The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Action of nonsteroidal anti-inflammatory agents on fibrinolysis and platelet aggregation

The influence of non-steroidal antiphlogistics (NSA, fluor derivatives of phenylanthranilic acid) on fibrinolysis, platelet function, prostaglandin metabolism and pharmacokinetics of indirect anticoagulants was studied in rats and rabbits in vitro and in vivo. NSA were found to shorten the euglobulin lysis time and to enlarge the lysis zones on fibrin plates. They potentiated the fibrinolytic activity of streptokinase and trypsin. Furthermore, they inhibited platelet aggregation induced by arachidonic acid in rabbits. NSA in combination with inhibitors of thromboxane synthetase potentiated inhibition of aggregation. After oral administration, NSA inhibited formation of thromboxane A2 and prostacyclin in rabbits in a dose-dependent manner. At comparatively low doses, thromboxane A2 synthesis was more effectively inhibited than prostacyclin formation. Due to pharmacokinetic interactions NSA enhanced the anticoagulant effect of indirect anticoagulants and accelerated their distribution and elimination.     

Effect of verapamil on platelet aggregation

The influence of the calcium channel blocker verapamil on the aggregation of human blood platelets was studied in vitro in comparison with the calcium channel blocker diltiazem and with the 5-HT antagonist cyproheptadine. Verapamil inhibited the 5-HT-potentiated. ADP-induced aggregation more effectively than the aggregation induced by adrenaline, ADP and collagen. Verapamil antagonized the 5-HT effect in a noncompetitive manner. The same was true of cyprohepatadine which was by more than one order of magnitude more potent than verapamil in inhibiting the 5-HT-induced aggregation. Diltiazem was much less effective than verapamil.     

Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in Myxococcus xanthus.

Exogenous cyclic nucleotide phosphodiesterase (PD) accelerated fruiting body (FB) formation and increased territory size of aggregates in Myxococcus xanthus. Both guanosine 3'5'-monophosphate (cGMP) and guanosine 5'-monophosphate (GMP) were antagonistic to the PD effect. Adenosine 3'5'-monophosphate (cAMP) increases FB numbers twofold in the absence but not in the presence of PD. PD induction is not affected by methionine or isoleucine, which inhibit, or by threonine, which stimulates, FB formation. There is an increase and subsequent decrease in cAMP levels during early glycerol-induced microcyst development but 10 mM theophylline or caffeine not only inhibited microcyst development but induced germination in the presence of glycerol. On the basis of these results and the reports of other investigators a tentative model is proposed based on a dual role for cyclic nucleotides in the development in M. xanthus.     

Effect of synthetic enantiomeric cannabinoids on platelet aggregation.

The effect of a synthetic pair of enantiomeric cannabinoids on platelet function was evaluated. The nonpsychotropic enantiomer, the 1,1-dimethylheptyl homolog of (+)-(3S,4S)-7-hydroxy-delta-6-tetrahydrocannabinol (HU-211), was found to be more active in inhibiting ADP-induced platelet aggregation than the highly psychotropic (-)-enantiomer (HU-210). The related (+)-(3R,4R) cannabinoid, HU-213, which lacks the 7-hydroxy moiety, exerted its inhibitory effect within a wider range of concentrations. The results indicate a differentiation between psychotropic activity and inhibition of platelet aggregation in the cannabinoid group of compounds.     

Effect of serotonin on cyclic nucleotides of human platelets.

Serotonin produced a 6 to 10 fold increase of cyclic GMP over baseline levels of this nucleotide in platelets. Maximum stimulation was reached within 30 sec to 1 min after addition of serotonin and was dependent upon its concentration in the medium. Inhibition of serotonin uptake by methysergide, dihydroergotamine and chloroimipramine did not influence the serotonin-induced stimulation of cyclic GMP but glutaraldehyde and formaldehyde blocked it completely. Cyclic AMP levels in platelets were not affected by serotonin. The serotonin-induced stimulation of cyclic GMP is independent of the uptake of this biogenic amine by platelets and is not due to platelet aggregation.     

Effect of cyclic nucleotides on the cyanide-insensitive respiration of polymorphonuclear leucocytes.

The effects of N6,O2'-dibutyryladenosine 3',5'-monophosphate (Bu2-cyclic AMP) and N2,O2'-dibutyrylguanosine 3',5'-monophosphate (Bu2-cyclic GMP) on the cyanide-insensitive respiration of guinea pig peritoneal exudate polymorphonuclear leucocytes were studied. Bu2-cyclic AMP inhibited the respiration induced both by phagocytosis of E. coli and by the interaction with trypsin-digested rat liver microsomes. The addition of theophylline gave rise to an inhibitory pattern similar to that with Bu2-cyclic AMP against both the respirations induced. On the other hand, Bu2-cyclic GMP did not affect the respiration induced by phagocytosis whereas it inhibited the respiration induced by the addition of myristic acid was inhibited by Bu2-cyclic AMP, which was similar to that with E. coli. The respiration induced by methylene blue was inhibited neither by Bu2-cyclic AMP nor by Bu2-cyclic GMP. These observations suggest that the cyanide-insensitive respiration of polymorphonuclear leucocytes may be classified into at least three types from the inhibitory pattern of cyclic AMP and cyclic GMP.     

