Modulation Of Cyclic Nucleotides Research Articles

Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin

Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3–5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5-fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals.

Vasodilating Agents and Platelet Function

The adenylate-cyclase activator forskolin, the guanylate-cyclase stimulator sodium nitroprusside, the phosphodiesterase inhibitor Ro 15-2041, different Ca-entry blockers, as well as various vasodilators, and the atrial natriuretic peptide were tested for antiplatelet activity. Thrombin, vasopressin, ADP, arachidonic acid, and the dihydropyridine Ca agonist CGP 28392 were used as platelet activators. The physiological and biochemical parameters of platelet function studied included shape-change reaction, intracellular free-Ca modulation, and cyclic nucleotide formation. When inhibition of the shape-change response occurred, it was accompanied by inhibition of the increase in intracellular free Ca. Furthermore, the results suggest a possible intracellular site of action of Ca entry blockers in platelets, and confirm the importance of modulation of cyclic nucleotides in the regulation of platelet function, regardless of the mechanism of platelet activation. Additional antiplatelet activity of antihypertensive agents may have a beneficial effect in reducing the associated risk of thrombo-embolic complications in essential hypertension.

ChemInform Abstract: A NEW GENERATION OF PHOSPHODIESTERASE INHIBITORS: MULTIPLE MOLECULAR FORMS OF PHOSPHODIESTERASE AND THE POTENTIAL FOR DRUG SELECTIVITY

With several notable exceptions, interest in the area of multiple molecular forms of phosphodiesterase remained relatively dormant during the decade following Thompson's discovery of more than one phosphodiesterase in brain in 1971. Within the last several years, however, over 20 novel agents have been identified that exert selective inhibitory effects on the various molecular forms of phosphodiesterase present within different cells. In addition, several studies have documented that such agents can produce discrete changes in cyclic AMP and cyclic GMP, an action that is not shared by first generation phosphodiesterase inhibitors such as theophylline. The purpose of this Perspective is to provide some clarity to this rapidly evolving area of selective phosphodiesterase inhibitors. Thus, we have attempted to characterize the different forms of phosphodiesterase present in various tissues and cells according to their kinetic properties, substrate specificity, etc. and also to characterize those major classes of agents that have been shown to inhibit phosphodiesterase activity, whether selectively or nonselectively. In addition, we have described several therapeutic areas wherein selective phosphodiesterase inhibitors might prove efficacious, paying particular attention to those areas in which selective phosphodiesterase inhibitors have already been shown to exert beneficial effects, namely, stimulation of myocardial contractility, inhibition of mediator release, and inhibition of platelet aggregation. Although focusing on these three areas, it is obvious that the potential therapeutic utility of selective phosphodiesterase inhibitors could conceivably extend to several other areas in which modulation of cyclic nucleotides can have desirable effects, including cancer chemotherapy, analgesia, the treatment of depression, Parkinson's disease, and learning and memory disorders. For example, the selective type III phosphodiesterase inhibitor rolipram has been shown to antagonize reserpine-induced hypothermia and also to potentiate yohimbine lethality, two tests that are indicative of antidepressant activity. In addition, microinjection of the selective PDE III inhibitor Ro 20-1724 into the rat brain stem has been shown to produce analgesia.(ABSTRACT TRUNCATED AT 400 WORDS)

A new generation of phosphodiesterase inhibitors: multiple molecular forms of phosphodiesterase and the potential for drug selectivity.

