Recombinant human NT-proBNP (>95%)

The study of ion channels has relied heavily on the use of pharmacological blocking agents. However, many of these agents have multiple effects, which may compromise interpretation of results when the affected mechanisms/pathways mediate similar functions. Volume regulated anion channels (VRAC) and connexin hemichannels can both mediate the release of glutamate and taurine, although these channels have distinct activation stimuli and hemichannels, but not VRAC, are permeable to Lucifer Yellow (LY). It has been reported that some anion channel blockers may inhibit connexin hemichannels. We further examined the effects of classic gap junction/hemichannel blockers and anion channel blockers on these channels. The typical VRAC blockers, NPPB, LAA-94, and tamoxifen blocked low divalent cation-induced glutamate and taurine release and LY loading, presumed due to hemichannel opening. The blocking action of these compounds on hemichannels was concentration dependent and fell within the same range where the drugs classically block VRACs. Conversely, carbenoxolone (CBX), the most widely used gap junction/hemichannel blocker, was an effective blocker of VRAC-mediated glutamate and taurine release, and blocked these channels at similar concentrations at which it blocked hemichannels. The CBX effect on VRACs was verified using astrocytes from connexin 43 knock out (Cx43 KO) animals. In these cells, the hypotonic induced amino acid flux was retained whereas the low divalent cation solution-induced flux was lost. These results extend our knowledge about "cross-inhibition" of VRACs and gap junctions/hemichannels by certain pharmacological agents. Given the overlap in function of these two types of channels, great care must be exerted in using pharmacological blockers to identify one channel from the other. (C) 2008 Wiley-Liss, Inc.

Soluble adenylyl cyclase (sAC) has been hypothesized to play a role in insulin secretion. The present study aimed to investigate the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), volume-regulated anion channels (VRACs) and the electrogenic sodium bicarbonate (Na+-HCO3-) cotransporter, NBCe1, in the regulation of nutrient- and hypotonicity-induced insulin release from rat pancreatic islets and tumoral insulin-producing BRIN-BD11 cells. In the islets, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 5-chloro-2-hydroxy-3-(thiophene-2-carbonyl) indole-l-carboxamide (tenidap) reduced glucose-stimulated insulin release, however, only NPPB suppressed the enhancing action of cAMP analogs upon such a release. Insulin output from the BRIN-BD11 cells was stimulated by 2-ketoisocaproate (KIC) or extracellular hypoosmolarity. cAMP analogs and 3-isobutyl-1-methylxanthine increased the insulin output recorded in the isotonic medium to a greater relative extent than that in the hypotonic medium. The secretory response to KIC or hypotonicity was inhibited by NPPB or tenidap, which both also opposed the enhancing action of cAMP analogs. Inhibitors of mitogen-activated protein (MAP) kinase decreased insulin output in isotonic and hypotonic media. The inhibitor of sAC, 2-hydroxyestriol, caused only a modest inhibition of insulin release, whether in the isotonic or hypotonic medium, even when tested at a concentration of 100 mu M. The omission of NaHCO3 markedly decreased the secretory response to KIC or extracellular hypotonicity. The omission of Na+ suppressed the secretory response to extracellular hypotonicity. The observations of the present study do not support the hypothesis of a major role for sAC in the regulation of insulin release.