Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

Purification and succinylation of cyclic GMP from large volume samples and radioimmunoassay of succinyl cyclic GMP.

Improved procedures for isolation of cyclic GMP and cyclic AMP and radioimmunoassay of cyclic GMP with succinylation are described. Procedures involved include modified chromatography on alumina and succinylation of cyclic GMP followed by purification of succinyl cyclic GMP on a Dowex AG 1×8 column. These procedures are convenient and applicable to any volume up to 50 ml of tissue extracts and especially for isotonic incubation mixtures. This assay system is sensitive to 6 femtomoles of cyclic GMP/tube. On radioimmunoassay, free and antibody bound [¹²⁵I]-labeled cyclic GMP are separated by Millipore filtration. Cyclic GMP levels in several tissue samples were determined in order to show the applicability of the procedures.     

Paradoxical stimulation by 1-methyl-3-isobutylxanthine of rat liver cyclic AMP phosphodiesterase activity

DNA newly synthesized in UV irradiated Escherichia coli B/r Hcr⁺ was 2 min pulse-labeled at various periods, then denatured and analysed by sucrose gradient centrifugation either in neutral or in alkaline conditions. Data indicate that in DNA of damaged cells alkali-labile sites are produced. In cells saturated with inducible proteins production of alkali-labile sites disappears in ～1 h. In the absence of inducible proteins production of alkali-labile sites continues.     

Subcellualr localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm

The subcellular localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 980% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucelotide phosphodiesterase in sperm flagella were also briefly described.     

Stimulation of guanylate cyclase activity by several fatty acids.

Guanylate cyclase of plasma membrane of isolated rat fat cells was activated 7 to 11 fold by oleic acid, linoleic acid, linolenic acid or arachidonic acid. The activation of the enzyme by linoleic acid or oleic acid was influenced by the concentration of enzyme protein and that of the fatty acid. At 158 μg/ml of enzyme protein, 0.6 mM linoleic acid produced maximal activation of 12 fold which was partially reversed by washing. Particulate guanylate cyclase of cerebral cortex and liver was also activated by linoleic acid.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.     

Effects of chymotrypsin and a calcium-dependent protein activator on guanosine 3′,5′-monophosphate phosphodiesterase from rat liver

Cyclic GMP phosphodiesterases from 100 00 × g rat liver supernatant were partially resolved by chromatography on DEAE-cellulose. Multiple forms of cyclic GMP phosphodiesterase(s) that were activated to different degrees by calcium plus a low molecular weight protein from rat liver and bovine brain supernantants, or by limited exposure to chymotrypsin, were identified. The cyclic GMP phosphodiesterase in some column fractions was activated over 10-fold by calcium plus activator or chymotrypsin. Activation by chymotrypsin was dependent both on the time of incubation with protease and its concentration. Prolonged exposure to chymotrypsin resulted in a decrease in s_20,w by sucrose density gradient centrifugation. The chymotrypsin-treated enzyme was no longer activated by exposure to calcium plus activator. The calcium- and protein activator-stimulated enzyme was inactivated by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA). Exposure of this activated enzyme to chymotrypsin did not result in further activation, but the chymotrypsin-treated enzyme was no longer inhibited by EGTA. The apparently irreversible effects of chymotrypsin and the reversible effects of calcium plus activator on cyclic GMP hydrolysis by the phosphodiesterase over a wide range of cyclic GMP concentrations appeared to be identical.     

Preparation of renal cortex basal-lateral and brush border membranes. Localization of adenylate cyclase and guanylate cyclase activities

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and γ-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na⁺ + K⁺)-ATPase. However, the specific activity of (Na⁺ + K⁺)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na⁺ + K⁺)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na⁺ + K⁺)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5′-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN₃ plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na⁺ + K⁺)-ATPase, be used as an enzyme “marker” for the renal basal-lateral membrane.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Comparison of cyclic nucleotide specificity of guanosine 3′,5′-monophosphate-dependent protein kinase and adenosine 3′,5′-monophosphate-dependent protein kinase

Guanosine 3′,5′-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3′,5′-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-folds less than that of cylic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic. AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophophorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.