Stimulation by cyclic nucleotides of prostaglandin E production in isolated Graafian follicles

Rat Graafian follicles isolated intact responded to 8-Br-cyclic AMP and 8-Br-cyclic GMP with increased prostaglandin E (PGE) production during a 6 h incubation. By contrast, 8-Br-cyclic IMP, 8-Br-5′ AMP and 8-Br-5′ GMP were inactive in this respect. The effect of 8-Br-cyclic AMP and 8-Br-cyclic GMP was noted only after a lag period of about 4 h. Choleragen, LH, and the phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine; IBMX) also stimulated PGE production. Actinomycin D and cycloheximide given simultaneously with 8-Br-cyclic AMP or LH prevented the stimulatory effect of these agents. Concomitant addition of arachidonic acid did not overcome the effect of these inhibitors.Administration of hCG $\text{in vivo}$ or incubation with LH $\text{in vitro}$ did not elevate endogenous ovarian free arachidonate, while PGE production was enhanced. Dexamethasone prevented this stimulatory effect of hCG.Collectively, the results suggest that stimulation of ovarian PGE production by cyclic mucleotides and LH is dependent on $\text{de novo}$ synthesis of one more components of the PG synthetase systme rather than on substrate availability. Cyclic nucleotides may mediate the stimulatory effect of gonadotropins on PGE production     

Cyclic-AMP inhibits neither A23187-stimulated [14C]-arachidonic acid release from prelabelled lipids nor phospholipase A2 activity in resident rat peritoneal macrophages

8-Bromo cyclic AMP inhibited A23187-stimulated PGE2 production in adherent resident rat peritoneal macrophages by 50% when this was assessed by radioimmunoassay. In contrast, neither exogenous 8-bromo cyclic AMP nor elevation of endogenous cyclic AMP with cholera toxin inhibited 14C-arachidonic acid release or labelled prostaglandin formation by [1-14C]-arachidonic acid-prelabelled macrophages stimulated with either A23187 or melittin. Inhibition by cyclic AMP appears to be confined to PGE2 originating from a pool of endogenous phosphoglyceride that does not readily exchange with isotopically-labelled arachidonic acid. Phospholipase A2 activity, assessed as calcium-dependent [1-14C]-arachidonic acid release from exogenous 1-stearoyl, 2-[1-14C]-arachidonyl phosphatidyl choline at pH 8.6, was activated by melittin but not by A23187 in 1000 x g supernates from sonicated cells. Neither melittin nor calcium activation of phospholipase A2 was inhibited by preincubation of the cells prior to breakage with 8-bromo-cyclic AMP, nor by inclusion of either 8-bromo cyclic AMP or the catalytic subunit of cyclic AMP-dependent kinase in the assay. The results are inconsistent with the hypothesis that inhibition of A23187-stimulated PGE2 production by cyclic AMP in peritoneal macrophages is due to inhibition of a calcium-stimulated phospholipase A2.     

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.     

Ethanol inhibition of antigen-induced histamine release from guinea pig lung: correlation with intracellular levels of cyclic nucleotides.

The effect of ethanol on histamine release from lungs of sensitized guinea pigs was studied in conjunction with measurements of tissue concentrations of cyclic AMP and cyclic GMP. Addition of antigen $\text{in vitro}$ elicited a rapid increase in cyclic AMP and cyclic GMP and stimulated release of histamine. Ethanol (2%) inhibited antigen-induced release of histamine over 95% and completely inhibited the increase in both cyclic nucleotides. The activity of cyclic AMP-dependent protein kinase was only slightly affected by ethanol.Metiamide blocked the ovalbumin stimulated increase in cyclic AMP but not cyclic GMP. Pyrilamine did not prevent the rise in either cyclic nucleotide. This suggests that the antigen-induced rise in cyclic AMP is an indirect result of histamine released from the tissue. The inability of H₁ and H₂ receptor antagonists to affect antigen-induced elevation of cyclic GMP in sensitized lung fragments suggests that an elevation in cyclic GMP might be either a primary event in the mediator release sequence or secondary to the release of a mediator other than histamine. The ability of ethanol to inhibit mediator release might be due to its capacity to attenuate the antigen-induced elevation of cyclic GMP in sensitized lung.     

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.     

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.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

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.     

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.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic GMP (0.3 and 1.0 mM) with increased prostaglandin E (PGE) production (4-fold and 8-fold, respectively) during a 6 h incubation. The effect of 8-Br-cyclic GMP was noted after a lag period of 2–4 h. 8-Br-cyclic AMP (1.0 mM) also stimulated PGE production (4-fold increase), while 8-Br-cyclic IMP, 8-Br-5′GMP and 8-Br-5′AMP were inactive in this respect. Actinomycin D (10 μg/ml) and cycloheximide (10 μg/ml) given simultaneously with 8-Br-cyclic GMP prevented the stimulatory effect of the cyclic nucleotide. The results suggest that cyclic GMP induces de novo synthesis of a macromolecular component of the ovarian prostaglandin synthetase system, and that this cyclic nucleotide, along with cyclic AMP, may play a role in the known stimulatory action of luteinizing hormone on follicular prostaglandin production.     

