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.

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.

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.

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.