With several notable exceptions, interest in the area of multiple molecular forms of phosphodiesterase remained relatively dormant during the decade following Thompson's discovery of more than one phosphodiesterase in brain in 1971. Within the last several years, however, over 20 novel agents have been identified that exert selective inhibitory effects on the various molecular forms of phosphodiesterase present within different cells. In addition, several studies have documented that such agents can produce discrete changes in cyclic AMP and cyclic GMP, an action that is not shared by "first generation" phosphodiesterase inhibitors such as theophylline. The purpose of this Perspective is to provide some clarity to this rapidly evolving area of selective phosphodiesterase inhibitors. Thus, we have attempted to characterize the different forms of phosphodiesterase present in various tissues and cells according to their kinetic properties, substrate specificity, etc. and also to characterize those major classes of agents that have been shown to inhibit phosphodiesterase activity, whether selectively or nonselectively. In addition, we have described several therapeutic areas wherein selective phosphodiesterase inhibitors might prove efficacious, paying particular attention to those areas in which selective phosphodiesterase inhibitors have already been shown to exert beneficial effects, namely, stimulation of myocardial contractility, inhibition of mediator release, and inhibition of platelet aggregation. Although focusing on these three areas, it is obvious that the potential therapeutic utility of selective phosphodiesterase inhibitors could conceivably extend to several other areas in which modulation of cyclic nucleotides can have desirable effects, including cancer chemotherapy, analgesia, the treatment of depression, Parkinson's disease, and learning and memory disorders. For example, the selective type III phosphodiesterase inhibitor rolipram has been shown to antagonize reserpine-induced hypothermia and also to potentiate yohimbine lethality, two tests that are indicative of antidepressant activity. In addition, microinjection of the selective PDE III inhibitor Ro 20-1724 into the rat brain stem has been shown to produce analgesia.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of canine heart sarcolemmal Ca 2+-pumping ATPase by cyclic GMP

Cyclic nucleotide modulation of the sarcoplasmic reticulum calcium pump has been recognized for some time. Little is known, however, of cyclic nucleotide effects on the sarcolemmal Ca 2+ pump. In sarcolemmal vesicles prepared from ventricular muscle by a recent technique (Jones, L.R., Maddock, S.W. and Besch, H.R. (1980) J. Biol. Chem. 255, 9971–9980) we have demonstrated via Millipore filtration that 10 −8 M and 10 −9 M cyclic GMP depressed the rate of ATP- and Mg 2+-dependent 45Ca 2+ uptake by 34% and 52%, respectively. Only at millimolar levels did cyclic AMP have any effect and the respective 5′-nucleotides had no effect at all. Parallel measurement of the associated (Ca 2+ + Mg 2+)-ATPase in the presence of either cyclic or 5′-nucleotides, however, revealed no concomitant depression in ATP hydrolysis. In another series of experiments, the cyclic GMP effect on 45Ca 2+ uptake was associated with a significant decrease in the pump V max , and at the most effective concentration of cyclic GMP increased the apparent K m for Ca 2+. These results suggest that cyclic GMP may depress ventricular Ca 2+ efflux by decreasing the enzyme turnover and to a limited extent, decreasing pump affinity for Ca 2+. This supports a hypothesis whereby cyclic GMP might modulate both local biochemical and electrophysiological events by an effect on a discrete, regional pool of intracellular Ca 2+.

Contribution of lipoxygenase and cyclooxygenase metabolites in the modulation of cyclic nucleotides in the guinea pig mymotrium. Differential effects of carbachol and ionophore A 23187

Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A 23186, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachiodonic acid, A 23187 and PGI 2. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A 23187 and arachidonate on cyclic AMP were accompanied by a parallel increase (2–3 fold) on the generation of PGI 2 by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI 2 was involved. Omission of Ca 2+ or addition of mepacrine of p-bromophenacylbromide abolished the stimulatory effects of A 23187 and carbachol on cyclic GMP as well as the A 23187-induced elevations in both PGI 2 and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A 2-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI 2 generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI 2 suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.

Cyclic nucleotide modulation of Na + and K + currents in the isolated node of Ranvier

The application of dibutyryl cyclic adenosine monophosphate (d-cAMP) to an isolated, voltage clamped node of Ranvier resulted in an increase in the peak inward and steady-state outward current amplitudes. Blocking K + channels with TEA abolished the effects of d-cAMP on Na + currents. Blocking Na + channels with TTX revealed an increase in K + current amplitude in response to d-cAMP. Application of a potent phosphodiesterase inhibitor, SQ20009, reproduced the effects of d-cAMP in the intact node, but with a latency in onset. These results suggest a physiological role for cAMP in the modulation of Na + and K + currents in myelinated peripheral nerve.

