Changes In Cyclic Nucleotide Levels Research Articles

Regulation of mitochondrial pyruvate carboxylation and gluconeogenesis in rat hepatocytes via an alpha-adrenergic, adenosine 3‘:5‘-monophosphate-independent mechanism.

Experiments were performed to determine if catecholamines can regulate control points in the gluconeogenic pathway, such as mitochondrial pyruvate carboxylation and pyruvate kinase activity, via an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism. Of a number of alpha agonists tested, only norepinephrine, epinephrine, and phenylephrine caused an increase in mitochondrial pyruvate metabolism. The effects of catecholamines on pyruvate carboxylation were not attenuated by 1-propranolol which abolishes changes in cyclic nucleotide levels but were blocked by alpha antagonists such as ergotamine, phenoxybenzamine, and phentolamine. Time course experiments demonstrated that the effects of catecholamines on the mitochondria and on carbohydrate metabolism correlated temporally with the concentration of epinephrine in the medium but not with the small changes in adenosine 3':5'-monophosphate. The effects of catecholamines appeared to require extracellular Ca2+ ion. The observation that catecholamines do not increase gluconeogenesis to the same extent as glucagon was not due to a differential effect on mitochondrial CO2 fixation. Rather, catecholamines caused a smaller inhibition of pyruvate kinase activity than did glucagon. The effects of catecholamines on pyruvate kinase also appeared to be mediated by an alpha-adrenergic, adenosine 3':5'-monophosphate-independent mechanism.

Effects of CuCl 2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart

CuCl 2 non-comepetitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg 2+, 10 μM Ca 2+ and phosphodiesterase activator with K i values of approximately 2 μM for both substrates. CuCl 2 inhibition was also non-competitive with Mg 2+, Ca 2+ and phosphodiesterase activator. Dialysis demonstrated that CuCl 2 inhibition in reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl 2 was not additive with that p-hydroxymercuribenzoate, therefore CuCl 2 may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl 2 also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I 50 value of 18 μM. Several effects of Cu 2+ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.

Regulation of antigen induced changes in cyclic nucleotide levels in NZB/WF 1 Mice

Normal C57BL/6J mice respond to the iv injection of antigen with an increase in splenic cAMP at 2 min. NZB/WF 1 mice are predisposed to autoimmune and immunological disorders upon aging. The ability of NZB/WF 1 mice to respond to antigen with an increase in their splenic cAMP level was found to diminish with age. This loss of responsiveness is antigen specific and not due to a loss of adenylate cyclase activity in spleen cells of old NZB/WF 1 mice. The adoptive transfer of spleen cells from unresponsive old mice into responder young mice inhibited the cAMP response to antigen by the recipients. Spleen cells from young responsive mice, on transfer into old nonresponsive NZB/WF 1 recipients, resulted in restoration of the cAMP response to antigen. In both cases, the activity of donor cells was dependent on the transfer of T cells. These results indicate that populations of T cells participate in the regulation of the cAMP response to antigen by NZB/WF 1 mice. The response of old mice could also be restored by treatment with indomethacin, and also the spleen cells of such treated donors failed to suppress the cAMP response of young recipients. Together, the results suggest a role for prostaglandins in regulating the cAMP response to antigen.

Dissociation of cAMP changes and myocardial contractility in taurine perfused rat heart

It has been determined that the inotropic effects of taurine in the rat heart are not necessarily mediated through changes in cyclic nucleotide levels. Taurine caused a transient 2-fold decrease in cAMP levels, and this was accompanied by a slight (5%), but significant positive inotropic effect. This cannot be explained by an antagonism of cAMP levels by 3′, 5′ -guanosine monophosphate (cGMP), since the latter remained essentially unchanged. Epinephrine, as is known, caused a rapid increase in cAMP levels. Perfusion with both taurine and epinephrine increased cAMP levels to the same extent as perfusion with epinephrine alone.