Effects of ionophore A23187 on in vitro rat adrenal corticosterone production.

The administration of the ionophore A23187 to superfused rat adrenal cortical tissue slices resulted in a significant elevation in corticosterone production. Removal of calcium from the superfusion medium prevented this ionophore induced corticosteroidogenesis. Threshold amounts of ionophore potentiated the steroidogenic action of 1 mU but not 10 mU ACTH under $\text{in vitro}$ conditions. This potentiation by ionophore on ACTH stimulated steroid production was not observed when calcium was omitted from the superfusing medium. Potentiation by the ionophore on dbCAMP or CAMP stimulated steroid formation was not observed for any dose of cyclic nucleotide employed.     

Neurotensin regulation of TSH secretion in the rat

The ionophore A23187 (6.7 microM) increased the rates of formation of prostaglandins and cyclic AMP in suspensions of thioglycollate-elicited rat peritoneal macrophages. Both effects were inhibited by the calmodulin blocker trifluoperazine (50 microM) and the calcium channel blocker verapamil (500 microM). Inhibitors of phospholipase A2 and cyclo-oxygenase also blocked both actions of A23187. The stimulated prostaglandin formation was markedly reduced when the cells were preincubated with 8-bromo-cyclic AMP (1mM), dibutyryl cyclic AMP (1mM) or cholera toxin (500ng/ml). Addition of exogenous arachidonic acid (30 microM) alleviated this inhibition. We propose that the effect of A23187 on macrophages includes a 'self-limiting' mechanism whereby newly-synthesized prostaglandins can inhibit, via cyclic AMP, a step(s) prior to the transformation of arachidonic acid and thus modulate their own production.     

Genetic manipulation of cyclic AMP levels in Drosophila melanogaster.

Cyclic AMP levels in $\text{Drosophila}$,$\text{melanogaster}$ adults can be altered genetically by changing the number of doses of chromomere 3D4 contained in the genome, a chromomere previously shown to control the activity of cyclic AMP phosphodiesterase in a dose-dependent manner. Flies completely deficient for chromomere 3D4 have 2–7 times the cyclic AMP level of flies with one or two doses of chromomere 3D4. Cyclic AMP levels are significantly depressed in flies carrying three doses of 3D4. Cyclic GMP levels are not influenced in a dose-dependent manner by chromomere 3D4. The effect on cyclic AMP levels may provide a useful system for investigating physiological and developmental consequences of aberrant cyclic AMP levels in the intact organism.     

Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists; cyclic AMP and corticosterone production

Isolated adrenal cortex cells respond to the addition of ACTH_1–39 or analogs with increased production of cyclic AMP and corticosterone. It is estimated that cyclic AMP production need proceed at less than 20% of maximum to induce maximum corticosterone production. ACTH_1–24, [Lys¹⁷, Lys¹⁸]ACTH_1–8 amide, and ACTH_1–16 amide induce a maximum rate of cyclic AMP and of corticosterone production equal to those of ACTH_1–39. The relative potencies as determined by cyclic AMP and by corticosterone production are in excellent agreement. The analog, ACTH_5–24, induces maximum cyclic AMP production equal to 45% of that of the natural hormone, but as predicted, induces maximum corticosterone production equal to that of ACTH_1–39. The derivative, [Trp(Nps)⁹]ACTH_1–39 induces 77% of maximum corticosterone production and less than 1% of maximum cyclic AMP production. The fragment ACTH_11–24 is a competitive antagonist of ACTH_1–39 for both cyclic AMP and corticosterone production. The observations on agonists, a partial agonist and a competitive antagonist are in harmony with the “second messenger” role assigned to cyclic AMP. A provisional model, based on the fit of the experimental observations to a set of equations, provides expressions of “intrinsic activity,” “receptor reserve”, “sensitivity”, and “amplification” in terms of maximum cyclic AMP production, concentration of ACTH which induces $\text{1}\text{2}$ maximum cyclic AMP production and concentration of cyclic AMP which induces $\text{1}\text{2}$ maximum corticosterone production.     

A selective defect in arachidonic acid release from macrophage membranes in high potassium media

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

Cyclic AMP production in isolated rat seminiferous tubule cell preparations: A potential in vitro assay for follicle stimulating hormone

A simple procedure for the isolation of rat seminiferous tubule cell preparations free of Leydig cells is described. Both ovine and human FSH stimulated cyclic AMP accumulation. in proportion to the concentration of the hormones. Other hormones, including ICSH and hCG, were inactive. It is proposed that stimulation of cyclic AMP accumulation in tubule cells may be a specific, sensitive and rapid $\text{in vitro}$ assay for FSH.     

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.     

Pentagastrin and adenosine 3',5'-cyclic monophosphate stimulated secretion of somatostatin from perfused rat gastric mucosa.

Immunoreactive somatostatin is secreted by rat gastric mucosa perifused $\text{in vitro}$. Somatostatin release is stimulated by pentagastrin and cyclic AMP with theophylline. These results suggest that gastric mucosal somatostatin may have a paracrine action as feedback inhibitor of gastrin secretion.