Cyclic nucleotides and intracellular-calcium homeostasis in human platelets.

The relationship between agonist-sensitive calcium compartments and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in human platelets. In this context, calcium mobilization from intracellular pools and manganese influx was investigated in relation to the effect of altered cyclic-nucleotide levels. For maximal calcium release from intracellular stores, thapsigargin, compared to a receptor agonist like thrombin, requires the platelet's self-amplification mechanism, known to generate thromboxane A2. With this lipid mediator formed, thapsigargin released calcium and stimulated manganese influx in a manner similar to thrombin. Blocking the thromboxane receptor by addition of sulotroban (BM13.177) or, alternatively, increasing platelet cAMP or cGMP using prostacyclin or sodium nitroprusside, dramatically reduced the ability of thapsigargin to release calcium from intracellular compartments. The same experimental conditions significantly reduced the rate of manganese influx initiated by thapsigargin compared to thrombin. The experiments indicate that thapsigargin-sensitive compartments play only a minor role in inducing manganese influx compared to the receptor-sensitive compartment. Cyclic nucleotides accelerate the redistribution of an agonist-elevated platelet calcium into the thapsigargin-sensitive compartment, from which calcium can be released by inhibition of the Ca(2+)-ATPase. In human platelets, thapsigargin-induced calcium increase and influx were responsible for only part the calcium release resulting from inhibition of the corresponding ATPase; another part results from the indirect effect of thapsigargin acting via thromboxane-A2-receptor activation. Cyclic nucleotides are therefore an interesting regulatory device which can modify the thapsigargin response by not allowing the self-amplification mechanism of platelets to operate.     

Cyclic nucleotides of human and animal glial tumors

Studies on the level of cyclic nucleotides (cAMP and cGMP) in human and animal glial tumours showed that the content of both nucleotides, especially that of cAMP, decreases in all the tumours. The cAMP/cGMP ratio also drops down. Concurrently it appears to be the most consistent parameter of nucleotide metabolism both in brain tissue and in human or animal glial tumours. The growing tumour affects cAMP and cGMP metabolism not only in the involved but also in the other hemisphere. No principal differences between human and animal tumours have been revealed in the content of cyclic nucleotides and its variation in tumour tissue.     

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.     

Cyclic nucleotides modulate store-mediated calcium entry through the activation of protein-tyrosine phosphatases and altered actin polymerization in human platelets

Agonists elevate the cytosolic calcium concentration in human platelets via a receptor-operated mechanism, involving both Ca(2+) release from intracellular stores and subsequent Ca(2+) entry, which can be inhibited by platelet inhibitors, such as prostaglandin E(1) and nitroprusside which elevate cAMP and cGMP, respectively. In the present study we investigated the mechanisms by which cAMP and cGMP modulate store-mediated Ca(2+) entry. Both prostaglandin E(1) and sodium nitroprusside inhibited thapsigargin-evoked store-mediated Ca(2+) entry and actin polymerization. However, addition of these agents after induction of store-mediated Ca(2+) entry did not affect either Ca(2+) entry or actin polymerization. Furthermore, prostaglandin E(1) and sodium nitroprusside dramatically inhibited the tyrosine phosphorylation induced by depletion of the internal Ca(2+) stores or agonist stimulation without affecting the activation of Ras or the Ras-activated phosphatidylinositol 3-kinase or extracellular signal-related kinase (ERK) pathways. Inhibition of cyclic nucleotide-dependent protein kinases prevented inhibition of agonist-evoked Ca(2+) release but it did not have any effect on the inhibition of Ca(2+) entry or actin polymerization. Phenylarsine oxide and vanadate, inhibitors of protein-tyrosine phosphatases prevented the inhibitory effects of the cGMP and cAMP elevating agents on Ca(2+) entry and actin polymerization. These results suggest that Ca(2+) entry in human platelets is directly down-regulated by cGMP and cAMP by a mechanism involving the inhibition of cytoskeletal reorganization via the activation of protein tyrosine phosphatases.     

Cyclic AMP phosphodiesterase in human lymphocytes and lymphoblasts.

Cyclic nucleotide phosphodiesterase activities were examined in lymphocytes from 12 transformed human B cell lines, two T cell lines, six patients with lymphocytic leukemia, and 10 normal donors. A consistent difference bwtween cells from the normal and leukemic state was observed. The cyclic AMP phosphodiesterase activity from normal lymphocytes is inhibited greater than 80% by muM cyclic GMP while this concentration of nucleotide has little or no effect on the enzyme from transformed lymphocytic cell lines or from lymphocytic cells of leukemia patients. The reported lack of cyclic GMP phosphodiesterase in human lymphocytes from several sources is confirmed. The apparent absence of a cyclic GMP degradation mechanism and of cyclic GMP control of cyclic AMP hydrolysis may be related to defective lymphocyte growth control.     

