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Biomedical subjects

G Brooker

Publications and source records attributed to G Brooker.

At least 19 recordsLinked to original sources
PubMed1979

Hormonal control of glycogen metabolism.

In summary, we have presented evidence which relates to the action pathway of hormonal control of glycogen metabolism. In the case of insulin, there are changes demonstrable in the cyclic AMP-dependent protein kinase and also in the phosphoprotein phosphatase, under conditions where no direct relationship to either cyclic AMP or cyclic GMP levels are measurable. Therefore, a new unknown intermediate or second messenger system is again proposed. An insulin-generated labile compound(s) which inhibits the protein kinase has been discovered. This may function as an intermediate. Finally, the fact that the glycogen synthase system clearly differs from phosphorylase in its regulation by covalent phosphorylation is discussed. Synthase is now accepted as a multiply phosphorylated subunit, in contrast to phosphorylase which is singly phosphorylated. The inherent theoretical advantages of multiple phosphorylation over single phosphorylation are considered. The advantages of a multistate over a two-state model of enzyme interconversion are mentioned. The importance of the multiple phosphorylations interacting in a nonlinear manner with the control by cellular metabolites is in the explanation of how a small change in covalent phosphorylation signalled by a hormone can be translated in the cell milieu into a much larger change in rate.

Animals↗
PubMed1979

Quantitative relationship between beta-adrenergic receptor number and physiologic responses as studied with a long-lasting beta-adrenergic antagonist.

The aminobenzyl analog of propranolol, 1- (p-amino-alpha,alpha-dimethylphenethylamino)-3-(1-naphthoxy)-2- propanol, was synthesized and found to be a potent beta-adrenergic blocking agent. The beta-adrenergic receptors of cultured rat C6 glioma cells (2B clone) as assessed by [(125)I]iodohydroxybenzylpindolol binding were decreased 50 and >95% after pretreatment with 8 nM and 1 muM aminobenzylpropranolol, respectively. Unlike propranolol, aminobenzylpropranolol displayed a prolonged blockade of receptors that was maintained during several hours of washing. [(125)I]Iodohydroxybenzylpindolol saturation binding experiments in cells exposed to aminobenzylpropranolol and subsequently washed indicated that the compound effectively diminished receptor number with no change in the affinity of the remaining receptors for iodohydroxybenzylpindolol. Aminobenzylpropranolol inhibited catecholamine-stimulated intracellular cyclic AMP accumulation; with increasing blockade, isoproterenol dose-response curves became progressively shifted to the right but the maximal response was unaltered. Aminobenzylpropranolol inhibited the beta-adrenergic contractile response in atria isolated from rats and guinea pigs. Treatment with 0.1 and 10 muM aminobenzylpropranolol produced decreases of 0.5 and 2 orders of magnitude in the contractile potency of isoproterenol. As in glioma cells, aminobenzylpropranolol failed to decrease the maximal response to isoproterenol. The effects of aminobenzylpropranolol persisted during extensive washing of atria (up to 17 hr). Repeated exposures to isoproterenol at concentrations sufficient to produce maximal tension development also failed to alleviate the blockade. The inotropic potency of histamine in guinea pig atria was not affected by aminobenzylpropranolol. These data suggest that catecholamines are capable of eliciting full biological responses in glioma cells and isolated atria even though the great majority of beta-adrenergic receptors are persistently blocked.

Adrenergic beta-Antagonists↗
PubMed1979

Induction of refractoriness to isoproterenol by prior treatment of C6-2B rat astrocytoma cells with cholera toxin.

Rat C6-2B astrocytoma cells responded to cholera toxin treatment with an 8-fold increase in intracellular cyclic AMP concentrations. Cyclic AMP levels began to rise 60--90 minutes after addition of the toxin and reached maximal concentrations in 3 hours. Cells exposed to cholera toxin and the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), displayed an increase in cyclic AMP of 15-fold. The peak isoproterenol response was reduced 80--90% in cells previously treated with cholera toxin. Cholera toxin-induced refractoriness was time dependent and was not altered by concurrent treatment with propranolol. Prolonged exposure of the cells to isoproterenol reduced the cyclic AMP response to cholera toxin by 80%. MIX augmented both cholera toxin-induced refractoriness and isoproterenol-induced refractoriness. Cycloheximide inhibited the full development of refractoriness to both cholera toxin and isoproterenol. These results indicate that C6-2B cell refractoriness to cholera toxin is mediated by cyclic AMP and requires new protein synthesis. Refractoriness in C6-2B cells does not appear to be agonist-specific and probably involves a common locus of action on adenylate cyclase beyond that of the membrane receptors for cholera toxin and isoproterenol.

1-Methyl-3-isobutylxanthine↗
PubMed1978

Sodium requirement for the positive inotropic action of isoproterenol on guinea pig atria.

