Role of K+ and Ca2+ fluxes in the cerebroarterial vasoactive effects of sildenafil.

The aim of this study was to assess the role of K(+) and Ca(2+) fluxes in the cerebroarterial vasoactive effects of the phosphodiesterase-5 inhibitor sildenafil. We used isolated rabbit basilar arteries to assess the effects of extracellular K(+) raising on sildenafil-induced vasodilatation, and studied the pharmacological interaction of sildenafil with selective modulators of membrane K(+) and Ca(2+) channels. Expression of Kv1 subunits of K(+) channels was assessed at messenger and protein levels. Parallel experiments were carried out with zaprinast for comparison. Sildenafil (10 nM-0.1 mM) induced concentration-dependent relaxation of endothelin-1 (10 nM)-precontracted arteries, which was partially inhibited by depolarization with KCl (50 mM), 3 mM tetraethylammonium (non-selective K(+) channel blocker) or 1 mM aminopyridine (inhibitor of K(v) channels), but not by 1 microM glibenclamide (inhibitor of K(ATP) channels) or 50 nM iberiotoxin (inhibitor of K(Ca) channels). Arterial smooth muscle expressed messengers for Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6, and proteins of Kv1.1, Kv1.2 and Kv1.4. CaCl(2) (10 microM- 10 mM) induced concentration-dependent contraction in Ca(2+)-free, depolarizing (50 mM KCl) medium. Sildenafil (0.1-100 microM) produced reversible concentration-dependent inhibition of the response to CaCl(2), which was completely abolished by the highest sildenafil concentration. By contrast, only 100 microM zaprinast inhibited the response to CaCl(2). The L-type Ca(2+) channel activator Bay K 8644 (0.1 nM-1 microM) induced concentration-dependent potentiation of the response to CaCl(2) inhibited by 100 microM sildenafil. Moreover, Bay K 8644 (0.1 nM-1 microM) induced concentration-dependent contraction in slightly depolarizing (15 mM) medium, which was inhibited to the same extent and in a concentration-dependent way by sildenafil (0.1-100 microM) and zaprinast (1 or 100 microM). These results show that sildenafil relaxes the rabbit basilar artery by increasing K(+) efflux through K(v) channels, which in turn may affect Ca(2+) signalling. Expression of Kv1 subunits involved in this pharmacological effect occurs at the messenger and, in some cases, at the protein level. In addition to this phosphodiesterase-5-related effect, sildenafil and zaprinast inhibit cerebroarterial vasoconstriction at least in part by directly blocking L-type Ca(2+) channels, although a decrease in the sensitivity of the contractile apparatus to Ca(2+) can not be discarded.

Distribution of Kv1-like potassium channels in the electromotor and electrosensory systems of the weakly electric fish Apteronotus leptorhynchus.

The electromotor and electrosensory systems of the weakly electric fish Apteronotus leptorhynchus are model systems for studying mechanisms of high-frequency motor pattern generation and sensory processing. Voltage-dependent ionic currents, including low-threshold potassium currents, influence excitability of neurons in these circuits and thereby regulate motor output and sensory filtering. Although Kv1-like potassium channels are likely to carry low-threshold potassium currents in electromotor and electrosensory neurons, the distribution of Kv1 alpha subunits in A. leptorhynchus is unknown. In this study, we used immunohistochemistry with six different antibodies raised against specific mammalian Kv1 alpha subunits (Kv1.1-Kv1.6) to characterize the distribution of Kv1-like channels in electromotor and electrosensory structures. Each Kv1 antibody labeled a distinct subset of neurons, fibers, and/or dendrites in electromotor and electrosensory nuclei. Kv1-like immunoreactivity in the electrosensory lateral line lobe (ELL) and pacemaker nucleus are particularly relevant in light of previous studies suggesting that potassium currents carried by Kv1 channels regulate neuronal excitability in these regions. Immunoreactivity of pyramidal cells in the ELL with several Kv1 antibodies is consistent with Kv1 channels carrying low-threshold outward currents that regulate spike waveform in these cells (Fernandez et al., J Neurosci 2005;25:363-371). Similarly, Kv1-like immunoreactivity in the pacemaker nucleus is consistent with a role of Kv1 channels in spontaneous high-frequency firing in pacemaker neurons. Robust Kv1-like immunoreactivity in several other structures, including the dorsal torus semicircularis, tuberous electroreceptors, and the electric organ, indicates that Kv1 channels are broadly expressed and are likely to contribute significantly to generating the electric organ discharge and processing electrosensory inputs.

Polar-localised poplar K+ channel capable of controlling electrical properties of wood-forming cells.

In previous studies, we have shown that annual expression profiles of cambial and wood tissue with respect to the Shaker K+ channel PTORK correlate with cambial activity. To follow PTORK-gene activity on the cellular level, we isolated the respective promoter regions and generated transgenic Arabidopsis plants expressing the GUS gene under the control of the PTORK promoter. Cross-sections of petioles showed PTORK-driven signals predominantly in the xylem parenchyma surrounding the vessels and in the phloem. Antibodies raised against a unique N-terminal region of PTORK in histo-immunochemical analyses recognised this K+-release channel in growth-active poplar plants only. PTORK labelling was found in differentiating xylem cells (young fibres) and mature xylem (vessel-associated cells of the ray parenchyma). Patch-clamp measurements on fibre cell protoplasts, derived from young poplar twigs, identified outward-rectifying K+ channels as the major K+ conductance of this cell type, which resembled the biophysical properties of PTORK when expressed in Xenopus oocytes.