Mutants of PC12 cells with altered cyclic AMP responses.

PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 microM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(beta, gamma-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, Ns. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PC12 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PC12 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

A cyclic adenosine monophosphate link in the catecholamine enhancement of transmitter release at the neuromuscular junction.

The frequency of miniature endplate potentials (mepps) in rat diaphragms was markedly increased by epinephrine and norepinephrine in preparations exposed to 15 mM K(+). The effect was rapid in onset but gradually declined during continued exposure to the catecholamines. N(6), O(2')-dibutyryl adenosine 3',5'-monophosphate (dibutyryl-cAMP) also caused transient frequency increases resembling in time-course those observed with catecholamines. Contrary to previous reports, catecholamines and dibutyryl-cAMP had little effect on mepp frequency in preparations not treated with K(+). Sustained increases with theophylline and decreases with adenosine were found in both K(+)-treated and untreated preparations. Analysis of the data obtained with catecholamines showed the intensity of the response to be a function of nerve terminal polarization. The inability of catecholamines and dibutyryl-cAMP to affect mepp frequency of untreated preparations argues against an obligatory role for cAMP in the neurosecretory mechanism. The findings are consistent with an action of catecholamines and cAMP in the regulation of transmitter release at fatigued preparations.

Potentiation of guanosine 3':5'-monophosphate accumulation in C-6 glial tumor cells.

C-6 glial tumor cells treated with norepinephrine and sodium azide accumulated cyclic GMP to concentrations approximately 10-fold greater than the sum of the separate responses. Isoproterenol, but not phenylephrine, was an effective substitute for norepinephrine, and the response was blocked by propranolol and sotalol. Nitroprusside, but neither cyanide nor isobutyl-methylxanthine, replaced azide. The potentiation was not affected by removal of CA2+ OR Na+ from the extracellular medium and was not blocked by cocaine. The potentiative accumulation of cyclic GMP in C-6 cells differs from the recently described stimulation by catecholamines of soluble guanylate cyclase of renal cortex. The potentiative phenomenon is compared with the few known instances in which cyclic AMP augments cyclic GMP formation and may be associated with synergistic modifications of cellular functions.

Calcium-dependent regulation of brain adenylate cyclase.

The adenylate cyclase activity of a particulate preparation of rat cerebral cortex is comprised of two contributing components, only one of which requires a CDR for activity. The CDR-dependent component was inhibited by high ratios of Mg2+ to Ca2+, responded in a biphasic manner (activation then inhibition) to increasing free Ca2+ concentrations, was inhibited by 0.1 to 0.4 mM chlorpromazine, and was activated by 1 to 100 micrometer cocaine. This enzyme form, which represented approximately 80% of tge basal activity of a cortex homogenate, was stable during pretreatment of homogenates at 45 degrees C but was completely deactivated by the removal of CDR during the preparation of the particulate fraction. Adenylate cyclase activity that did not depend on CDR was unaffected by the removal of CDR during the preparation of the particulate fraction, had elevated activity at high ratios of Mg2+ to Ca2+, was inhibited by Ca2+, was unaffected by 0.1 to 0.4 mM chlorpromazine and was slightly inhibited by 1 to 100 micrometer cocaine, and was not stable during pretreatment of homogenates at 45 degrees. The CDR-dependent component of adenylate cyclase activity was activated by 5 mM NaF to varying degrees depending on the concentration of CDR present in the assay. NaF decreased the concentration of CDR required to produce half-maximal velocity obtained at optimal concentrations of CDR. Activation by NaF required the presence of Ca2+ and was immediately and completely reversed by EGTA. In contrast, the component that did not respond to CDR was activated four- to fivefold by NaF. This activation was not influenced by Ca2+ or CDR and was not reversed by EGTA. The observed effects of effects of divalent cations on the CDR-dependent enzyme are discussed in relation to the cation-binding properties of CDR. The relationship of the CDR-dependent form of adenylate cyclase to other forms of this enzyme remains to be determined.

Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase.

An activating factor of adenylate cyclase (EC 4.6.1.1) HAS BEEN OBTAINED FROM DETERGENT-DISPERSED PREPARATIONS OF PORCINE CEREBRAL CORTEX BY COLUMN CHROMATOGRAPHY ON ECTEOLA-cellulose. The factor was identified by acrylamide gel electrophoresis and by enzyme activation studies as the Ca2+-binding protein that regulates the activity of a brain cyclic nucleotide phosphodiesterase. This Ca2+-binding protein confers a Ca2+-dependent activation upon the adenylate cyclase, which is reversed by the subsequent addition of egta in excess of the free Ca2+. It is proposed that this Ca2+-dependent regulator controls enzymatic activities responsible for the synthesis of adenosine 3':5'-monophosphate and for the hydrolysis of guanosine 3':5'-monophosphate.

Regulation of adenylate cyclase from glial tumor cells by calcium and a calcium-binding protein.

A biphasic response to changes in Ca2+ concentration was observed for basal and norepinephrine-stimulated adenylate cyclase activity in homogenates of C-6 glioma cells. The enzyme was stimulated approximately 40% by low concentrations of free Ca2+ (less than or equal to 1 muM) and inhibited to successively greater extents as free Ca2+ concentrations were increased to approximately 100 muM. Ca2+ did not alter the concentration of norepinephrine required for enzyme activation. Homogenates of C-6 cells were separated into particulate and supernatant fractions by centrifugation at 27,000 X g for 20 min. The particulate fraction contained nearly all of the adenylate cyclase activity. This activity was stimulated approximately 40% by the addition of untreated supernatant fraction, by boiled or dialyzed supernatant fraction, and by a homogenous Ca2+-binding protein (calcium-dependent regulator (CDR) prepared from brain. Addition of either the supernatant fraction or CDR lowered the Ca2+ concentration required for maximal stimulation of the adenylate cyclase. The factor in the supernatant fraction which activated the particulate enzyme was subsequently identified in acrylamide gel electrophoretic studies to be CDR. The amount of CDR required for maximal activation of the enzyme was found to be lowered as the Ca2+ concentration in the assay was increased. High amounts of added CDR (100 to 1000 ng) were inhibitory. Use of the monionic detergent, Lubrol PX, to prepare dispersed adenylate cyclase from the particulate fraction resulted in large losses of activity. The resultant preparation of enzyme contained some CDR which could not be removed by chromatography of the preparation on anion exchange columns. Addition of homogeneous CDR to the assay activated the enzyme several-fold at low Ca2+ concentrations. At higher Ca2+ concentrations the enzyme was activated fully by the CDR endogenous to the preparation and added Ca2+. CDR was inhibitory.