Isoproterenol doses not elicit its characteristic positive inotropic action in contracting guinea pig atria suspended in sodium-free media. However, the ability of isoproterenol to decrease the time to peak tension development during an individual contraction cycle is still present in sodium-free solutions. Removal of sodium diminished but did not eliminate the tissues' ability to elevate adenosine 3',5'-monophosphate in response to isoproterenol. The striking absence of an inotropic action by isoproterenol on atria in sodium-free media suggests that sodium (and possibly a sodium-calcium exchange across the sarcolemma) plays an important role in the inotropic action of catecholamines.

Animals↗
PubMed1978

Temperature sensitivity of cyclic AMP production and catecholamine-induced refractoriness in a rat astrocytoma cell line.

Intracellular cyclic AMP was increased more than 100-fold when rat C6-2B astrocytoma cells were treated with isoproterenol in the cold (4 degrees C). When the cells were treated with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and isoproterenol, cyclic AMP levels rose more than 150-fold. The levels achieved compared favorably with the 250-fold increase in cyclin AMP produced by (-)isoproterenol at 37 degrees C.(-)Isoproterenol at 5 nM stimulated half-maximal cyclic AMP production at 4 degrees C and at 37 degrees C and was blocked by (-)propranolol at both temperatures. The concentrations of cyclic AMP attained by these cells after (-)isoproterenol stimulation in the cold may be accounted for, in part, by alterations in the efflux of the nucleotide from the cells since extracellular cyclic AMP, an indicator of cyclic AMP efflux, was found to be dramatically reduced in the cold. The cells, when exposed to (-)isoproterenol for up to 6 hr at low temperature, maintained normal responsiveness to this agent when rechallenged at 4 degrees or 37 degrees C. Thus, they did not display agonist-induced refractoriness during that period of exposure at 4 degrees C, although refractoriness is always seen within 90 min at 37 degrees C. Refractoriness, once established by (-)isoproterenol treatment at 37 degrees C, was not reversed by exposure of the cells to cold. These data suggest that the development of catecholamine refractoriness requires a temperature-sensitive step that lies distal to the hormone-receptor interaction and cyclic AMP generaton.

1-Methyl-3-isobutylxanthine↗
PubMed1977

Cardiac adenosine 3':5'-monophosphate. Free and bound forms in the isolated rat atrium.

Adenosine 3':5'-monophosphate (cyclic AMP), a mediator of hormone action in a variety of tissues, has been measured in its free and bound forms in intact cardiac tissue. We have used a rapid high dilution technique which involves tissue homogenization, subcellular fractionation, and separation of bound from free cyclic AMP by Millopore filtration. The precision of this method is dependent upon minimization of binding and dissociation of cyclic AMP that occur during the preparation and handling of tissue homogenates. In each experiment, a tracer of cyclic [3H]AMP prebound to isolated cardiac binding protein was freed of unbound cyclic [3H]AMP by Sephadex gel filtration and added to the tissue just prior to homogenization in cold EDTA buffer. This tracer was therefore treated identically to the sample through all subsequent dilution, fractionation, and filtration procedures, and provided an acurate internal monitor for total cyclic AMP dissociation during the course of the free-bound determination. Each tissue sample was then individually corrected for dissociation. Rapid dilution to produce a 1:1000 homogenate was found to lower endogenous cyclic AMP levels sufficiently to make binding (or rebinding) during the procedure negligible (less than 5%). Spontaneously beating rat right atria (controls) contained 5.96 +/- 0.28 pmol of cyclic AMP/mg of protein (n = 19) of which 41 and 14% were bound to soluble and particulate proteins, respectively. The remaining cyclic AMP was free. Pretreatment of the tissue with 1 muM isoproterenol (30 s at 30 degrees) increased both the bound and free forms of cyclic AMP (n = 8). While free cyclic AMP increased 420% with the catecholamine, the bound forms increased 240% (soluble) and 60% (particulate). Similar results were obtained when atria (n = 6) were treated with the phosphodiesterase inhibitor, methylisobutylxanthine (0.5 mM, 10 min at 30 degrees). When both agents were used together, cyclic AMP bound to soluble proteins was elevated 4-fold over control while free cyclic AMP increased 27-fold (n = 7), indicating saturation of the soluble sites. It could be calculated that less than one-third of these sites are occupied in the unstimulated cell. These sites may represent the R subunit of cyclic AMP-dependent protein kinase. The data suggest that half-maximal binding in vivo occurs at an intracellular free cyclic AMP concentration of about 1 muM.

Animals↗
PubMed1977

Dissociation of cyclic GMP from the negative inotropic action of carbachol in guinea pig atria.

The relationship between the negative inotropic action of carbachol and its ability to elevate cyclic GMP was determined in isolated paced guinea pig atria. A clear dissociation was observed between that concentration of carbachol which depressed contractility and that which elevated tissue cyclic GMP content. Doses as low as 0.03 micronM caused a negative inotropic effect while cyclic GMP was not elevated until concentrations nearly 100-fold higher were used. Thus a correlation between tissue cyclic GMP content and the negative inotropic action of carbachol was not found to exist in guinea pig atria.

Animals↗