), followed by a Ca2+-activated Cl- current, and, on stepping back to -80 mV, large Cl- tail currents were observed. Both currents were abolished when the cells were superfused with Ca2+-free bath solution, suggesting that Ca2+ influx was necessary for activation of the Cl- current. The Cl- current was also abolished when Ba2+ was substituted for Ca2+ in the bath or the cell was dialyzed with EGTA (2 mM). The Cl- current was also reduced by cyclopiazonic acid, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), and xestospongin C, suggesting that Ca2+-induced Ca2+ release (CICR) involving both ryanodine and inositol 1,4,5-trisphosphate receptors contributes to its activation.