Cyclic nucleotide levels in activated human T lymphocytes

Phytohemagglutinin (PHA)-MR69 was used to activate human peripheral blood lymphocytes. The PHA concentration in the range of 1 to 4 micrograms/ml was optimal for lymphocyte stimulation. Cell activation occurred only in the presence of Ca ions and 5 min after it was followed by an increase in cGMP but not in cAMP. Immunomodulator, methylene bisphosphonic acid (10(-7) M and 4.10(-5) M), did not influence in culture. The cAMP and cGMP levels in PHA activated cells. Methylene bisphosphonic acid similar to 1-hydroxyenthylidene-1,1-bisphosphonic acid, aminomethylene bisphosphonic acid and phosphoneacetic acid on its addition to the culture (in the range from 10(-8) to 10(-4)M) 60 min before PHA or 24 or 48 hours after PHA administration produced no effect on the [3H]-incorporation into PHA-activated human blood lymphocytes.     

Cyclic nucleotides in experimental glaucoma

cAMP and cGMP contents were studied in various eye tissues of rabbits with experimental glaucoma induced by chronic intravenous adrenaline administration. Cyclic nucleotide level was measured in the retina, choroid, iris and ciliary body. An increase in the tissue cAMP level was found especially in the iris and ciliary body. An increase in tissue cAMP content is explained by an enhanced beta-adrenergic regulation in the eyes of rabbits with experimental glaucoma. No consistent changes were found in cGMP content in eye tissues.     

Cyclic nucleotides and cell growth

Growth induction in resting fibroblast cultures by serum or growth factors induces a fast, transient cGMP peak which may constitute the intracellular signal for growth. A similar cGMP peak occurs when 3T3 cells arrested at the restriction point or in G₀ by starvation for certain amino acids are induced for growth by readdition of the lacking nutrients. Both 3T3 and SV3T3 cells which are arrested randomly all around the cell cycle do not exhibit major changes in cyclic nucleotides after growth induction. Determination of intracellular cAMP and cGMP levels in normal and transformed fibroblasts under different growth conditions shows that the transition between growing and resting state (G₀ arrest) is accompanied and probably induced by characteristic changes in cAMP to cGMP ratios. cGMP is decreased 2-5-fold in resting as compared to growing cultures, and increased 10-20-fold in activated cultures 20 min after serum induction. No major cGMP change was observed in growing, confluent, or serum-activated cultures of transformed cells. Measurement of guanylcyclase under unphysiological conditions (2 mM Mn⁺⁺) in crude and purified membranes from 3T3 and SV3T3 cultures did not show increased enzyme activity in the transformed cells. Significant differences may only show up when synchronized cells pass through the restriction point in G₁ phase. As a hypothesis it is proposed that transformed cells have an activated guanylcyclase system or a relaxed cGMP-pleiotypic response mechanism at the restriction point of their cell cycle.     

Cyclic nucleotides and neuroblastoma differentiation

We have shown that intracellular cGMP levels increase during retinoic acid- and mycophenolic acid-induced neuroblastoma differentiation and that a 6 days treatment with 1 mM dbcGMP lead LAN5 cell to elaborate a network of neuritic processes suggesting an involvement of cGMP in neuroblastoma differentiation. We have also investigated the effects of some specific inhibitors of phosphodiesterases (PDE1, PDE3, PDE4 and PDE5) on human neuroblastoma (LAN5 and SHEP) growth and differentiation. After six days of incubation in the presence of each specific inhibitor at 10 x IC50 levels a cytostatic and differentiating effect was only observed with the PDE5 inhibitors Zaprinast and MY-5445. The cytostatic effect of these compounds increased increasing their concentrations far above their IC50 levels for PDE5, suggesting that these compounds could act by interfering with other molecular events than direct cGMP-PDE inhibition. No appreciable effect was observed using Dipyridamole, another specific PDE5 inhibitor.     

Cyclic nucleotides, calcium, bicarbonate, and protein secretion in canine pancreatic juice in response to cholecystokinin-pancreozymin.

The secretion of cyclic AMP, cyclic GMP, protein, calcium, and bicarbonate in the pancreatic juice of three nonanesthetized dogs with chronic gastric and duodenal Thomas cannulae has been studied. Intravenous infusions of increasing doses of cholecystokinin-pancreozymin (CCK) (1.5, 3, 6, 12, 24 Crick Harper-Raper (CHR) U kg-1 h-1) were administered together with a continuous submaximal dose of secretin (1 clinical unit (CU) kg-1 h-1). Doubling CCK doses every 45 min induced a parallel increase in the output of both cyclic nucleotides. Cyclic AMP output peaked at between 15 and 30 min for 3 and 6 U kg-1 h-1 of CCK and later for 12 and 24 U kg-1 h-1 of CCK whereas cyclic GMP output increased more constantly. Calcium output followed a pattern similar to that of cyclic GMP secretion. Flow rate and protein output attained their peaks at between 30 and 45 min. A strong linear correlation was found between the quantities of cyclic AMP, cyclic GMP, and the quantities of protein secreted in response to each CCK dose. This study demonstrates the presence of cyclic GMP in the canine pancreatic juice and the dose-dependent stimulation of the secretion of cyclic GMP and cyclic AMP by CCK in the presence of secretin.     