Dynamics of renal histamine in normal rat kidney and in nephrosis induced by aminonucleoside of puromycin.

Histamine is known to have a profound effect on capillary permeability in nonrenal tissues and this effect is presumably mediated by cyclic (c)AMP. Because in our previous experiments we found that histamine stimulates cAMP accumulation in glomeruli (Torres, V. E., T. E. Northryn, R. M. Edwards, S. V. Shah, and T. P. Dousa. 1978. Modulation of cyclic nucleotides in isolated rat glomeruli. J. Clin. Invest.62: 1334.), we now explored whether this amine is formed in renal tissue, namely in glomeruli, and whether its renal metabolism is altered in experimental nephrosis induced by puromycin aminonucleoside (PA) in rats. In normal rats, histamine content was higher (Delta + 240%) in cortex than in medulla. In glomeruli isolated from renal cortex, histamine content was significantly higher (Delta + 260%) than in tubules. Incubation of isolated glomeruli with l-histidine resulted in a time-dependent increase of histamine content in glomeruli, but no change was found in tubules. The increase in glomerular histamine was blocked by the histidine decarboxylase inhibitor bromocresine. In rats with PA nephrosis induced by a single intraperitoneal injection of PA (15 mg/100 g body wt) urinary excretion of histamine was markedly increased (>Delta + 200%), but control rats did not differ from rats with PA nephrosis in urinary excretions of l-histidine and of creatinine. At the peak of proteinuria (day 9 after injection of PA) the plasma level of histamine was slightly elevated, and plasma histidine slightly decreased in animals that developed PA nephrosis. The content of histamine was markedly higher and the level of histidine was significantly lower in the renal cortex of PA-nephrotic rats as compared with controls; PA-nephrotic and control rats did not differ in the content of histidine and histamine in the liver. In addition, the content of histamine was higher in glomeruli isolated from PA-nephrotic rats; lesser difference was found in cortical tubules. The results further indicate that PA-nephrotic rats have higher content of histamine in the renal cortex, predominently in glomeruli with increased urinary histamine excretion. The elevated renal cortical histamine is not due to higher availability of histamine precursor l-histidine. Results thus show that glomeruli are a major site of intrarenal histamine synthesis and accumulation, and also suggest that abnormal renal metabolism of this amine in PA nephrosis may be related, as a cause or as a consequence, to the pathogenesis of this disease.

Cyclic Nucleotide Levels in Psoriatic and Normal Keratomed Epidermis

Changes in the cyclic nucleotide modulation of the epidermis could be involved in the altered state psoriatic lesional tissue. Since several conflicting values for cyclic AMP levels in the diseased state have been reported and cyclic GMP levels have been determined in only a small number of psoriatic patients, we have attempted to define the changes in epidermal cyclic nucleotide levels that can occur between psoriatic and normal epidermis. In this study, 34 normal volunteers and 28 persons with classical psoriasis vulgaris were biopsied using a Castroviejo keratome. The cyclic AMP and cyclic GMP levels of the snap frozen sample, which consists almost entirely of epidermal tissue, were determined by acetylated RIA of partially purified cyclic nucleotide fractions obtained by ion-exchange chromatography. The results indicate that cyclic GMP levels are increased 132% (NDA date base) in the lesional tissue when compared to the uninvolved areas. This is in agreement with our previously reported values obtained from a small number of biopsies. However, in contrast to our previous work, no decrease in lesional cyclic AMP in comparison with uninvolved epidermis was detected, although the content of cyclic AMP in all psoriatic tissue (both involved and uninvolved) was now observed to be decreased when compared to the normal values that have been obtained in our past and this present study. We conclude that cyclic AMP and cyclic GMP assays of biopsied epidermal tissue obtained form a large and varied human population can only suggest the involvement of cyclic nucleotides in the pathophysiology of the disease. Other systems and techniques are presently being used and developed to define the role of cyclic nucleotides in epidermal function and to formulate hypothesis on which to base further investigations of the misregulation of cyclic AMP and cyclic GMP in psoriatic tissue.

