Protein kinase modulation of GABA, currents in rabbit retinal rod bipolar cells

1. Protein kinase modulation of gamma-aminobutyric acid-A (GABA(A))- and glycine-activated Cl- currents in freshly dissociated, morphologically identified rabbit retinal rod bipolar cells was studied under voltage clamp with the use of the whole cell patch-clamp technique, Responses to pulses of GABA and glycine were monitored before, during, and after application of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC) activators, inactive analogues, and inhibitors. 2. Bath perfusion with either forskolin, an adenylate cyclase activator, or its inactive analogue, 1,9 dideoxyforskolin, reduced the GABA-activated Cl- currents by 30-50%; coapplication of N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-8), a PKA inhibitor, did not prevent the forskolin effects. The: membrane-permeable cAMP analogues, 8-bromo-cAMP and 8-(4-Chlorophenylthio)-cAMP, and intracellularly dialyzed cAMP, did not modulate either the GABA- or glycine-activated Cl- current. Perfusion of the phosphodiesterase inhibitor 3-isobutyl-L-methylxantine (IBMX) had no direct effect on the GABA-activated current and did not alter the results with cAMP or Its membrane-permeable analogues. Collectively, these results make it very unlikely that PKA represents an important mechanism of either GABA(A) or glycine channel modulation in the rabbit rod bipolar cell. 3. Although the isoquinoline sulfonamide protein kinase inhibitor H-8 was without discernible effect, the related compounds 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochlorine (H-7) and N-(2-Aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9) both dramatically reduced the GABA response. H-7 also strongly reduced the response to glycine, whereas II-X had no effect and H-9 had an intermediate effect. Because only certain members of this inhibitor class of agents proved effective, and their effectiveness appeared unrelated to the established activity profiles, these agents probably inhibit the Cl- currents in a phosphorylation-independent manner. Direct interaction of these inhibitors with binding sites in the GABA(A) receptor-channel complex has been previously reported in some other preparations. 4. The phorbol ester and PKC activator phorbol 12,13 dibutyrate (PDB) led to a 35-55% reduction in the GABA-actiwated Cl- current of the rod bipolar cell. The broad-spectrum kinase inhibitor staurosporine, and the more PKC-specific inhibitor calphostin C, had no direct effect on GABA responses, but prevented Cl- current reduction when coapplied with PDB. Phorbol 12-myristate 13-acetate (PMA) reduced the GABA-activated current in a fashion very similar to PDB. Staurosporine and calphostin C blocked the PMA effect, No reduction of Cl- current was seen with the inactive analogue, 4-alpha-PMA, used as a control for PKC-independent phorbol ester effects. 5. PDB effectively reduced the GABA-activated Cl- current of the rod bipolar cell at low concentrations, whereas PMA had a diminished effect at low concentrations. This is consistent with the reported concentration-dependent abilities of these agents to promote translocation of PKC-alpha immunoreactivity from the membrane to the cytosolic compartment in the rabbit retinal rod bipolar cell. Collectively, the data from phorbol esters, inactive analogues, and kinase inhibitors support the existence of a PKC-mediated mechanism for GABA-activated Cl- current reduction in these cells. 6. The naphthalenesulfonamide PKC activator N-(n-Heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) also potently and reversibly reduced the GABA-activated current. Staurosporine and calphostin C eliminated this effect When the nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTP-gamma-S) replaced GTP in the recording pipette, the SC-10-mediated GABA current reduction became irreversible. When guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) was substituted for GTP, the SC-10 effect was blocked. GTP-gamma-S and GDP-beta-S had no independent effects on the GABA-activated current. These data are consistent with the involvement of G proteins in the PKC-mediated GABA(A) current modulation, suggesting that they may play a role ''downstream'' from the initial transduction event. However, the synthetic membrane-permeable diacyglycerol analogue 1,2-oleoylacetyl-glycerol did not modulate the GABA-activated Cl- current of the rod bipolar cell. 7. We have previously reported that the neuropeptide vasoactive intestinal peptide (VIP) reduced the GABA(A) current of the rod bipolar cell. Experiments were conducted to determine whether this effect was mediated by the identified PKC mechanism. GTP-gamma-S tended to prolong the VIP effect, but did not make it irreversible; GDP-beta-S tended to reduce the VIP effect, but did not prevent it. Thus, although the impact of GTP analogues on the VIP effect was in the same direction as with PKC activators, it was both smaller and less consistent. Furthermore, the potent and reliable PKC inhibitor, calphostin C, failed to prevent GABA current reduction by VIP in almost half of the cells tested. These data make it unlikely that PKC represents the sole or principal mechanism of VIP action in the rabbit retinal rod bipolar cell.