cyclic-gmp and 1-2-dioctanoylglycerol

The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.

Topics: Biological Transport; Cations; Cell Membrane; Cloning, Molecular; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Diglycerides; Dose-Response Relationship, Drug; Fluorescent Dyes; HeLa Cells; Humans; Kidney Tubules; Kinetics; Potassium; Protein Kinase C; Pyridinium Compounds; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Fluorescence

The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment plasma membrane. Although cGMP appears to be the primary light-regulated second messenger, cellular levels of other substances, including Ca2+ and phosphatidylinositol-4,5-bisphosphate, are also sensitive to the level of illumination. We now show that diacylglycerol (DAG) analogs reversibly suppress the cGMP-activated conductance in excised patches from frog rod outer segments. This suppression did not require nucleoside triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 microM 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) reduced the current with an IC50 of approximately 22 microM (Hill coefficient, 0.8), whereas with 1.2 microM 8-Br-cGMP, only approximately 1 microM 1,2-DiC8 was required to halve the current. DAG reduced the apparent affinity of the channels for cGMP: 4 microM 1,2-DiC8 produced a threefold increase in the K1/2 for channel activation by 8-Br-cGMP, as well as a threefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC8 was not relieved by supersaturating concentrations of 8-Br-cGMP, suggesting that DAG did not act by competitive inhibition of cGMP binding. Furthermore, DAG did not seem to significantly reduce single-channel conductance. A DAG analog similar to 1,2-DiC8--1,3-dioctanoyl-sn-glycerol (1,3-DiC8)--suppressed the current with the same potency as 1,2-DiC8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective. Our results suggest that DAG allosterically interferes with channel opening, and raise the question of whether DAG is involved in visual transduction.

Topics: Ambystoma; Animals; Biophysical Phenomena; Biophysics; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Diglycerides; Electrophysiology; In Vitro Techniques; Ion Channel Gating; Ion Channels; Kinetics; Light; Phosphorylation; Rana pipiens; Rod Cell Outer Segment; Second Messenger Systems

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Calcimycin; Carcinogens; Cells, Cultured; Cyclic GMP; Diglycerides; Enzyme Induction; Kinetics; Lipopolysaccharides; Liver; Nitric Oxide Synthase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Rats; Tetradecanoylphorbol Acetate