Cyclic nucleotides and calcium: their role in the control of cell communication in the heart

The effects of theophylline and di-butyryl cyclic adenosine 3',5'-monophosphate (db-cAMP) on the electrical coupling of heart cells were investigated in rat trabeculae. Theophylline (4 X 10(-4) M) and db-cAMP (5 X 10(-5) M) increased both the space constant and conduction velocity. The time constant of the membrane was not changed by either drug. Measurements of the time constant of the foot of the action potential and conduction velocity were used to calculate the intracellular longitudinal resistance. Both theophylline and db-cAMP were found to enhance cell-to-cell communication in the heart by decreasing the intracellular longitudinal resistance.     

Cyclic nucleotides in spinal cells.

The most striking effects of intracellular injections of adenosine 3'5'-cyclic monophosphate (cAMP) into spinal mononeurons in cats are a speeding-up of the action potential, both its rising and falling phase, and a potentiation of the after-hyperpolarization; the latter porbably indicates a marked enhancement of Ca2+ influx. In this respect, cAMP and guanosine 3'5'-cyclic monophosphate (cGMP) have similar actions, though cAMP appears to be more potent. It is suggested that through this mechanism, cyclic nucleotides may play an important role in synaptic facilitation. Changes in resting membrane potential and resistance are less conspicuous or predictable. By contrast, both agents, when injected into unresponsive cells, presumed to be neuroglia, regularly cause a drop in membrane resistance; this is associated with hyperpolarization and therefore likely to reflect an increase in membrane K+ conductance.     

Cyclic nucleotides and neuromuscular transmission.

A review of the research on cyclic nucleotides and neuromuscular transmission suggests that cAMP is involved in the release of transmitter from motor nerve endings. Lipid-soluble derivations of cAMP cause depolarization of unstimulated nerve endings and prolong the after potentials of stimulated nerve endings. They also increase the frequency of miniature end plate potentials and increase the quantal content of stimulus evoked end plate potentials. Similar effects are produced by compounds that activate adenylate cyclase or inhibit phosphodiesterase. The responses to the derivatives of cAMP and activators of cyclase are enhanced by inhibitors of phosphodiesterase and prevented by compounds that block the flux of calcium into nerve endings. There is no evidence that suggests that cyclic nucleotides are involved in the postjunctional response to transmitter. Thus, it seems likely that cAMP is involved in the regulation of calcium in motor nerve endings and the exocytosis of transmitter. Additional study should expand our knowledge of neuromuscular transmission and contribute to an understanding of the functions of cyclic nucleotides in other synapses.     

Cyclic nucleotides and calcium ions in the activation of B-lymphocyte movement in mice by an anti-immunoglobulin serum

It was shown that B lymphocyte motility activated by anti-immunoglobulin serum may be abrogated in a Na+-deficient medium and in a 10(-5)M trifluoperazine-containing medium but not in a Ca2+-deficient medium. The tetracycline fluorescence test demonstrated Ca2+ efflux from isolated B lymphocyte mitochondria due to Na+ exposure. The radioimmunoassay demonstrated the cGMP level to rise after exposure to anti-immunoglobulin serum. The Na+-dependent Ca2+ efflux from the mitochondria might be the main mechanism in anti-immunoglobulin serum activation of B lymphocytes and in the cGMP level rising.     

Cyclic nucleotides in stroke and related cerebrovascular disorders

Evidence has steadily accumulated to indicate that the rapid fluctuations in cyclic nucleotides during primary and secondary stroke are more than epiphenomena of the disease. During acute phases of ischemia, anoxia or hypoxia cyclic AMP rapidly accumulates in cerebral tissue, cerebrospinal fluid (CSF) and venous plasma, while cyclic GMP either remains unchanged or declines. The massive release of transmitters (catecholamines and adenosine) or ionic fluxes (Na+ and K+) may account for these observations. If reflow is established through a previously occluded vessel cyclic AMP content rises even higher in conjunction with a sharp rise in cyclic GMP. It is during this reflow period subsequent to longer term stroke (30-60 min) that the synaptic membrane enzyme, adenylate cyclase, is especially vulnerable. Presumably the cause of injury to cell membrane systems results from excess lactic acid accumulation and/or Ca++ entry through the damaged blood-brain barrier. The latter initiates breakdown of membrane phospholipids with resultant synthesis of vasoactive prostaglandins and formation of free radicals causing further insult to membrane phospholipids. Thus drugs acting to inhibit formation of prostaglandins, scavenge free radicals, reduce lactate formation, inhibit Ca++ entry or stabilize cell membranes have been shown to possess varying degrees of protective action toward adenylate cyclase. Moreover, cyclic AMP has been found to reverse stroke-induced vasospasm in central vessels. Reduced cyclic AMP content in CSF has been used to monitor the severity of coma, whereas clinical improvement was associated with predictable increases in the cyclic nucleotide. Therefore, cyclic nucleotides and related membrane enzyme systems might be used as target molecules in which to develop future therapeutic strategies for prevention or treatment of stroke.