Regulation of hepatic phosphoenolpyruvate carboxykinase (GTP) Role of dietary proteins and amino acids in vivo and in the isolated perfused rat liver

The effect of protein feeding and the addition of amino acids on the activity of hepatic phosphoenolpyruvate carboxykinase (GTP: oxalacetate carboxylyase (transphosphorylating), EC 4.1.1.32) was investigated in vivo and in the isolated perfused rat liver. Protein feeding resulted in a considerable increase in phosphoenolpyruvate carboxykinase activity within 6 h. This rise was independent of the presence of glucocorticoids.In the isolated perfused liver system amino acids per se had a small effect on phosphoenolpyruvate carboxykinase activity and led to an increase by 20% when glucocorticoids were present, but resulted in a rise by 100% when glucocorticoids plus dibutyryl cyclic AMP were added to the perfusion medium. The effect of amino acids in the presence of dibutyryl cyclic AMP could also be observed in the liver of glucocorticoid-deprived rats.Cycloheximide, a translational inhibitor, totally blocked all effects of amino acids on enzyme activity.These results indicate that the concentration of amino acids in the portal vein modify the regulation of phosphoenolpyruvate carboxykinase by cyclic AMP.     

Role of the microfibrillar system in knob action of transformed cells

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5–4 μm in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15–60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: (1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; (2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; (3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reversetransformed cells or normal fibroblasts; (4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; (5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.     

Superoxide dismutase activity of macrocyclic polyamine complexes

Copper(II) and nickel(II) complexes of macrocyclic polyamine derivatives possessing partial oligopeptide-like structures are found to suppress the xanthine-xanthine oxidase-mediated reduction of nitroblue tetrazolium and also to suppress formazan formation by potassium superoxide. The activity in the superoxide dismutase assay is dependent on ring size, type and number of donor atoms, metal ion, and substituents on the macrocycles. Some of those are more active than the known O₂^? scavengers such as copper(II)-salicylate and copper(II)-amino acid (or peptide) complexes. Nickel (II)-naphthylmethyl-dioxo-[16]ane N₅, 13, 1 : 1 complex (NiH_?2L) is the most active among the 30 chelates examined.     

Activation of cyclic AMP-dependent protein kinase A common step in vasopressin- and hypertonicity-induced water flow

Water flow across the amphibian urinary bladder can be induced by either vasopressin or serosal hypertonicity. In an effort to determine the common intracellular steps mediating both responses, we determined the in situ activation of cyclic AMP-dependent protein kinase in bladders stimulated by vasopressin or hypertonicity. Treatment of bladders with vasopressin (1 mU/ml) caused in situ activation of cytosolic cyclic AMP-dependent protein kinase of epithelial cells, with a rise in the kinase ratio and cyclic AMP content. Similarly, hyperonicity increased the kinase ratio, but this occured without a measurable increase in cyclic AMP content per mg protein. Because of the hypertonicity-induced cell shrinkage, epithelial cell water decreased by 20%, which may result in a proportionate increase in cyclic AMP concentration (per ml cell water). Furthermore, cell shrinkage also increases intracellular electrolyte concentration, which, in turn, should delay reassociation and consequent inactivation of the predominant Type II cyclic AMP-dependent protein kinase of the epithelial cells. Thus activation of cyclic AMP-dependent protein kinase during hypetonicity may be the result of cell shrinkage, with an associated increase in cyclic AMP and electrolyte concentrations. Studies with prostaglandin synthesis inhibitors and colchicine, a microtubule disrupting agent, also indicated common pathways for vasopressin and hypertonicity. Both naproxen and meclofenamate significantly enhanced the hypertonicity response. Colchicine pretreatment, on the other hand, caused a small (18%) but significant inhibition of the hypertnicity response, similar to its effect on the vasopressine response (25% inhibition). Thus, the increased water permeability of the toad bladder in response to both vasopressin and hypertonicity follows a similar pathway. Activation of cyclic AMP-dependent protein kinase represents the first common step yet identified.     