Effects of zinc chloride on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart

In the presence of 10 μM Ca 2+ and 5 mM Mg 2+ (or 0.25 mM Mg 2+), the addition of 100 μM Zn 2+, Ni 2+, Co 2+, Fe 2+, Cu 2+ or 1 mM Mn 2+ resulted in varying degrees of stimulation or inhibition of 10 −6 M cyclic GMP and cyclic AMP hydrolysis by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart in the absence or presence of phosphodiesterase activator. The substrate specificity of the enzyme was altered under several conditions. The addition of Zn 2+ in the presence of 5 mM Mg 2+ and the absence of activator resulted in the stimulation of cyclic GMP hydrolysis over a narrow substrate range while reducing the V 65% due to a shift in the kinetics from non-linear with Mg 2+ alone to linear in the presence of Zn 2+ and Mg 2+. Zn 2+ inhibited the hydrolysis of cyclic GMP and cyclic AMP in the presence of activator with K i values of 70 and 100 μM, respectively. Zn 2+ inhibition was non-competitive with substrate, activator and Ca 2+ but was competitive with Mg 2+. In the presence of 10 μM Ca 2+ and activator, a K i of 15 μM for Zn 2+ vs. Mg 2+ was noted in the hydrolysis of 10 −6 M cyclic GMP. Several effects of Zn 2+ are discussed which have been noted in other studies and might be due in part to changes in cyclic nucleotide levels following phosphodiesterase inhibition.

Interaction between cyclic nucleotides and herpes simplex viruses: productive infection

Infection of human fibroblasts and HEp-2 cells with herpes simplex virus type 1 (HSV-1) produced a decrease in the intracellular levels of cyclic adenosine 5'- monophosphate (cAMP) and a concomitant increase in the cyclic guanosine 5'- monophosphate (cGMP) levels. In both cell cultures, changes in cyclic nucleotide levels were first observed at 6 h after viral inoculation and were maximal at 12 h. In human fibroblasts, the addition of theophylline, dibutyryl cAMP, or papaverine (cAMP-enhancing compounds) decreased significantly the yield of HSV-1, whereas the addition of insulin or dibutyryl cGMP (cGMP-enhancing compounds) increased the viral yield. In HEp-2 cells, only theophylline decreased the yield of HSV-1, and the cGMP-enhancing compounds had no apparent effect. Cyclic nucleotide enhancing compounds exhibited their effect only if added to either cell culture within the first 3 h after inoculation with HSV-1.

DISTRIBUTION AND REGULATION OF CYCLIC NUCLEOTIDE LEVELS IN CEREBELLUM, IN VIVO1

Abstract— In mouse cerebellum, in vivo. cyclic GMP levels are 7 pmol/mg protein in the vermis and 40% lower in the hemispheres, whereas cyclic AMP levels are 7 9 pmol/mg protein in both regions. In the vermis. most of the cyclic GMP is contained in the molecular layer; cyclic AMP levels are highest in the granular layer. Amphetamine, harmaline. pentylenetetrazol and physical shaking elevate, and diazepam and reserpine depress levels of cyclic GMP in both vermis and hemispheres. Oxotremorine and atropine, respectively, increase and decrease cyclic GMP levels only in vermis. Regardless of the agent used, most of the change (67 89%) in cyclic GMP levels occurs in the molecular layer of the vermis; the remainder occurs in the granular layer. Of the drugs tested, only pentylenetetrazol affects cyclic AMP levels, and this drug increases cyclic AMP levels in both vermis and hemispheres and causes equal elevations in the molecular and granular layers of the vermis. In incubated slices of mouse cerebellum, none of the drugs produces changes in cyclic nucleotide levels which are similar to those in vivo. These data indicate that many drugs and conditions that alter cyclic GMP levels in cerebellum act via a common, but indirect, process.We suggest that cyclic GMP levels in cerebellum are regulated by the activity of both the climbing fiber and mossy fiber cerebellar afferent systems. Increased activity in these afferent pathways causes elevation of cyclic GMP levels in Purkinje cells and perhaps in other cells; decreased activity leads to depressed cyclic GMP levels.

Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods. Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio. Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.

