Adenosine 3′, 5′-cyclic monophosphate in Schistosoma mansoni

Homogenates of adult $\text{Schistosoma mansoni}$ (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E₁, E₂ or B₁. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1×10}{}^{\mathrm{?7}}\text{M}$) over cyclic GMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5×10}{}^{\mathrm{?6}}\text{M}$). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen.     

Activation of protein kinase in the guinea pig fundic gastric mucosa by histamine.

The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa. Histamine induced a significant elevation of tissue cyclic AMP levels and also $\text{in situ}$ activation of the protein kinase. 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these two parameters. Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa.     

Histamine H2 receptors and cyclic AMP in brain

The intravenous injection of histamine to 2–3 day old chicks resulted in a rapid and marked increase in the cyclic AMP content of the cerebral hemispheres that had been removed and frozen within 0.5s using a freeze-blowing technique. This response was not antagonized by pretreatment of the chicks with the histamine H₁-receptor antagonists mepyramine and diphenhydramine but was blocked by the H₂-receptor antagonists burimamide and metiamide. Parallel $\text{in vitro}$ experiments on slices of chick cerebral hemispheres demonstrated that the H₁ antagonists only produced a weak and non-competitive antagonism of the effects of histamine on cyclic AMP production. On the other hand the H₂ antagonists at low concentrations competitively blocked the histamine response. It is suggested that increased cyclic AMP formation in chick cerebral hemispheres can be mediated through stimulation of histamine H₂-receptors.     

Cyclic nucleotide metabolism and reactive oxygen production by macrophages

The production of reactive oxygen species by elicited rat peritoneal macrophages was assessed by $\text{in vitro}$ measurement of chemiluminescence in the presence of luminol. The divalent ion ionophore A23187 stimulated the production of reactive oxygen species. This action was inhibited by monobutyryl and dibutyryl derivatives of cyclic AMP but was not affected by derivates of cyclic GMP. Cyclic AMP and cyclic GMP concentrations increased rapidly in macrophages exposed to A23187 or zymosan. Indomethacin (20 μmol/1) inhibited the increase in cyclic AMP concentration but not the increase in cyclic GMP concentration. Neither A23187 nor zymosan stimulated adenylate cyclase activity in broken cell preparations of macrophages. The observations are consistent with the hypothesis that PGE produced by macrophages after phagocytotic stimuli may inhibit certain macrophage functions and perform a regulatory role in these cells. This action of PGE may be mediated by cyclic AMP.     

Antigen-induced histamine release from sensitized tissue and the measurement of calcium ion fluxes

Antigen stimulated release of histamine from sensitized guineapig lung was inhibited by the exclusion of calcium ions from the incubation fluid. Subsequent addition of calcium ions induced release, but the magnitude of this release decreased with time. When the releasing potential had declined to zero, addition of an alternative antigen together with calcium ions induced further release. If the primary challenge was inhibited by the presence of an antiallergic agent, challenge by a second antigen was similarly inhibited, in contrast to the effect when there was no primary antigen challenge. Antigen challenge induced a flux of ${}^{45}\text{Ca}$ into cells, these fluxes were inhibited by compounds which inhibited histamine release. Inhibition of release did not correlate with inhibition of calcium flux with some agents, suggesting that the measured flux is the sum of at least two fluxes, one secondary to release. These results are explained in a scheme for antigen-induced histamine release.     

Inactivation of cyclic GMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethylketone

Cyclic GMP-dependent protein kinase from bovine lung and cyclic AMP-dependent protein kinase from bovine heart are inactivated by N^α-tosyl-L-lysine chloromethylketone (TLCK) in the presence of cyclic GMP and cyclic AMP, respectively. The inactivation of both protein kinases is pseudo-first order, suggesting the rate limiting step is beyond the binding of TLCK. Cyclic GMP-dependent protein kinase is inactivated less than $\text{1}\text{4}$ as rapidly as cyclic AMP-dependent protein kinase, although it shows a higher apparent affinity for TLCK. Cyclic AMP stimulated the rate of inactivation of cyclic AMP-dependent protein kinase 10-fold but cyclic GMP stimulated the rate of inactivation of cyclic GMP-dependent protein kinase only 1.5-fold. The rate of inactivation of cyclic GMP-dependent protein kinase by TLCK is sufficiently rapid (half-time of about 30 min at 37°C with 2 mM TLCK) to account for the effects of TLCK on cell growth observed by others.     