Nitric oxide stimulates embryonic somatotroph differentiation and growth hormone mRNA and protein expression through a cyclic guanosine monophosphate-independent mechanism

In the pituitary gland, NO is locally synthesized by gonadotroph and folliculo-stellate cells. Many reports have shown that NO can modulate the growth hormone (GH) secretion. However, its role on mice embryo GH regulation remains unclear. In addition, it is unknown whether the regulation is associated with the proliferation of pituitary cells. In this study, we have investigated the regulatory effects of NO on somatotroph differentiation, proliferation and GH mRNA and protein expression using primary cell cultures of mice fetal pituitaries (embryonic days 16.5, ED 16.5). Our results show that incubation of pituitary cells in the presence of sodium nitroprusside (SNP; 1 mM), a NO donor, for 4.5 h resulted in a significant increase in GH mRNA and protein expression (P < 0.05) and the stimulation of SNP can be inhibited by hemoglobin, a NO scavenger. But the addition of cyclic guanosine monophosphate (cGMP; 3.0 mM), the second messenger of multiple NO actions cannot influence GH mRNA and protein expression. The cyclic nucleotide cellular efflux pumps existed in the pituitary cells can transport the majority of de novo-produced cGMP and effectively block cGMP accumulation. For maintaining intracellular concentration of cGMP, probenecid (0.5 mM), a blocker of cGMP efflux pump, combined with cGMP (3.0 mM) was used to treat the pituitary cells. This also cannot influence GH mRNA and protein expression. In addition, the ratio of GH-positive cells is increased significantly after the stimulation of SNP (P < 0.05). However, SNP cannot modulate the pituitary cell proliferation. From these results we conclude that NO can increase GH mRNA and protein expression in fetal pituitary cells and cGMP is not involved in this hormonal regulation. Stimulation of NO on the somatotroph differentiation does not occur due to pituitary cell proliferation.     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

Phosphate inhibition of secondary metabolism in Streptomyces hygroscopicus and its reversal by cyclic AMP

Inorganic phosphate inhibited the biosynthesis of the macrolide antibiotic turimycin in different strains of Streptomyces hygroscopicus. In the wild type strain a depression was observed with increasing phosphate concentrations. A total inhibition was found at 0.1 M phosphate. In a high producing mutant a minimum of turimycin production occured when the phosphate concentration was between 5 mM and 10 mM. Above this concentration the antibiotic synthesis increased again but the production period shifted to a later period of cultivation. Addition of inorganic phosphate resulted in an initial increase of intracellular cyclic AMP content. But a second elevation characterizing the normal level of cyclic AMP throughout the growth phase was prevented by phosphate. Exogenous cyclic AMP as well as positive effectors of the adenylyl cyclase system were able to overcome the phosphate suppression. Cyclic AMP abolished the reduction of protein synthesis following phosphate addition and caused the reappearance of a protein band which may be responsible for the turimycin biosynthesis.     

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2–3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low K_m (0.17 μM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study. Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.     

Highly Activatable Adenylate Cyclase in [2-3H]Adenine-Prelabeled Vesicles Prepared from Guinea Pig Cerebral Cortex by a Simplified Procedure

An improved procedure utilizing a simple fractionation technique is described for the preparation and use of [2-3H]adenine-prelabeled vesicles from guinea pig cerebral cortex containing highly responsive adenylate cyclase activity. Adenosine consistently increased activity 1500--2000%, contrasted with activations of 200--300% previously reported by other investigators. Adenosine at 5 microM was more active in our system than at 20 times this concentration in studies by other investigators, increasing activity by 580--840%. Experimental conditions were explored, and Ca2+ was found to be necessary during tissue homogenization, but not during subsequent vesicle incubation. However, neither the higher Ca2+ concentration used by us (2.5 mM) nor the method of tissue homogenization could adequately explain the high activity of our preparations. The size of the incubation vessel was critical for both low basal activity and high activity in the presence of adenosine. Our preparations were similar to others in that combinations of neurohormones, which included histamine and epinephrine, elicited higher activities than individual neurohormones. Inspection of our vesicle preparations by scanning electron microscopy also showed them to be compatible with previously described preparations.