We have cloned cDNAs encoding the alpha- and beta-subunits of a large-conductance Ca(2+)-activated K+ channel (BK channel) from canine colonic smooth muscle (cslo-alpha and cslo-beta). Nucleotide sequence homology of cslo-alpha with mslo and dslo suggests that it is the canine homologue of these genes. The carboxy-terminal end of the protein is the most diverse between species, and we have also found alternative exons in cslo-alpha in this region. We have identified a unique splice site in the carboxy-terminal region of cslo-alpha, which we term site 5. Northern analysis demonstrates expression of both alpha- and beta-subunits in all canine vascular and visceral smooth muscles tested. Expression of alpha-1 alone and alpha + beta-subunit cRNA in Xenopus oocytes results in a Ca(2+)- and voltage-dependent conductance. The activity of alpha/beta-channels, measured as either changes in the voltage of half-maximal activation (V0.5) in open probability (NP0) or in the normalized conductance (G/Cmax), was more sensitive to [Ca2+]free than channels composed of the alpha-subunit alone. Neither alpha- nor alpha/beta-channels expressed in membrane patches of Xenopus oocytes were found to be regulated by protein kinase G.

Kv2.2, homologous to the shab family of Drosophila voltage-gated K+ channels, was isolated from human and canine colonic circular smooth muscle-derived mRNA. Northern hybridization analysis performed on RNA prepared from tissues and RT-PCR performed on RNA isolated from dispersed and selected smooth muscle cells demonstrate that Kv2.2 is expressed in smooth muscle cells found in all regions of the canine gastrointestinal (GI) tract and in several vascular tissues. Injection of Kv2.2 mRNA into Xenopus oocytes resulted in the expression of a slowly activating K+ current (time to half maximum current, 97 ± 8.6 ms) mediated by 15 pS (symmetrical K+) single channels. The current was inhibited by tetraethylammonium (IC50 = 2.6 mM), 4-aminopyridine (IC50 = 1.5 mM at +20 mV), and quinine (IC50 = 13.7 μM) and was insensitive to charybdotoxin. Low concentrations of quinine (1 μM) were used to preferentially block the slow component of the delayed rectifier current in native colonic myocytes. These data suggest that Kv2.2 may contribute to this current in native GI smooth muscle cells.

Atrial natriuretic factors, peptide hormones originally found in the heart, slowly but strongly elevate the level of cyclic GMP in primary astrocyte-rich cultures derived from brains of newborn rats or mice but not in neuron-rich cultures prepared from embryonic rat brain. In the absence of a phosphodiesterase inhibitor, a plateau level of cyclic GMP is obtained within 10 min. In the presence of the inhibitor 3-isobutyl-1-methylxanthine, the concentration of cyclic GMP continues to rise, even after 30 min. The elevation of the level of cyclic GMP in response to atrial natriuretic factor is much more pronounced in the rat cultures than the mouse cultures. Even at peptide concentrations of 1 microM, plateaus of the concentration-response curves are not yet reached. The potencies of the active peptides vary over a range of approximately 1.5 orders of magnitude, with atriopeptins II and III and auriculin A being the most potent ones. These results suggest (a) that atrial natriuretic factors may regulate functions of glial cells, most likely of astrocytes, in brain and (b) that such cultures may be useful tools in defining such astroglial functions.