Early changes in splenic DNA synthesis induced by thymic-dependent and thymic-independent antigens in vivo

Antigen-induced early alterations in splenic [ 3H]Tdr incorporation were studied in normal and athymic mice. Antigens which have been demonstrated to be thymus dependent (sRBC and BA), as well as a thymic-independent antigen (PVP), were employed. The results confirm that thymic-dependent antigens induce an early decrease in DNA synthetic rate (between 4 and 8 hr). Also, an increase in the rate of DNA synthesis between 10 and 20 hr has been detected. These alterations appear to be dependent on the synthesis of prostaglandin, and may be linked to antigen-induced changes in cyclic nucleotide levels. The T-cell-independent antigen PVP does not induce an early suppression of DNA synthesis, but instead stimulates an increase in DNA synthesis between 0 and 8 hr in both normal and athymic mice. Experiments performed using athymic “nude” mice and cortisone-treated mice support the hypothesis that antigen-induced suppression is dependent on a cortisone-resistant T cell. Lastly, the increased DNA synthesis observed using PVP is delayed in nude mice, suggesting that more than one cell type may be involved.

Cell-cycle-related changes of 3':5'-cyclic GMP levels in Novikoff hepatoma cells.

Intracellular and extracellular levels of 3':5'-cyclic GMP and 3':5'-cyclic AMP were studied in synchronized Novikoff rat hepatoma cells. Intracellular levels of cyclic GMP increased spontaneously from 2-fold (without colcemid) to 10-fold (with colcemid), in proportion to the number of cells in mitosis. As cells entered mitosis, cellular cyclic AMP declined simultaneously with the rise in cyclic GMP. These reciprocal changes in cyclic nucleotide levels were reversed as cells passed out of metaphase and through anaphase. Maximum cyclic AMP and minimum cyclic GMP concentrations occurred during G-1. Less marked reciprocal fluctuations in both cyclic nucleotides were also found in S-phase and early G-2, where the ratio of cyclic AMP to cyclic GMP concentrations first fell and then increased. These changes in cyclic nucleotide ratios were closely correlated with major cell-cycle transitions at the boundaries between G-1/S-phase, S-phase/G-2, G-2/prophase, and metaphase/anaphase. Most, but not all, of the extracellular cyclic nucleotides were extruded when cells traversed mitosis. Colcemid or vinblastine completely prevented the appearance of extracellular cyclic AMP but augmented the appearance of extracellular cyclic GMP in parallel with the accumulation of mitotic cells. These results reflected changes in intracellular cyclic nucleotides and indicated that increased intracellular turnover of cyclic GMP and cyclic AMP occurred before and after metaphase, respectively. Elevated cyclic GMP levels during mitosis and S-phase are consistent with potential modulatory roles for this cyclic nucleotide in proliferation.

199) - Changes in cyclic nucleotide levels in the catecholamine release from dog adrenal medulla

199) - Changes in cyclic nucleotide levels in the catecholamine release from dog adrenal medulla

Alterations in splanchnic cyclic nucleotide levels in splanchnic artery occlusion shock and their modification by dexamethasone

The effects of dexamethasone sodium phosphate (DSP), 5 mg/kg, administration on the biochemical alterations in hepatic tissue subsequent to the production of splanchnic artery occlusion (SAO) shock was investigated. Following the induction of SAO shock, DSP-treated dogs exhibited a significantly improved cardiovascular status compared to placebo-treated shocked dogs. 2 hr after release of the occlusion, biopsies of liver were taken and analyzed for β-glucuronidase (BG), adenosine-3′,5′-cyclic monophosphate (cAMP) and guanosine-3′,5′-cyclic monophosphate (cGMP) content. SAO shock produced a significant increase in hepatic free BG activity which was reversed by DSP pretreatment. Additionally, SAO shock decreased hepatic cAMP levels, increased cGMP levels and significantly lowered the hepatic ratio of cAMP/cGMP. These changes in cyclic nucleotide levels were reversed by DSP administration and were found to be inversely related to changes in hepatic free BG activity. Thus, the ratio of cellular cAMP/cGMP may function as a regulatory mechanism for lysosomal enzyme release secondary to ischemia and hypoxia. Further, DSP may act to maintain lysosomal integrity in ischemic tissues by preservation of cAMP/cGMP ratios.

