Effects of Phenylephrine on Spontaneous Activity and L-Type Ca2+ Current in Isolated Corpus Cavernosum Myocytes

Abstract

Introduction Norepinephrine is important in maintaining detumescent tone in the corpus cavernosum, although the mechanism is incompletely understood. As α-adrenoceptor-induced tone is antagonized by L-type Ca2+ channel blockers, it is usually assumed that direct modulation of this current is involved. However, the effects of α-adrenoceptor agonists have never been directly examined on L-type current in corpus cavernosum myocytes (CCSMC), leaving open other possibilities. In particular, CCSMC are now known to develop spontaneous tone via a pacemaker mechanism involving spontaneous Ca2+ waves that activate Cl- currents, causing depolarization and voltage-dependent activation of L-type channels. We hypothesized that phenylephrine modulates tone via this system, rather than by directly activating L-type channels. Aims Examine in freshly isolated CCSMC the effect of phenylephrine on: (i) spontaneous Cl- currents and depolarizations; (ii) cytosolic Ca2+ waves; and (iii) L-type current. Methods CCSMC were enzymatically dispersed from male New Zealand White rabbits for patch clamp recording and real time Ca2+ imaging. Main Outcome Measures Spontaneous Cl- currents, spontaneous depolarizations, cytosolic Ca2+ and L-type current. Results Phenylephrine enhanced the amplitude and frequency of spontaneous Cl- currents, increased the duration and frequency of spontaneous depolarizations and increased the frequency of spontaneous Ca2+ waves. These effects were blocked by 2-aminoethoxy diphenylborate (2-APB), suggesting that they were mediated by IP3-induced Ca2+ release from intracellular stores. In contrast, 2-APB had no effect on Ca2+ transients evoked by releasing stored Ca2+ with caffeine, suggesting that it had little effect on store Ca2+ content. Phenylephrine depressed L-type current by around 30%. This effect was removed by blocking with 2-APB. Notably, phenylephrine failed to enhance the current, even in the presence of 2-APB. Furthermore, the phorbol ester, phorbol 12-myristate 13-acetate, had no effect on L-type current. Conclusion Phenylephrine effects on the corpus cavernosum are mediated by modulation of the spontaneous pacemaker mechanism, rather than by direct stimulation of L-type channels.

ATP Evokes Inward Currents in Corpus Cavernosum Myocytes

“…In the present study, we have provided the first functional evidence that ATP induces an inward cationic current in corpus cavernosum myocytes. Ion substitution experiments showed that this is distinct from the inward current mediated by I ClCa and therefore different from the spontaneous inward currents generated by these cells [17,18] and from the previously reported inward currents generated by phenylephrine [19]. As P2X receptors are known to be ligand‐gated cation channels [6], it was highly likely that the cation current described here was mediated by P2X receptors.…”

“…When isolated corpus cavernosum smooth cells were held under voltage clamp at −60 mV, they often exhibited spontaneous Ca 2+ ‐activated Cl − currents (I ClCa , Figure A–C) as described previously [18,19]. In 84% of cells, a 20‐second application of ATP (20 μM) evoked a single transient inward current that decayed despite the continued presence of ATP (Figure A).…”

“…As P2X receptors are known to be ligand‐gated cation channels, it was of interest to identify the main ion species responsible for the inward current. As rabbit corpus cavernosum smooth muscle cells (CCSMC) have previously been shown to express an I ClCa , and this can be activated as a result of intracellular Ca 2+ ‐release in response to stimulation by agonists such as phenylephrine or caffeine [18,19], it was important to distinguish between I ClCa and any potential cation current (I cat ) mediated by P2X receptors. Figure A shows a comparison of the effect of ATP and caffeine under the same conditions as all of experiments described so far (holding potential = −60 mV, E Cl = 0 mV, extracellular [Na + ] = 130 mM).…”

“…Patch clamp recordings were made as described previously [18,19] using the amphotericin B perforated patch method, where electrical access between the pipette and cell interior is achieved by inclusion of the pore forming compound amphotericin B (600 μg/mL) in the pipette solution [20]. Voltage clamp commands were delivered via an Axopatch 1D patch clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) connected to a Digidata 1322A AD/DA converter (Molecular Devices) interfaced to a computer running pClamp software (Molecular Devices).…”

“…Intracellular Ca 2+ was imaged as described previously [19]. Cells were plated and incubated in 0.4 μM fluo‐4AM (Molecular Probes, Life Technologies, Grand Island, NY, USA) in “Hanks Ca 2+ ‐free solution” (see below) but with added 100 μM Ca 2+ for 6–8 minutes at 20°C.…”