dibutyryl-cyclic-gmp and Pheochromocytoma

cAMP-dependent protein kinase (PKA) and phospholipid-dependent protein kinase (PKC) play a role in nerve growth factor (NGF)-mediated differentiation. In PC12 cells, NGF causes neurite outgrowth and increases the number of voltage-gated Na+ channels. Neurite outgrowth involves in part activation of PKC. How NGF regulates Na+ channel number is unknown. Using patch-clamp techniques, we find that agents activating PKC, including phorbol esters and a ras oncogene product (p21) that induces neurites, caused little increase in channel number. In contrast, agents increasing intracellular cAMP were as effective as NGF. A specific protein inhibitor of the PKA catalytic subunit blocked increases by NGF or cAMP. Thus, NGF increases Na+ channel number in PC12 cells in part by activating PKA but apparently not PKC.

Topics: 1-Methyl-3-isobutylxanthine; Adrenal Gland Neoplasms; Animals; Bucladesine; Cell Differentiation; Cell Line; Colforsin; Cyclic CMP; Dibutyryl Cyclic GMP; Dimethyl Sulfoxide; Electric Conductivity; Electrophysiology; Gene Expression; Genes, ras; Kinetics; Nerve Growth Factors; Pheochromocytoma; Protein Kinases; Rats; Sodium Channels; Tetrodotoxin

Nerve growth factor (NGF) induces in 2 to 10 min the redistribution of F-actin in rat pheochromocytoma PC12 cells. The NGF specificity of this phenomenon was shown by blocking it with anti-NGF antibodies. We used the rapid F-actin redistribution as an assay to study NGF second messenger systems and their inhibition or activation by specific agents. The results show that the NGF-induced effect on the microfilament system of PC12 cells can be specifically inhibited by lithium chloride and neomycin, inhibitors of the phosphoinositol system, but cannot be mimicked by TPA and acetylcholine, the activators of the phosphoinositol system. An increase in the intracellular concentration of cyclic AMP by addition of dBcAMP (but not dBcGMP) caused rapid F-actin redistribution that nonetheless differed from the NGF-induced effect. Changes in the intracellular calcium level did not have any influence on the microfilament system of PC12 cells. The specificity of the inhibition of NGF-induced effects by methylase inhibitors was questionable, since MTA- or SAH-treated PC12 cells acquired an altered morphology even in the absence of NGF or dBcAMP. Using the microfilament- and microtubule-disrupting drugs cytochalasin B and colchicine, we showed that the microtubule system in PC12 cells is required for the initiation of neurite outgrowth and that microfilament-associated filopodial activity does not appear to be necessary.

Topics: Actin Cytoskeleton; Actins; Adrenal Gland Neoplasms; Animals; Axons; Bucladesine; Calcium; Colchicine; Colforsin; Cyclic AMP; Cyclic GMP; Cytochalasin B; Cytoskeleton; Dibutyryl Cyclic GMP; Kinetics; Methyltransferases; Nerve Growth Factors; Pheochromocytoma; Phosphatidylinositols; Rats; Second Messenger Systems; Tumor Cells, Cultured

This study tests the hypothesis that atrial natriuretic factor (ANF) inhibits catecholamine release from rat pheochromocytoma cells by increasing levels of intracellular cyclic GMP (cGMP). Rat differentiated pheochromocytoma cells are a model of adrenergic nerves and allow the exploration of the effects of various hormones, autacoids, drugs and neuromodulators on adrenergic neurotransmission in cell culture. Synthetic rat ANF (99-126) inhibited K+-induced norepinephrine and dopamine release, as measured by high-performance liquid chromatography, in a concentration-dependent manner over the concentration range of 10(-11) to 10(-8) M. ANF stimulated intracellular cGMP accumulation, as measured by specific radioimmunoassay, in a concentration-dependent manner over the same concentration range. The cGMP analog, N2-2'-O-dibutyryl cGMP also inhibited K+-induced norepinephrine and dopamine release in a concentration-dependent manner. The results of this study are consistent with the hypothesis that ANF acts as an inhibitory neuromodulator in adrenergic nerves via the second messenger, cGMP.

Topics: Animals; Atrial Natriuretic Factor; Catecholamines; Cyclic GMP; Dibutyryl Cyclic GMP; Pheochromocytoma; Potassium; Rats; Tumor Cells, Cultured