Reversal of the epinephrine stimulation of Cl- transport in bullfrog cornea by phorbol esters.
The effects of protein kinase C (PKC) stimulation with phorbol dibutyrate, PDBu, were determined on transepithelial net Cl- transport (Isc) across the isolated bullfrog cornea. Between 1 nM and 1 microM, PDBu had no effect on either the Isc or the conductance, gt, as well as the membrane voltage (Vsc) but it decreased the fractional apical membrane resistance (fR0). PDBu caused a dose-dependent decline and reversal of the stimulatory effect of the mixed alpha and beta adrenergic agonist, epinephrine, on the Isc. With 1 microM PDBu, 100 microM epinephrine decreased the Isc by 20% below its control value. As without PDBu, epinephrine decreased fR0 and depolarized the Vsc. Evidence that this reversal by PDBu stems from a selective stimulation of PKC is: (1) neither its vehicle nor the inactive phorbol analog, 4 alpha-phorbol didecanoate, PDD (1 microM), decreased either the fR0 or altered the stimulatory effect of epinephrine on the Isc. (2) After preincubation with 30 microM H-7, which inhibited PKC stimulation in subcellular membrane and cytosolic fractions of frog corneal epithelium, the stimulatory effect of epinephrine on the Isc was unchanged by 1 microM PDBu. Preincubation with PDBu completely inhibited the stimulatory effect of the beta adrenergic agonist, isoproterenol (10 microM), on the Isc but did not affect the stimulatory effect of 1 microM forskolin on the Isc, indicating that PKC stimulation results in uncoupling of adrenergic regulation of adenylate cyclase activity. Epinephrine did not reverse the Isc in corneas that were preincubated with either the alpha 2- -adrenergic antagonist, yohimbine (100 microM), or the general alpha adrenergic antagonist, phenoxybenzamine (100 microM). With yohimbine, epinephrine had no effect on the Isc whereas with phenoxybenzamine the stimulation by epinephrine was fully restored. These effects suggest that stimulation of PKC is dependent upon the stimulation of alpha 1-adrenergic receptors by epinephrine as well as the presence of PDBu. Reversal and stimulation of the Isc by epinephrine were both associated with a depolarization of the Vsc and similar decreases in fR0. These similar effects on fR0 suggest that PKC stimulation as well as alpha 2 adrenergic activation by epinephrine are associated with increases in basolateral membrane resistance.
Reinach
P S
PS
Department of Ophthalmology, Washington University School of Medicine, St. Louis, MO 63110.
Thurman
C
C
Bazan
H E
HE
Bazan
N G
NG
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D004594:Electrophysiology; D004837:Epinephrine / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D011892:Rana catesbeiana
D002712:Chlorides; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D004837:Epinephrine