PROTEIN-KINASE MODULATION OF GABA, CURRENTS IN RABBIT RETINAL ROD BIPOLAR CELLS

1. Protein kinase modulation of gamma-aminobutyric acid-A (GABA(A))- a nd glycine-activated Cl- currents in freshly dissociated, morphologica lly identified rabbit retinal rod bipolar cells was studied under volt age clamp with the use of the whole cell patch-clamp technique, Respon ses to pulses of GABA and glycine were monitored before, during, and a fter application of adenosine 3',5'-cyclic monophosphate (cAMP)-depend ent protein kinase (PKA) and protein kinase C (PKC) activators, inacti ve analogues, and inhibitors. 2. Bath perfusion with either forskolin, an adenylate cyclase activator, or its inactive analogue, 1,9 dideoxy forskolin, reduced the GABA-activated Cl- currents by 30-50%; coapplic ation of N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide hydrochlor ide (H-8), a PKA inhibitor, did not prevent the forskolin effects. The : membrane-permeable cAMP analogues, 8-bromo-cAMP and 8-(4-Chloropheny lthio)-cAMP, and intracellularly dialyzed cAMP, did not modulate eithe r the GABA- or glycine-activated Cl- current. Perfusion of the phospho diesterase inhibitor 3-isobutyl-L-methylxantine (IBMX) had no direct e ffect 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 e ither GABA(A) or glycine channel modulation in the rabbit rod bipolar cell. 3. Although the isoquinoline sulfonamide protein kinase inhibito r H-8 was without discernible effect, the related compounds 1-(5-Isoqu inolinesulfonyl)-2-methylpiperazine dihydrochlorine (H-7) and N-(2-Ami noethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9) both dramatic ally 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 ef fect. Because only certain members of this inhibitor class of agents p roved effective, and their effectiveness appeared unrelated to the est ablished activity profiles, these agents probably inhibit the Cl- curr ents in a phosphorylation-independent manner. Direct interaction of th ese inhibitors with binding sites in the GABA(A) receptor-channel comp lex has been previously reported in some other preparations. 4. The ph orbol eater 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 resp onses, but prevented Cl- current reduction when coapplied with PDB. Ph orbol 12-myristate 13-acetate (PMA) reduced the GABA-activated current in a fashion very similar to PDB. Staurosporine and calphostin C bloc ked the PMA effect, No reduction of Cl- current was seen with the inac tive analogue, 4-alpha-PMA, used as a control for PKC-independent phor bol ester effects. 5. PDB effectively reduced the GABA-activated Cl- c urrent 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. Collecti vely, the data from phorbol esters, inactive analogues, and kinase inh ibitors support the existence of a PKC-mediated mechanism for GABA-act ivated Cl- current reduction in these cells. 6. The naphthalenesulfona mide PKC activator N-(n-Heptyl)-5-chloro-1-naphthalenesulfonamide (SC- 10) also potently and reversibly reduced the GABA-activated current. S taurosporine and calphostin C eliminated this effect When the nonhydro lyzable guanosine 5'-triphosphate (GTP) analogue guanosine 5'-O-(3-thi otriphosphate) tetralithium salt (GTP-gamma-S) replaced GTP in the rec ording pipette, the SC-10-mediated GABA current reduction became irrev ersible. When guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP- beta-S) was substituted for GTP, the SC-10 effect was blocked. GTP-gam ma-S and GDP-beta-S had no independent effects on the GABA-activated c urrent. These data are consistent with the involvement of G proteins i n the PKC-mediated GABA(A) current modulation, suggesting that they ma y play a role ''downstream'' from the initial transduction event. Howe ver, the synthetic membrane-permeable diacyglycerol analogue 1,2-oleoy lacetyl-glycerol did not modulate the GABA-activated Cl- current of th e rod bipolar cell. 7. We have previously reported that the neuropepti de 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; GD P-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 sa me direction as with PKC activators, it was both smaller and less cons istent. 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 bi polar cell.