Interaction of cyclic nucleotides and ecdysterone in breaking the pupal diapause of the bertha armyworm, Mamestra configurata WLK.

Cyclic GMP and cyclic AMP have distinct and opposite effects upon the action of ecdysterone in diapausing pupae of the Bertha armyworm, $\text{Mamestra}$$\text{configurata}$. Cyclic GMP enhanced the effectiveness of suboptimal doses of ecdysterone in breaking diapause; the amount of cyclic GMP required to lower the ED50 of ecdysterone by half was 80 μg/g. Dibutyryl cyclic GMP had no apparent effect on the action of ecdysterone over a wide dose range (0.07 – 70 μg/g). On the other hand, cyclic AMP and dibutyryl cyclic AMP effectively blocked the diapause-breaking action of ecdysterone when administered simultaneously with the steroid hormone. The amount of cyclic AMP required to reduce the incidence of diapause termination from 100% to 50% was 60 μg/g; for dibutyryl cyclic AMP the amount required was only 14 μg/g. No cyclic nucleotide tested in the study could by itself break the pupal diapause of $\text{M.}$$\text{configurata}$. The concept that cyclic GMP and cyclic AMP provide at least different if not opposing regulatory influences in certain insect systems is discussed briefly in the light of these observations.     

IV. Immunologic mechanisms for release of chemical mediators of anaphylaxis from human lung tissue

The antigen-induced, IgE-dependent release of chemical mediators from human lung tissue in vitro is modulated by a variety of pharmacologic maneuvers involving alterations in the intracellular levels of cyclic nucleotides. Increase in the level of cyclic AMP inhibits the immunologic release of histamine, SRS-A and ECF-A; β-adrenergic agents, prostaglandins, cholera toxin and methylxanthines all produce accumulations of cAMP in human lung tissue. Depletion of cAMP after α-adrenergic, low-dose prostaglandin and imidazole stimulation is associated with enhancement of the release of mediators. Studies involving purified preparations of rat peritoneal mast cells confirm that alterations in the cAMP levels of a homogeneous population of target cells indeed influence histamine release in a fashion analogous to that of human lung tissue.Furthermore, cholinergic stimuli produce a marked enhancement of the antigen-induced release of mediators from human lung through an apparently independent mechanism, presumably acting through alterations in the tissue concentration of cyclic GMP. This latter observation suggests an important interaction between endogenously released parasympathetic neurohormones and the immunologic release of the chemical mediators of asthma.     

Purification and characterization of guanylate cyclase from Caulobacter crescentus

Guanylate cyclase has been purified 60-fold from cell extracts of the bacterium $\text{Caulobacter crescentus}$. It has a molecular weight of approximately 140,000 and is dependent upon Mn²⁺ for activity. Enzymic activity is unaffected by cyclic AMP, cyclic GMP or N⁶,O^2′-dibutyryl cyclic AMP but is stimulated by N²,O^2′-dibutyryl cyclic GMP. The partially purified preparation of guanylate cyclase does not contain detectable adenylate cyclase activity.     

Adenosine 3',5' cyclic monophosphate in Euglena gracilis

$\text{Euglena gracilis}$ contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. $\text{Euglena}$ also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.     

Vasoactive intestinal polypeptide: increased tone, enhancement of acetylcholine release, and stimulation of adenylate cyclase in intestinal smooth muscle

