BETA-ADRENERGIC STIMULATION OF NA- COTRANSPORT IN FETAL NONPIGMENTED CILIARY EPITHELIAL-CELLS(,K+,CL)

Purpose. The effects of adrenergic agonists and antagonists on Na+,K+, Cl(-)cotransport in fetal human nonpigmented ciliary epithelial (NPE) cells were investigated. Methods. Rb-86(+) as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive Rb-86(+) uptake in cultured NPE monolayers. Cyclic adenosine monophosphate (cAMP) format ion in NPE cells was determined by radioimmunoassay. Results. 1 mu M i soproterenol caused a 1.65-fold stimulation in Na+,K+,Cl(-)cotransport measured as bumetanide-sensitive, ouabain-insensitive Rb-86(+) uptake . The half-maximal concentration for this effect was 6.4 nM, with maxi mal stimulation at 100 nM isoproterenol. Epinephrine stimulated Na+,K,Cl(-)cotransport similarly to isoproterenol, whereas norepinephrine s timulated at much higher concentrations (half-maximal effective concen tration = 1.4 mu M). Stimulation of Na+,K+,Cl(-)cotransport by 1 mu M isoproterenol was inhibited completely by the beta(2)-adrenergic antag onist ICI-118,551 at 100 nM, with a half-maximal inhibitory concentrat ion of 5 nM. Neither atenolol, a beta(1)-specific adrenergic antagonis t, prazosin, an alpha(1)-adrenergic antagonist, nor yohimbine, an alph a(2)-specific antagonist, was as effective. These four antagonists inh ibited isoproterenol-stimulated cAMP formation with potencies similar to those observed against stimulated Na+,K+,Cl(-)cotransport. The hypo tensive adrenergic antagonists timolol, propranolol, and betaxolol als o inhibited Na+,K+,Cl(-)cotransport stimulated by isoproterenol in the order timolol > propranolol > betaxolol. Na+,K+,Cl(-)cotransport coul d be maintained in a stimulated state for at least 2 hours in the pres ence of agonist, but activity returned to basal levels within 20 minut es of isoproterenol removal. Adrenergic stimulation of Na+,K+,Cl(-)cot ransport was blocked 80% to 85% by 70 mu M H-89, a protein kinase A in hibitor. Conclusions. These data suggest that beta(2)-adrenergic recep tor activation results in increased cAMP formation and sustained stimu lation of Na+,K+,Cl(-)cotransport in fetal human NPE cells. Protein ki nase A activation is required for maximal stimulation of Na+,K+,Cl(-)c otransport by adrenergic agonists.