A new solid-state microelectrode for measuring intra-cellular chloride activities

Solid-state microelectrodes for measuring intracellular Cl^? activity (aⁱCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 μm), coating them under vacuum with a 0.2–0.3 μm thick layer of spectroscopic grade silver, and sealing them (except for the terminal 2–5 μm of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0 ± 0.6 mV (S.E.) per decade change in α_Cl. Tip resistance was (77.1 ± 3.1 × 10⁹ω (n=30). Electrode response was rapid (10–20 s), was unaffected by HCO₃^?, H₂PO₄^2? or protein, and remained essentially unchanged over a 24-h period. αⁱ_Cl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, αⁱ_Cl significantly exceeded the value corresponding to equilibrium distribution of Cl^? across the cell membrane.     

Studies on the inhibition of glycogenolysis by D-galactosamine in rat liver hepatocytes.

Effect of galactosamine on glycogenolysis was studied in isolated hepatocytes. It was found that addition of galactosamine strongly inhibited glycogenolysis in normal hepatocytes. Galactosamine-inhibited glycogenolysis was not stimulated by epinephrine or glucagon. This inhibition was specific as no such inhibition was observed with galactose, 2-deoxy-glucose or glucosamine. The glucagon-stimulated cyclic AMP formation in galactosamine-treated hepatocytes was the same as in normal cells; Glc-1-P and Glc-6-P did not accumulate nor was lactate formation enhanced. The glucose production by hepatocytes from regenerating liver was only slightly inhibited by galactosamine and glucagon addition stimulated glycogenolysis in the presence of the amino sugar.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats

In order to ascertain the possible involvement of cyclic GMP in the physiological regulation of the function and development of brown fat of the rat, we have determined its tissue concentration in vivo under a variety of conditions. The steady-state concentration of cyclic GMP in interscapular brown adipose tissue of late foetus was about 80 pmol per g fresh weight. The concentration gradually declined during the first 2 weeks after birth to reach 40 pmol/g fresh weight and then remained constant into adulthood. The cyclic GMP content of brown fat was decreased by chemical sympathectomy and was increased after complete acclimatization of the animals to the cold. The activity of cyclic GMP-dependent protein kinase was also highest in tissue from newborn and cold-acclimatized rats.Both acute cold stress and injection of norepinephrine resulted in a significant but temporary increase in the concentration of cyclic GMP in brown fat, which was followed by a depression of the concentration below values in untreated animals. The concentration of cyclic AMP showed similar pattern of changes. Injection of phenylephrine was followed by a pronounced increase in the cyclic GMP content of brown fat, with little effect upon cyclic AMP. Injection of isoproterenol raised the content of cyclic AMP but not that of cyclic GMP. The ability of norepinephrine and phenylephrine to increase the concentration of cyclic GMP was abolished by pre-treatment of the animals with phenoxybenzamine, but not by pre-treatment with propranolol. Conversely, propranolol but not phenoxybenzamine abolished the effects of norepinephrine on the cyclic AMP content of the tissue.Thus we have established the responsiveness of the cyclic GMP content of brown fat to physiological and pharmacological stimuli and have evidence of the possible participation by cyclic GMP in the α-adrenergic stimulation and in the regulation of proliferative processes in the tissue.     

Effects on N6, O2′-dibutyryladenosine-3′,5′ cyclic monophosphate on calcium accumulation by mouse mastocytoma cells

The accumulation of ⁴⁵Ca²⁺ by intact mouse mastocytoma cells was examined before and after treatment of the cells with N⁶,O^2′-dibutyryladenosine 3′,5′, cyclic monophosphate and theophylline to inhibit growth. In the presence of phosphate either glycolysis, respiration or ATP supported ⁴⁵Ca²⁺ uptake by the cells and in each case the accumulated ⁴⁵Ca²⁺ appeared to be retained by mitochondria. Inhibition of growth by drug treatment for 20h increased subsequent ⁴⁵Ca²⁺ accumulation when cells were incubated with ⁴⁵CaCl₂, succinate and phosphate. Since prior drug treatment did not increase ⁴⁵Ca²⁺ accumulation with glucose, ATP or malate the drugs appeared to increase ⁴⁵Ca²⁺ accumulation by affecting succinate metabolism.     

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.     

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.     

Stimulation of rat liver adenylate cyclase by halothane.

Rat liver membranes prepared by a modification of the procedure of Neville were exposed to clinical and toxic concentrations of the general anesthetic, halothane, for 10 minutes. Basal, glucagon (5 × 10⁻⁵M) and sodium fluoride (20 mM) stimulated adenylate cyclase activity was assayed. Clinical and toxic concentrations of halothane augmented basal adenylate cyclase activity. Glucagon and sodium fluoride stimulated adenylate cyclase activity was enhanced at greater than clinically useful halothane concentrations only. The study provides direct evidence that halothane stimulates adenylate cyclase, the extent of augmentation of enzyme activity is halothane concentration dependent, and modified by other drugs.     

Effect of norepinephrine and dibutyryl cyclic AMP on S-100 protein level in C6 glioma cells

S-100 Protein level was determined in C6 glioma cells after treatments by norepinephrine. In growing cells norepinephrine induces an important increase of S-100 protein level continuing during the stationary phase to reach a level higher than in untreated quiescent cells. In quiescent, low density, thymidine blocked cells, S-100 protein level is also enhanced by norepinephrine. In high density, contact inhibited cells, S-100 protein level is not modified although cAMP level is much more stimulated by norepinephrine than is low density cells. Exogenous addition of dibutyryl cyclic AMP mimics the effects of norepinephrine.Our results suggest that cyclic AMP level can mudulate S-100 protein level in C6 cells but that in density inhibited cells, a subsequent step involved in the regulation is no more operative.     

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.     

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.     

Non-dependence on native structure of pig liver pyruvate kinase when used as a substrate for cyclic 3',5'-AMP-stimulated protein kinase.

Alkali-inactivated pig liver pyruvate kinase, type L, and a cyanogen bromide fragment from the same enzyme were shown to be phosphorylated by (³²P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In both cases the rate of phosphorylation was higher than with the native enzyme. Pyruvate kinases types A and M were not phosphorylated under the same conditions. From the ³²P-labelled cyanogen bromide fragment (³²P)phosphorylserine was isolated. The electrophoretic pattern of (³²P)phosphopeptides obtained on partial acid hydrolysis of the fragment indicated that the phosphorylated site of the fragment was identical with that of the native pyruvate kinase.     

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.     

Antagonistic effect of methadone on steroidogenesis and the adenylate cyclase system in isolated rat adrenocortical cells

Methadone exhibits an antagonistic effect toward steroidogenesis which lies prior to progesterone in the biosynthetic pathway in isolated rat adrenal cells. Levels of adenosine cyclic 3′–5′ monophosphate are depressed in a dose dependent fashion in ACTH stimulated cells as is steroidogenesis in cells stimulated with N⁶O²-dibutyryl adenosine cyclic 3′–5′ monophosphate. Stimulation produced by the ACTH analog, O-nitrophenyl sulfenyl ACTH, is also inhibited by methadone. The participation of adenosine cyclic 3′–5′ monophosphate as an obligatory messenger in ACTH stimulated steroidogenesis is discussed with respect to the pharmacological properties of methadone in this system.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.