Vasoactive intestinal polypeptide (VIP) was examined $\text{in vitro}$ for effects on tone and neuronal release mechanisms in intestinal smooth muscle since this is a site of high peptide concentration. VIP contracted the guinea pig ileum and rabbit jejunum in concentrations ranging from 10^?9 to 10^?7 M. Increased tone in the guinea pig ileum was partially antagonized by the anticholinergic agent, atropine (4.38 × 10^?6 M) suggesting that one component of the contractile response was due to the indirect release of acetylcholine. The H₁ receptor antagonist, pyrilamine, did not alter the increased tone produced by VIP indicating that histamine release did not contribute to the ileal contractile response and that VIP exerted a selective effect to enhance neuronal release of acetylcholine. The ability of VIP to modulate acetylcholine release was confirmed in field stimulated ileal preparations where VIP increased the force developed to endogenously released acetylcholine without altering the direct response to acetylcholine. In rabbit jejunum and ileal smooth muscle, VIP related cyclic AMP levels. However, inhibition of phosphodiesterase with papaverine did not potentiate either the VIP-induced ileal contraction or enhancement of the field stimulated response. This raises the possibility that increases in intestinal cyclic AMP may be involved more in VIP-induced alterations in ion transport or secretory phenomenon than in intestinal motility. These studies describing the ability of VIP to modulate acetylcholine release and to increase ileal tone are consistent with the proposed role of VIP in intestinal patholgies involving excessive mucous secretion and motility.     

Formation of guanosine 3′,5′-cyclic monophosphate by thyroliberlin in cultured rat pituitary cells a possible role in stimulation of prolactin synthesis

In a clonal strain of rat pituitary tumour cells (GH₄C₁ cells), thyroliberin stimulated prolactin secretion and synthesis: effects that could be demonstrated after 5 min and 4–5 h of treatment, respectively. Within 0.5–5 min after addition of thyroliberin, maximal increases (2–4 hold) in cellular cyclic GMP concentrations were observed, and this rise preceded or occurred simultaneously with that of cyclic AMP. After 60 min of treatment the concentrations of the cyclic nucleotides had returned to control values. Half maximal and maximal stimulation of cyclic GMP elevations were obtained with approx. 2·10⁹ and approx. 27·10^?9 thyroliberin, respectively. Aminophylline increased both cyclic GMP and cyclic AMP, and potentiated the stimulatory effects of thyroliberin on both cyclic nucleotides. The dibutyryl derivative of cyclic GMP (10^?4–10^?6 M) stimulated prolactin synthesis, but not hormone release. Prostaglandin E₂ (3·10^?7 M) stimulated cellular cyclic AMP concentrations, but did not affect cyclic GMP levels. We conclude that thyroliberin in the GH₄C₁ ccell strain stimulates cyclic GMP formation, in addition to elevate cyclic AMP concentrations. The stimulatory effect on cyclic GMP is probably not secondary to the rise in cyclic AMP concentration, since prostaglandin E₂ elevates only cyclic GMP is involved in the action of thyroliberin on prolactin, the present results suggest a role on hormone synthesis.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Effect of ethanol upon isoproterentol stimulation of growth and secretion in the mouse parotid gland

Isoproterenol (0.3 mmole/kg body wt.), when injected into the mouse intraperitoneally, increases the weight by 35% and stimulates DNA synthesis 30-fold in the parotid gland. The induction of both hypertrophy and hyperplasia is completely inhibited by ethanol at a dose of 200 mmole/kg body wt. but is almost unaffected by 60 mmole/kg. The full inhibiton of both growth parameters is observed when ethanol is administered up to 5 hr after isoproterenol. Partial inhibition is observed when ethanol is given as long as 15 hr after isoproterenol. It contrast ethanol did not alter the secretion of α-amylase in response to isoproterenol. Ethanol had no effect upon the rise in cyclic GMP level caused by isoproterenol but augmented the rise in cyclic GMP In agreement with these $\text{in}$$\text{vivo}$ observations, low concentrations of ethanol activated adenylate cyclase $\text{in}$$\text{vitro}$, however guanylate cyclase activity was quite strongly inhibited. Although high levels of ethanol (300 mmole/kg) inhibited the induction of both ornithine decarboxylase and S-adenosylmethionine decarboxylase little inhibition was seen at 200 mmole/kg suggesting that the interference with polyamine metabolism is not the mechanism of the ethanol effect upon isoproterenol-induced parotid growth.     