Changes in cyclic nucleotide levels and contractile force in the isolated hypoxic rat heart during perfusion with glucagon.

Isolated rat hearts were perfused with hormonal concentrations of glucagon during a hypoxic perfusion to determine whether it would enhance recovery after reoxygenation. Rat hearts were divided into two groups: (1) those perfused with glucose-free Tyrode's solution and (2) those perfused with Tyrode's solution containing glucose. During 3 minutes of exposure to hypoxia both untreated hearts and hearts perfused with glucagon demonstrated a decrease in contractile force to 10-20% of control. When glucose was present in the perfusion medium, cardiac performance was better during both the periods of hypoxia and reoxygenation. During reoxygenation, recovery of contractile force was significantly better (P less than 0.05) in glucagon-perfused hearts than in untreated hearts; this improved recovery occurred regardless of whether glucose was included in the medium. The enhanced recovery of the glucagon-perfused hearts was associated with decreases in myocardial levels of guanosine, 3',5'-monophosphate (cyclic GMP) both during the periods of hypoxia and reoxygenation. At the end of the hypoxic period, cyclic GMP levels in the glucagon-perfused hearts were 20-64% of the levels in untreated hearts. Similarly, after 5 minutes of reoxygenation cyclic GMP levels in the glucagon-perfused hearts were 21% of the levels in the untreated hearts. The effect of glucagon on adenosine 3',5'-monophosphate (cyclic AMP) concentrations in untreated hearts and in hearts receiving glucagon was not significantly different either after 3 minutes of hypoxia or during reoxygenation. The rate of anaerobic glycolysis after 3 minutes of hypoxia was higher in untreated hearts than in glucagon-perfused hearts, as determined by the lactate content of coronary perfusates. These studies suggest that hormonal concentrations of glucagon exert a protective effect on the hypoxic rat heart which involves a modulation of cardiac cyclic GMP accumulation.

Mediation of serum-induced changes in cyclic nucleotide levels in cultured fibroblasts by cyclic nucleotide phosphodiesterase

CELL cultures of untransformed fibroblasts can be maintained in a stationary or relatively quiescent state by adjusting serum concentrations to suboptimal levels1. Addition of excess serum to quiescent cell cultures causes a rapid decrease in the intracellular concentration of cyclic AMP and a rapid increase in cyclic GMP levels2–5. These alterations in cyclic nucleotide concentrations precede the changes in macromolecular synthesis (such as RNA, protein and DNA synthesis) and growth induced by serum, and constitute one of the earliest steps in the sequence of events before the initiation of growth. The mechanism responsible for altering cyclic nucleotide concentrations in response to serum or other growth stimuli has not been elucidated however. We show here that a rapid alteration in cyclic nucleotide phosphodiesterase activity occurs when serum is added to quiescent fibroblasts and demonstrate that this alteration could mediate the changes in cyclic nucleotide levels.

Control of mitogen-induced lymphocyte activation: II. Analysis of cell populations and metabolic events involved in cyclic AMP-mediated recovery of DNA synthesis suppressed by mitogens

Exposure of human peripheral blood lymphocytes to increased doses of PHA (0–2000 μg/ml PHA) leads to a decrease in thymidine incorporation. The addition of exogenous cyclic AMP (10 −3–10 −4 M) to lymphocyte cultures exposed to such high levels of PHA results in a recovery of the ability to incorporate both thymidine and uridine. Using this system as a model for the study of cyclic nucleotide metabolism, experiments have demonstrated a differential sensitivity to changes in cyclic nucleotide levels among lymphocyte subpopulations exposed to PHA, concanavalin A, or pokeweed mitogen. Further evidence for differential sensitivity is also obtained using lymphocyte populations separated on the basis of rosette formation. Measurement of intracellular cAMP and cGMP levels in lymphocyte cultures exposed to PHA, concanavalin A, or pokeweed mitogen suggests that while cGMP is the mitogenic signal, cAMP may control deactivation, thereby exerting an overide effect on cGMP mediated events. In addition, metabolic studies suggest that exogenous cAMP may influence PHA-induced transformation up to 67 hr after culture initiation and that the effects of Ca 2+ ions and cyclic nucleotides are interrelated in the activation of lymphocytes by PHA.