Modulation of Cyclic Nucleotides in Isolated Rat Glomeruli

Because glomerular functions are modulated by numerous humoral agents, probably acting through cyclic nucleotides, the effects of some polypeptide hormones and biogenic amines on cyclic AMP (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) were studied in glomeruli isolated from rat renal cortex. Glomeruli and cortical tubules were prepared by a combination of sieving and density-gradient centrifugation. Under basal conditions, the contents of cAMP and cGMP in glomeruli were significantly higher than in tubules and unfractionated renal cortical tissue.Histamine caused a striking increase in cAMP in glomeruli (+Delta% 675+/-87) and, to a lesser degree, increased cAMP in tubules (+Delta% 103+/-25) or in tissue slices. This stimulation was dose-dependent in the range of 1 muM-1 mM histamine. Metiamide (an H(2)-antagonist), but not pyrilamine (an H(1)-antagonist) blocked the effect of histamine on cAMP, which indicates that histamine causes its effect via interaction with H(2) receptors. Histamine caused less extensive increases in cGMP in both glomeruli and tubules. Carbamylcholine caused a marked increase in cGMP in glomeruli (+Delta 295+/-7) and a much lower increase in tubules (+Delta% 70+/-20); these effects were blocked by atropine. Parathyroid hormone (1 mug/ml) increased cAMP and, to a much lesser degree, also cGMP in glomeruli. In tubules, parathyroid hormone caused much more extensive increases in cAMP than in glomeruli; no changes, or rather a small decline in cGMP, was observed. Angiotensin-II (2 muM) markedly lowered cAMP in glomeruli (-Delta% -45+/-8) and in tubules (-Delta% 33+/-7) but had no effect on cGMP. Bradykinin (20 muM) did not consistently influence either cAMP or cGMP in glomeruli or tubules. Present results demonstrate that cAMP and cGMP metabolism in glomeruli are controlled independently by humoral agents known to alter glomerular functions in vivo. Our findings are consistent with the view that histamine and cholinergic agents generated and (or) released locally in glomeruli or in their vicinity may play important roles as mediators of immunopathological injury of glomeruli, and that these effects are mediated by cAMP and (or) cGMP through interaction with H(2) receptors and muscarinic receptors. Likewise, our results suggest that the effects of angiotensin-II and parathyroid hormone on glomerular dynamics may be mediated by cyclic nucleotides.Thus, we surmise that extrarenal as well as intrarenal humoral agents may play an important role in the pathology and physiology of glomeruli through mediation of either cAMP, cGMP, or both.

Neurotransmitter and cyclic nucleotide modulation of frog cardiac contractility

The experiments performed in this study were designed to test the hypothesis that cyclic nucleotides mediate the effects of adrenergic and cholinergic neurotransmitters on frog ventricle. A physiological approach was to measure the twitch tension and contracture tension of ventricular strips in the presence of exogenous neurotransmitter agonists. These observations were correlated to biochemical measurements of cyclic AMP and cyclic GMP levels in ventricular strips exposed to the same physiological conditions. Epinephrine augmented twitch tension, depressed contracture tension, and augmented cyclic AMP levels. Phosphodiesterase inhibitors potentiated the effects of epinephrine. Exogenous cyclic AMP usually mimicked the epinephrine effect on both twitch and contracture tension. Carbachol and acetylcholine depressed twitch tension without affecting contracture, and carbachol transiently augmented cyclic GMP while depressing cyclic AMP levels. These muscarinic agonists inhibited the effects of epinephrine in depressing contracture tension and increasing cyclic AMP levels. Cyclic GMP did not mimic the twitch depressant effect of carbachol but did tend to mimic the cholinergic antagonism of epinephrine. Our observations generally support the idea that cyclic AMP mediates the effects of epinephrine but do not clearly relate cyclic GMP to the action of cholinergic agonists.

