Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

Stimulation of DNA synthesis and mitosis of hepatocytes in primary cultures of neonatal rat liver by arachidonic acid and prostaglandins

At low concentrations (i.e. 10⁻¹²–10⁻⁹ mol/l) arachidonic acid intensely stimulated both DNA synthetic and mitotic activities of hepatocytes in 4-day-old primary cultures of neonatal rat liver. This effect of arachidonate was completely suppressed by the simultaneous administration to the cultures of a high dose (i.e. 10⁻⁴ mol/l) of indomethacin. A similar, but much weaker proliferogenic activity was exerted on neonatal hepatocytes by quite low concentrations of some of the main products of arachidonic acid metabolism, namely prostaglandins A₁, E₁, and E₂. Although these data support the possibility that arachidonate and prostaglandins are involved in the regulation of hepatocytic proliferative activation, the exact role of prostaglandins remains to be ascertained, because such agents might as well have acted by inducing intracellular surges of known mitogenic compounds, such as cAMP and cGMP.     

Cyclic GMP stimulation and inhibition of cyclic AMP phosphodiesterase from thymic lymphocytes

A particulate preparation of cyclic AMP phosphodiesterase from rat thymic lymphocytes exhibited two apparent Km's at 0.9×10⁻⁶M and 8.0×10⁻⁶M. The enzyme with the higher Km was stimulated by cyclic GMP by a mechanism involving an increase in the Vmax of the enzyme with no change in the Km. Cyclic GMP competitively inhibited the enzyme with the low apparent Km which had a Ki for cyclic GMP of 4×10⁻⁵M. The modulation of cyclic AMP phosphodiesterase activity by cyclic GMP in the control of cyclic AMP-mediated lymphocyte proliferation is discussed.     

Mechanism of stimulation of DNA synthesis induced by epinephrine in primary culture of adult rat hepatocytes

Addition of epinephrine to primary cultured adult rat hepatocytes stimulated their DNA synthesis dose-dependently, especially in presence of insulin and epidermal growth factor. This effect of epinephrine was strongly inhibited by an alpha 1-antagonist, prazosin, but not by a beta-antagonist, propranolol, and was also slightly inhibited by an alpha 2-antagonist, yohinbin. These results indicate that the stimulation of DNA synthesis of hepatocytes by epinephrine is mediated predominantly by an alpha 1-action. 12-o-Tetradecanoylphorbol-13-acetate (TPA) or Ca2+-ionophore A-23187 stimulated DNA synthesis of Swiss 3T3 cells, but did not induce DNA synthesis of hepatocytes either singly or in combination. The fact that pretreatment of hepatocytes with TPA caused down-regulation of the stimulatory effect of epinephrine on DNA synthesis of hepatocytes within 15 min suggested that the effect of epinephrine on hepatocytes is mediated by its alpha 1 receptor and that TPA activated protein kinase c in the hepatocytes. Addition of dibutyryl cGMP did not induce DNA synthesis of hepatocytes. Therefore, the alpha 1-action of epinephrine that induce stimulation of DNA synthesis of primary cultured adult rat hepatocytes was apparently not mediated by either activation of phospholipid-dependent protein kinase or Ca2+ mobilization. Possible alternative mechanism was discussed.     

Cyclic AMP, cyclic GMP and glucose utilization by diabetic rat fat cells

Fat cells from epididymal adipose tissue from normal and streptozotocin-diabetic rats were studied to determine glucose utilization and cyclic nucleotide levels. Diabetic rat fat cells present a higher cAMP content (P less than 0.05) compared with controls. Addition of insulin decreases within 10-min incubation the cAMP content in both normal and diabetic cells (P less than 0.05). However, the value obtained in the latter remains by 25% higher than that of normal cells not exposed to insulin. No changes in cGMP were detected. Pretreatment of the diabetic animals during two days with propranolol (1 mg kg body wt-1 day-1) induces the decrease to normal levels of the fat cell cAMP content. However, it persists the impairment on glucose utilization observed in fat cells from diabetic animals. It seems that the increase in the intracellular amount of cAMP found in fat cells from diabetic rats is not involved, at least directly, to the impaired glucose utilization found in the diabetic state. Furthermore, through an unknown mechanism, pretreatment with propranolol can induce a drop in fat tissue cAMP toward normal values without normalizing glucose utilization.     

Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes

Separate or combined addition of cyclic AMP-dependent and Ca2+-linked hormones to isolated rat hepatocytes suspended in a low Ca2+ medium reduced the total cellular Ca. When the hormones were administered together, their effects were not additive. This suggests that both types of hormones mobilize Ca2+ from a common intracellular pool. In the presence of 1.8 mM extracellular Ca2+, the Ca2+ influx counterbalanced or even exceeded the hormone-induced Ca2+ loss, depending on the ability of the hormones to stimulate the Ca2+ influx.     

The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin

A low concentration (10(-11) mol/l) of epidermal growth factor (EGF) and/or an equimolar (10(-14) mol/l) mixture of glucagon and insulin stimulated DNA synthesis in hepatocytes in 4-day-old primary cultures of neonatal rat liver. EGF seems to have acted by inducing quiescent hepatocytes to begin cycling, while the glucagon-insulin combination seems to have acted mainly by shortening the cell cycle time. Incubation in low calcium medium blocked untreated hepatocytes in the G1 phase of their cycle and prevented EGF and the glucagon-insulin mixture from stimulating DNA synthesis. Nevertheless, hepatocytes in calcium-deficient medium did respond to these agents, as they reached a late stage of prereplicative development before being blocked: in fact, they initiated DNA synthesis soon after the addition of calcium. EGF, but not the glucagon-insulin combination, also enabled the already cycling hepatocytes (but not the newly activated ones) to overcome the block imposed by the extracellular calcium deficiency after a delay of several hours.     

Cyclic GMP metabolism in macrophages. I. Regulation of cyclic GMP levels by calcium and stimulation of cyclic GMP synthesis by NO-generating agents

Levels of guanosine 3′,5′-cyclic monophosphate (cGMP) were determined by radioimmunoassay in adherence-purified, oil-induced guinea pig peritoneal exudate macrophages, after extraction of the cells with perchloric acid, purification on Dowex AG1-X8, and acetylation. We found that: (i) Basal cGMP levels were strictly dependent on the concentration of extracellular Ca²⁺ (0.33 ± 0.03 pmol/mg macrophage protein in Ca²⁺-free medium and 2.49 ± 0.42 pmol/mg in 1.8 mM Ca²⁺). (ii) The stimulatory effect of Ca²⁺ on cGMP levels was prevented by tetracaine. (iii) The cGMP content of macrophages was not elevated by incubation with the ionophore A23187 at extracellular Ca²⁺ concentrations varying between 0 and 1.8 mM. (iv) Macrophage cGMP levels were increased markedly (up to 40-fold) by incubation of the cells with the nitric oxide (NO)-generating agents, sodium azide, hydroxylamine, sodium nitrite, and sodium nitroprusside. (v) Stimulation of cGMP accumulation by NO-generating agents occurred within 30 sec, was Ca²⁺-independent, and developed in the presence and absence of the phosphodiesterase inhibitor, isobutyl-methylxanthine. (vi) A minimal elevation in the macrophage cGMP level (less than 2-fold) was induced by ascorbic acid but no significant increases were induced by the following agents, found effective in other cells: serotonin, acetylcholine, carbamylcholine, phorbol myristate acetate, arachidonic acid, Superoxide dismutase, and nitrate reductase.     

Cyclic AMP and cyclic GMP control of synthesis of constitutive enzymes in Escherichia coli

Escherichia coli was grown in chemostat culture under glycerol-limited and ammonium-limited conditions at growth rates between 0.1 and 0.5 h-1. At steady state, the concentrations of cyclic AMP and cyclic GMP and the activities of four constitutive enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, NADH oxidase and cyclic phosphodiesterase) were determined in the organism. Addition of exogenous cyclic AMP, cyclic GMP or phencyclidine perturbed the steady state and caused inhibition or stimulation of synthesis of phosphodiesterase and isocitrate dehydrogenase. A novel hypothesis is proposed to account for the ability of bacteria to regulate the synthesis of constitutive enzymes with cyclic nucleotides and possibly other small molecules.     

Regional sensitivity of cyclic AMP and cyclic GMP in rat brain to central cholinergic stimulation

We determined cyclic AMP and cyclic GMP levels in 18 regions of rat brain following administration of two different centrally active cholinergic agonists. Administration of oxotremorine (2 mg/Kg IP), a muscarinic agonist, 10 minutes prior to sacrifice by exposure to high power microwave irradiation resulted in significant increases in cyclic GMP in cerebellum, brainstem, hippocampus, midbrain, thalamus and septal region. Cyclic AMP levels were significantly elevated in substantia nigra, nucleus interpeduncularis, hypothalamus, brainstem, midbrain and in the pituitary where a greater than tenfold increase was observed. Levels of plasma prolactin and corticosterone did not differ in any of the groups examined, but growth hormone was significantly lower in animals exposed to oxotremorine. Physostigmine (0.5 mg/Kg) a cholinesterase inhibitor, administered IP also produced elevations in cyclic AMP and cyclic GMP in several of the brain regions examined. These results indicate that multiple regions of the brain are responsive to central cholinergic activation of not only cyclic GMP, but also cyclic AMP system.     

Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue.

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.     

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.     

Changes in levels of cyclic AMP and cyclic GMP in various tissues of the rat induced by cold acclimation

In the rat, the effects of cold acclimation on the content of cyclic AMP and cyclic GMP were studied in various tissues concerned with increased heat production: brown and white adipose tissue, liver, heart, diaphragm, lungs, adrenals, thyroid. Significant cold-induced variations were observed only in those tissues in which the lipid metabolism is enhanced by cold (adipose tissues and liver). In these tissues, decrease in the cAMP/cGMP ratio indicates a role of cGMP in the regulation of the increased lipid metabolism.     

Cyclic GMP and cyclic AMP induced changes in control and hypertrophic cardiac myocyte function interact through cyclic GMP affected cyclic-AMP phosphodiesterases.

We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) would be greater after increasing cyclic AMP (cAMP), because of the action of cGMP-affected cAMP phosphodiesterases in cardiac myocytes and that this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. Myocyte shortening data were collected using a video edge detector, and O2 consumption was measured by O2 electrodes during stimulation (5 ms, 1 Hz, in 2 mM Ca2+) from control (n = 7) and LVH (n = 7) dog ventricular myocytes. cAMP and cGMP were determined by a competitive binding assay. cAMP was increased by forskolin and milrinone (10(-6) M). cGMP was increased with zaprinast and decreased by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxilin-1-one (ODQ) both at 10(-6) and 10(-4) M, with and without forskolin or forskolin + milrinone. Zaprinast significantly decreased percent shortening in control (9 +/- 1 to 7 +/- 1%) and LVH (10 +/- 1 to 7 +/- 1%) myocytes. It increased cGMP in control (36 +/- 5 to 52 +/- 7 fmol/10(5) myocytes) and from the significantly higher baseline value in LVH (71 +/- 12 to 104 +/- 18 fmol/10(5) myocytes). ODQ increased myocyte function and decreased cGMP levels in control and LVH myocytes. Forskolin + milrinone increased cAMP levels in control (6 +/- 1 to 15 +/- 2 pmol/10(5) myocytes) and LVH (8 +/- 1 to 18 +/- 2 pmol/10(5) myocytes) myocytes, as did forskolin alone. They also significantly increased percent shortening. There were significant negative functional effects of zaprinast after forskolin + milrinone in control (15 +/- 2 to 9 +/- 1%), which were greater than zaprinast alone, and LVH (12 +/- 1 to 9 +/- 1%). This was associated with an increase in cGMP and a reduction in the increased cAMP induced by forskolin or milrinone. ODQ did not further increase function after forskolin or milrinone in control myocytes, despite lowering cGMP. However, it prevented the forskolin and milrinone induced increase in cAMP. In hypertrophy, ODQ lowered cGMP and increased function after forskolin. ODQ did not affect cAMP after forskolin and milrinone in LVH. Thus, the level of cGMP was inversely correlated with myocyte function. When cAMP levels were elevated, cGMP was still inversely correlated with myocyte function. This was, in part, related to alterations in cAMP. The interaction between cGMP and cAMP was altered in LVH myocytes.     

Ultrastructural localization of cyclic GMP and cyclic AMP in rat striatum

The subcellular localization of cyclic GMP and cyclic AMP in the rat caudate-putamen has been studied using horseradish peroxidase immunocytochemistry. Both of the putative neurotransmitter second messengers were visualized in neurons and glial cells at light microscopic resolutions, but not all cells of either category gave detectable staining. This was confirmed at the ultrastructural level where both stained and unstained elements of the same cell type were found within the same field. A striking variation was seen in cyclic nucleotide staining intensity within individual neural and glial cells. Both of the cyclic nucleotides were detected within postsynaptic terminal boutons and within astroglial processes. Cyclic GMP postsynaptic staining was stronger than glial staining, whereas the localization pattern was reversed for cyclic AMP. The synaptic localization of cyclic AMP and cyclic GMP immunoreactivity adds support to the idea that these compounds have an influential role in synaptic function within the striatum.