Atrial natriuretic factor regulation of cyclic GMP levels and steroidogenesis in isolated fasciculata cells of rat adrenal cortex

Isolated fasciculata cells of rat adrenal cortex, when incubated with atrial natriuretic factor (ANF), stimulated the levels of cyclic GMP and corticosterone production in a concentration-dependent manner without a rise in the levels of cyclic AMP. The ANF-dependent elevation of cyclic GMP was rapid, with a detectable increment in 30 s. ANF also stimulated the particulate guanylate cyclase. These results not only indicate the coupling of cyclic GMP and corticosterone production with ANF signal, but also demonstrate that, like the ACTH signal, cyclic AMP is not the mediator of ANF-induced adrenocortical steroidogenesis.     

Modification of radiosensitivity of mammalian cells by cyclic nucleotides

Some $\text{in vitro}$ and $\text{in vivo}$ studies suggest that adesosine 3′,5′-cyclic monophosphate (cyclic AMP) may be one of the important factors in determining the radiosensitivity of certain mammalian cells; however, the role of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in radiosensitivity of mammalian cells is completely unknown. Recent data also suggest that the mechanism of radiation protection afforded by moderate hypoxia and SH-containing compounds may involve an alteration in the intracellular level of cyclic AMP. At least one $\text{in vivo}$ study shows that cyclic AMP protects hair follicles and gut epithelial cells against radiation damage; however, it does not protect lymphosarcoma and breast carcinoma in mice. If a similar phenomenon is found in humans, an elevation of the intracellular level of cyclic AMP during radiation exposure may improve the effectiveness of radiation therapy in those cases where the radiation damage of normal tissue becomes the limiting factor for a continuation of the therapy program. More $\text{in vitro}$ and $\text{in vivo}$ studies on normal and cancer cells are needed to substantiate the role of cyclic nucleotides in radiosensitivity.     

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. $\text{N,N′-}\text{Dicyclohexylcarbodiimide}$, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, $\text{N-1-}\text{napthylphthalamate}$, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H⁺ transport across their respective membranes.     

Comparison of circulating and peritoneal leukocyte cationic proteins in release of histamine from rat mast cells

Lysosomal cationic proteins which release histamine from rat peritoneal mast cells were prepared from circulating as well as peritoneal leukocytes of rabbits. The release of histamine by cationic proteins and by compound $\text{48}\text{80}$ was compared as a function of temperature, pH and concentration. Cationic protein-mediated histamine release appears to be a non-cytotoxic energy requiring process similar to compound $\text{48}\text{80}\text{-}\text{mediated release}$. It was inhibited by iodoacetate, n-ethylmaleimide, 2,4-dinitrophenol, malonate, oxamate, glutamate and slightly inhibited by 2-deoxyglucose. Pharmacologic inhibition of release by isoproterenol, aminophylline, dibutyryl cyclic AMP and prednisone was also demonstrated.     

Prostaglandins and cardiac anaphylaxis.

Manifestations of cardiac anaphylaxis include sinus tachycardia and arrhythmias, both of which result from histamine release. The marked decrease in coronary flow, which also occurs during cardiac anaphylaxis, cannot be attributed solely to histamine release.To indirectly assess the possible role of prostaglandins in cardiac anaphylaxis, hearts from sensitized guinea pigs were challenged $\text{in}$$\text{vitro}$ in the presence of indomethacin. This resulted in a marked increase in histamine release, which caused a greater tachycardia and an increase in the incidence of arrhythmias. Moreover, for the same degree of histamine release sinus rate increments were larger in the presence of indomethacin. However, despite the enhanced cardiac dysfunction, coronary flow rate did not decrease.The results suggest that, during cardiac anaphylaxis, prostaglandins modulate histamine release and the effects of released histamine. Furthermore, since we have found that PGF_2α is released from the anaphylactic heart, we tentatively ascribe the anaphylactic reduction in coronary flow to the elaboration of PGF_2α.     

The effect of various secretagogues on accumulation of cyclic AMP and secretion of α-amylase from rat exocrine pancreas

The relationship between accumulation of cyclic AMP and the secretion of α-amylase was investigated in the rat pancreas $\text{in vitro}$. Theophylline and secretin induced an increase in tissue cyclic AMP levels, however, only secretin stimulated secretion of α-amylase. Pancreozymin caused a release of α-amylase and had a biphasic effect on nucleotide levels — stimulation followed by inhibition. Carbachol, which induced a secretory response in the rat pancreas, reduced tissue levels of the cyclic nucleotide.