Agents That Increase Cyclic AMP Inhibit Accumulation of cGMP and Depress Human Monocyte Locomotion

Abstract Ascorbic acid and serotonin increased the 3′,5′-guanosine monophosphate (cGMP) content and enhanced the chemotactic responsiveness of mononuclear cells from human peripheral blood. The ionophore A23187, PGE1, and polystyrene beads increased the 3′,5′-adenosine monophosphate (cAMP) content of mononuclear cells and had no effect on basal cGMP content. A23187, PGE1, and beads caused significant increases in cAMP content in preparations of adherent cells (chiefly monocytes); PGE1 and beads also increased cAMP content in the nonadherent cells (chiefly lymphocytes). A23187 and PGE1 each inhibited mononuclear cell locomotion. The chemically unrelated agents, A23187, beads, and PGE1, that raised cAMP of monocytes inhibited the accumulation of cGMP in response to serotonin and asorbic acid. PGE1 at a concentration that caused a 349% increase in cAMP inhibited the effect of serotonin on cGMP accumulation and the amplification of chemotaxis by serotonin only 37% and 40%, respectively. In contrast A23187, which increased monocyte cAMP by only 71%, inhibited the effect of serotonin on cGMP accumulation and locomotion by 85 and 79%, respectively. Although there is no clear quantitative relationship between the increase in cAMP content and the degree of inhibition of cGMP accumulation, it is possible that the elevation of cAMP in monocytes interferes with their capacity to accumulate cGMP in response to serotonin and ascorbic acid, perhaps by increasing cGMP degradation and/or inhibiting synthesis. In any case cyclic nucleotide modulation of monocyte locomotion appears to be more closely related to intracellular cGMP content than to cAMP content.

Antigen-, anti-F(ab')2- and anti-IgE-induced histamine release from rat mast cells.

The existence of IgE antibody molecules on peritoneal mast cells from rats immunized with dinitrophenylated ascaris extract was demonstrated by double antibody immunofluorescence staining and by histamine release induced by anti-IgE antibodies. Cross-linking of the IgE molecules, which are fixed to mast cells, by interaction with either the homologous antigen or with antibodies to the Fab or Fc regions of IgE, triggered in the presence of calcium ions a chain of intracellular reactions which involve cyclic nucleotide modulation and energy-requiring processes leading ultimately to the release of histamine from the granules of these cells. Although there was no apparent difference in the mechanism(s) underlying the reactions triggered by any of these three agents, the extent of histamine release caused by antibodies to the Fc region of the IgE was significantly lower than that induced by cross-linking of IgE molecules through their Fab regions by reactions with either anti-Fab antibodies or antigens.

Effects of A and B series prostaglandins on cAMP, cortisol and aldosterone production by the human adrenal

PGA 1 and PGA 2 (10, 100 μg/ml) significantly increased human adrenal cAMP levels and cortisol output but low doses (1 μg/ml) depressed both parameters. Only 1 μg/ml PGA 1 significantly increased aldosterone output while higher doses depressed same. The low PGA 2 dose (1 μg/ml) depressed aldosterone output. The glucocorticoid and mineralocorticoid outputs appear to be inversely modulated by prostaglandins. PGB 1 and PGB 2 behaved similarly to E type prostaglandins. However, like PGA 1, 1 μg/ml of PGB 1 or PGB 2 significantly increased aldosterone output. Higher doses were ineffective. The present findings reveal an increased complexity of prostaglandin modulation of cyclic nucleotides and steroid output.