Antigen-stimulated changes in cyclic nucleotide levels in the mouse.

Mice injected intravenously with sheep erythrocytes (sRBC) demonstrate a transient increase in splenic cAMP levels (4-fold), which peak at 2 min after injection and return to basal levels by 20 min. In addition to the change in cAMP, an increase in splenic cGMP levels (1.5-fold) occurs beginning 5-10 min after sRBC injection, and persists for up to 7 days. During this period cAMP levels remain at or below control levels in the spleen. There is no change in 3':5' cyclic nucleotide levels in the liver and a small increase (1.2- to 1.3-fold) in the thymus at the time when splenic cyclic nucleotide levels are elevated. The changes in splenic cyclic nucleotide levels appear to be dependent on the presence of thymus-derived (T) lymphocytes, since little change occurs in cAMP and changes in cGMP are absent in athymic nude mice. In addition, cAMP levels were increased by pretreatment of normal mice with cortisone acetate, which selects for mature T lymphocytes. Agents that block autonomic nervous system functions have no effect on the early sRBC-induced changes in cyclic nucleotide levels.Indomethacin, an inhibitor of prostaglandin synthesis, reduces the change in cAMP level by 50% and blocks the change in cGMP levels completely. Secondary stimulation with sRBC results in a larger increase in cAMP levels than that seen with a primary injection of sRBC, indicating the presence of specific antigen-sensitive memory cells. Changes in splenic cyclic nucleotide levels cannot be detected at early times after the injection of soluble protein antigens such as bovine serum albumin and keyhole limpet hemocyanin. Salmonella H antigen stimulates a 2-fold increase in cAMP levels, the increase occurring more slowly than with sRBC. The in vivo changes incyclic nucleotide levels are correlated with known changes in cyclic nucleotide levels which have been documented in vitro in both T-cells and T-cell-dependent-B-cell (bone-marrow derived) antibody responses.

Effects of norepinephrine on cyclic nucleotide levels in the ductus deferens of the rat

The effect of l-norepinephrine on cyclic nucleotide levels was studied in rat ductus deferens. Norepinephrine (0.01–0.3 m M) increased cyclic AMP and cyclic GMP levels in a concentration-dependent manner. Cyclic GMP was increased by about two-fold and cyclic AMP by 30%–40% by 0.1 m M norepinephrine after 3 min. Phenylephrine (0.1 m M) increased cyclic GMP levels about twofold without affecting cyclic AMP. Cyclic GMP was maximally increased by norepinephrine or phenylephrine after 3–10 min, but was not significantly changed after 20 sec, when contraction was fully developed. Atropine did not affect the norepinephrine-induced changes in cyclic nucleotide levels; phentolamine blocked the effect of norepinephrine on cyclic GMP, and propanolol blocked that on cyclic AMP. These findings indicate that in the ductus deferens α-adrenergic receptors are involved in the effect of catecholamines on cyclic GMP, and β-adrenergic receptors are involved in the effect on cyclic AMP levels. The presence of Ca 2+ was required for the effect of norepinephrine on cyclic GMP, but not on cyclic AMP.

Cyclic nucleotide levels during spontaneous uterine contractions.

Tissue levels of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP) were determined in uterine muscles frozen at various points during spontaneous contraction–relaxation cycles. No significant changes in cyclic nucleotide levels were detected at any of the stages of contraction studied. Exposure of uterine segments to 1 μM isoproterenol resulted in an eightfold increase in cyclic AMP levels but no change in cyclic GMP, whereas exposure to 2 mM theophylline resulted in a doubling of cyclic GMP levels and a 42% increase in cyclic AMP content. Thus, the methods used were capable of detecting changes in cyclic nucleotide levels when they did occur. It was concluded that changes in cyclic nucleotide levels do not play a role in the initiation or regulation of spontaneous contractions of isolated rat uterus.

Daily fluctuation (circadian and ultradian) in biogenic amines of the rat brain.

Daily fluctuation (circadian and ultradian) in biogenic amines of the rat brain.