On the interrelationship of prostaglandin endoperoxide G2 and cyclic nucleotides in platelet function.

The prostaglandin endoperoxide G2 caused rapid aggregation and relase of ADP and [14C]serotonin in human platelets. Since the presence of the ADP phosphorylating system creatine phosphate/creatine phosphokinase markedly inhibited the aggregation caused by the endoperoxide, this effect seemed to be mediated mainly by ADP, which is instantaneously released by the endoperoxide. The prostaglandin G2 counteracted the increasing effect of prostaglandin E1 on the adenosine 3':5'-monophosphate (cAMP) levels in platelet-rich plasma. This effect of prostaglandin G2 was only observed when ADP was released by the endoperoxide. This finding indicates that the effect of prostaglandin G2 on the cAMP levels in platelet-rich plasma is principally mediated by ADP. The rapid release of ADP by prostaglandin G2 and the time courses for the effects of the endoperoxide and ADP on the level of cAMP give further evidence for this hypothesis. ADP also caused primary aggregation in the presence of indomethacin, and prostaglandin synthesis inhibitors did not influence the decreasing effect of ADP on the cAMP levels. N2,O2-Dibutyrylguanosine 3':5'-monophosphate did not influence the aggregation and release-reaction caused by ADP and no changes of the cGMP levels were observed after addition of prostaglandin G2.     

Effect of heparin and heparinoids on platelet aggregation

Biological effectiveness of bovine lung heparin and porcine mucosal heparin was tested in vitro and compared with the effectiveness of 12 semisynthetic heparinoids obtained by sulfation of waste heparin mucopolysaccharides and other biomolecules. Equieffective concentrations of these substances were determined in the sense of prolongation of the thrombin time and the APTT, inhibition of the thrombin- and collagen-induced aggregation, and potentiation of the primary ADP-induced aggregation. The influence of sulfation was proved on the biological effectiveness as well as the significance of the proper choice of the parent structure. Some polycondensates of the polysaccharide type were effective altogether with the sulfated waste heparin mucopolysaccharides. On the contrary, protein structures and low molecular weight glycosides exhibited little or no activity. The effective substances were related to heparin not only by the anticoagulant activity but also by the inhibitory action on the thrombin- and collagen-induced platelet aggregation. Contrary to heparin, higher concentrations of the studied heparinoids strongly potentiated the ADP-induced aggregation response. Strong inhibition of the collagen-induced aggregation was proved after administration of heparin to patients with end-stage renal failure on days without haemodialysis. Less significant changes in the secondary aggregation were observed also after administration of S-heparin (one of the studied heparinoids) to volunteers in the form of the rectal suppositories.     

Cyclic amp-mediated phosphorylation reactions in the regulation of blood platelet aggregation

• 1.1. Cyclic AMP (cAMP), an inhibitor of platelet aggregation, is pointed out to exert its effect on platelet function solely by stimulating platelet protein kinase.
• 2.2. Data from our laboratory and others indicate that in the presence of physiological concentrations of cAMP, ATP and Mg⁺, platelet protein kinase(s) phosphorylates and alters the activity of key platelet membrane and cytoplasmic enzymes. These enzymes include those involved in platelet cAMP synthesis and degradation, ATP→ADP hydrolysis coupled with either monovalent ion transport or contractile function, glycogen synthesis and degradation, prostaglandins and endoperoxides production, and other protein substrates such as microtubules whose function in platelets is yet to be determined.
• 3.3. A scheme is presented in which an increase in platelet cAMP concentration and phosphorylation correlates with an inhibition of platelet aggregation.

     

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.     

Saturation of anti cyclic nucleotides antibodies by endogenous cyclic nucleotides.

Rabbits immunized against cyclic AMP or cyclic GMP produce antibodies which are fully saturated by their respective endogenous cyclic nucleotides. This was proved a) in comparing radioimmunological measurements of cyclic nucleotides in antiserum and the binding site concentration determined by equilibrium dialysis, b) in showing the ineffectiveness of serum phosphodiesterase to hydrolyze the cyclic AMP present in the anti-cyclic AMP antiserum. Immunological and radioimmunological implications of this phenomenon are discussed.     

Effect of bencyclan and theophylline on changes of platelet aggregation and factor 3 activity]

The ADP- and adrenaline-induced platelet aggregation and platelet factor 3 availability were studied in patients before and eight hours after intravenous administration of bencyclan (100 mg) and/or theophylline (240 mg). Aggregation was primarily inhibited by bencyclan, the availability of factor 3 was inhibited by theophylline. Combination of both drugs exerted additive effects on both parameters. The combined use of drugs as inhibitors of aggregation is recommended, since they inhibit primary haemostasis simultaneously at two points of attack.