Stimulation of guanylate cyclase by EDTA and other chelating agents.

The partially purified soluble guanylate cyclase (GTP pyrophosphatelyase(cyclizing), EC 4.6.1.2) from human caudate nucleus is stimulated from 2 to 4-fold by metal chelating agents. EDTA (K 1/2 - 4.8 microM) is more potent than CDTA (K 1/2 = 13.2 microM) or EGTA (K 1/2 = 21.8 microM) at stimulating activity. Stimulation by chelating agents is apparently not due to removal of inhibitory divalent cations which contaminate the enzyme or reaction mixture. EDTA increases guanylate cyclase activity in part by increasing the affinity of the enzyme for the substrate (MgGTP) 10-fold. Dopamine inhibits partially purified guanylate cyclase in the presence or absence of EDTA. Dopamine increases the Ka of guanylate cyclase for the activator, free Mn2+, more than 50-fold, from 3 to 150 microM.

A comparison of peripheral and central human muscarinic cholinergic receptor affinities for psychotropic drugs.

A peripheral model offers a valuable research tool for the investigation of central cholinopathic disorders. The in vitro affinities of several psychotropic drugs for the muscarinic cholinergic binding sites of human caudate and erythrocyte were compared in competition with a tritiated antagonist (quinuclidinyl benzilate). The relative affinities of the drugs for both tissues were strikingly similar. Thus, the erythrocyte muscarinic receptor may represent an accessible in vitro assay for the characterization of central cholinopathic states.

Inhibition of estrogen-induced increases in uterine guanosine 3',5'-cyclic monophosphate levels by inhibitors of protein and RNA synthesis.

Uterine guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels are elevated significantly from 2 to 12 h after a single injection of estradiol-17 beta or diethylstilbestrol to mature, ovariectomized, or immature rats. The accumulation of cyclic GMP is greater in endometrial- than myometrial-enriched uterine tissue. The estrogen-induced increase in cyclic GMP can be prevented by administration of the protein synthesis inhibitors, cycloheximide and puromycin, or by relatively large doses of the RNA synthesis inhibitor, actinomycin D, but not by the muscarinic antagonist, atropine. The requirement for a protein with a relatively rapid rate of turnover is suggested by the demonstration that cycloheximide, when administered after estrogen, can within a 3-h period restore the estrogen-elevated levels of cyclic GMP to those of the non-estrogen-treated tissue.

IL-1 beta modulates the concanavalin-A-induced expression of proenkephalin A mRNA in murine thymocytes.

We have previously shown that proenkephalin A (PEA) messenger RNA (mRNA) is induced in murine thymocytes by the T cell-specific mitogen concanavalin-A (Con-A). We now show that this Con-A-induced expression of PEA mRNA is modulated by the cytokine murine interleukin-1 beta (mIL-1 beta) in a biphasic, dose-dependent manner. Murine thymocytes were cultured for 72 h with Con-A and with varying concentrations of mIL-1 beta. PEA mRNA expression was analyzed by Northern gel and solution hybridization techniques. Concentrations of mIL-1 beta of 10(-14) and 10(-13) M enhanced the Con-A-induced expression of PEA mRNA in cultured murine thymocytes up to 2.5-fold, whereas higher concentrations of mIL-1 beta (10(-11) and 10(-10) M) inhibited its expression 60 and 85%, respectively. Both the enhancing and inhibiting effects of mIL-1 beta in the Con-A-induced expression of PEA mRNA were reversed by a 100-fold excess of interleukin-1 receptor antagonist protein, but not by a 10-fold excess of interleukin-1 receptor antagonist protein. The effects of mIL-1 beta on PEA mRNA expression in Con-A-activated thymocytes are different from its effects on Con-A-stimulated thymocyte proliferation. In the latter case, only enhancement of thymocyte proliferation was seen, as measured by [3H]thymidine incorporation. The present study demonstrates that PEA mRNA expression is regulated by IL-1 beta, which is thought to play a role in thymocyte maturation.

Catecholamine-sensitive guanylate cyclase from human caudate nucleus.

Partial purification of soluble guanylate cyclase on DEAE-Sephacel yields two separate peaks of guanylate cyclase activity. After 10-fold purification of the soluble enzyme, guanylate cyclase is markedly inhibited by micromolar concentrations of dopamine (I50 = 0.2 microM). Dopamine inhibition is observed whether the reaction is conducted with Mn2+ or with Mg2+, under atmosphere or N2(g), and using enzyme from either peak from the DEAE-Sephacel column. Other catecholamines also inhibit partially purified guanylate cyclase with an order of potency at 1 microM of: dopamine = L-DOPA > norepinephrine = isoproterenol = adrenochrome > epinephrine. The structural requirements for inhibition are two free hydroxyl groups on the phenyl ring and an ethylamine side chain. Dopamine also inhibits the Triton X-100-solubilized microsomal guanylate cyclase after partial purification on DEAE-Sephacel. Neither chlorpromazine, propranolol, nor phentolamine at 20 microM effectively block the dopamine inhibition of partially purified soluble guanylate cyclase. Micromolar concentrations of the reducing agents dithiothreitol and glutathione also inhibit partially purified guanylate cyclase, but unlike these agents, catecholamines can inhibit whether added in the reduced or the oxidized forms. Inhibition of enzyme activity by micromolar concentrations of dopamine, adrenochrome, or dithiothreitol is rapidly reversed by dilution and the dopamine inhibition is competitive with MgGTP. Inhibition does not appear to involve covalent binding or to result from the ability of catecholamines to reduce the concentrations of oxygen or free radicals in solution.

Postmortem stability of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in rat striatum.

The stability of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase was measured in homogenates of rat striatal tissue frozen from 0 to 24 h postmortem. ATPase, GTPase, and Mg2+-dependent guanylate cyclase activities showed no significant change over this period. Mn2+-dependent guanylate cyclase activity was stable for 10 h postmortem. Basal and dopamine-stimulated adenylate cyclase activity decreased markedly during the first 5 h. However, when measured in washed membrane preparations, these adenylate cyclase activities remained stable for at least 10 h. Therefore, the postmortem loss of a soluble activator, such as GTP, may decrease the adenylate cyclase activity in homogenates. These results are not consistent with an earlier suggestion that there is a postmortem degradation of the enzyme itself. Other kinetic parameters of dopamine-sensitive adenylate cyclase can also be measured independently of postmortem changes. Thus, it is possible to investigate kinetic parameters of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in human brain obtained postmortem.

Estrogen-related increases in uterine guanosine 3':5'-cyclic momophosphate levels.

Endogenous uterine cyclic GMP and cyclic AMP concentrations were monitored at each stage of the estrus cycle; cyclic GMP concentrations were found to be highest and cyclic AMP concentrations lowest during proestrus when plasma estrogen levels have been shown to be maximal. After administration of estradiol-17beta or diethylstilbestrol, the concentrations of uterine cyclic GMP in ovariectomized rats underwent a progressive and significant elevation and the levels of cyclic AMP declined significantly below control values when near maximal accumulation of tissue cyclic GMP was achieved. The increase in uterine cyclic GMP concentrations produced by estradiol benzoate administration was prevented when progesterone and the estrogen were co-administered. These results raise the possibility that an enhanced cellular accumulation of cyclic GMP may be involved in the expression of some of the actions of estrogen in uterine tissue.