Comparison of sulfinpyrazone and BL-3459 with S - 20344, a potent new antithrombotic agent

We wish to report the discovery of a new class of compounds, the nitrooxazoles, which demonstrate potent antithrombotic activity. One of those compounds, S-20344, was compared with sulfinpyrazone and BL-3459 for its ability to block arterial thrombosis in the rat, and in other $\text{in}$$\text{vitro}$ tests designed to assess antiplatelet activity and a possible biochemical mechanism. Results indicate S-20344, given orally 4 hours prior to testing, blocks electrically induced arterial thrombosis in the rat at doses as low as 25 mg/kg (p<0.001). Similar activity was observed with BL-3459 at 10 mg/kg and sulfinpyrazone at 150 mg/kg. $\text{In}$$\text{vitro}$, both BL-3459 and S-20344 were potent inhibitors of platelet aggregation induced by ADP, thrombin or arachidonic acid. In contrast, sulfinpyrazone failed to block aggregation $\text{in}$$\text{vitro}$. Studies on a possible biochemical mechanism show BL-3459 to be a potent phosphodiesterase inhibitor and to act synergistically with PGE₁ in stimulating platelet cyclic AMP synthesis. However, neither S-20344 nor sulfinpyrazone exert an effect on platelet cyclic nucleotide metabolism. Thus, the mechanism underlying the potent anti-thrombotic activity of the nitrooxazole, S-20344, is still unknown, while it appears BL-3459 may exert its antithrombotic effect by increasing platelet cyclic AMP.     

Stimulation of human platelet adenylate cyclase by prostaglandin D2

Prostaglandin D₂ (PGD₂) stimulates the formation of cyclic AMP in human platelets measured as the incorporation of radio-activity from previously labelled intracellular nucleotides. In this action it is similar to, and more powerful than PGE₁. Both inhibition of platelet aggregation and stimulation of cyclic AMP accumulation by PGD₂ and by PGE₁ are potentiated by an inhibitor of platelet phosphodiesterase. A number of minor differences in the response of platelets to PGD₂ and PGE₁ suggest the existence of at least two prostaglandin receptors influencing a single adenylate cyclase.     

Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3′,5′-monophosphate (cyclic AMP)

D,L-Octopamine elevates the cyclic AMP content of the lateral oviduct of the locust, Locusta migratoria, in a dose-dependent manner with a threshold of about 10(-8) M. The effect of octopamine is potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The response is specific for octopamine and synephrine with an order of potency being octopamine = synephrine greater than metanephrine greater than tyramine greater than norepinephrine = dopamine = 5-hydroxytryptamine and the effect of octopamine is inhibited by the alpha-adrenergic receptor antagonist phentolamine. The diterpene adenylate cyclase activator forskolin also elevates cyclic AMP levels and IBMX potentiates the action of forskolin. Stimulation of the two identified octopaminergic neurons which project to the lateral oviducts results in an elevation in cyclic AMP and again this effect is blocked by phentolamine. Elevation of cyclic AMP levels in the lateral oviducts by means of IBMX, forskolin or dibutyryl cyclic AMP mimics the physiological effects of octopamine on this preparation. The results indicate that the octopaminergic control of this insect's visceral muscle is mediated via cyclic AMP.     

INVOLVEMENT OF CYCLIC GMP IN THE MODE OF ACTION OF A NEW ANTITHROMBOTIC AGENT PCA-4230; INHIBITION OF THE PLATELET CYCLIC GMP DEPENDENT PHOSPHODIESTERASE

The effect of PCA-4230, a new dihydropyridine derivative with a potent antithrombotic activity, on cyclic nucleotide phosphodiesterase in platelets was studied. PCA-4230 inhibited (54%) cyclic GMP hydrolytic activity of a platelet cytosolic fraction, whereas it did not affect that of cyclic AMP. Results suggested that PCA-4230 inhibited a cyclic GMP-dependent phosphodiesterase, known as cGB PDE or type V, on a purified enzyme from rabbit platelets by a non-competitive-uncompetitive type inhibition. In addition, PCA-4230 potentiated the increase in both cyclic GMP and cyclic AMP levels evoked by sodium nitroprusside. Furthermore, PCA-4230 and forskolin caused a synergistic effect in cyclic AMP, and also potentiated the phosphorylation of 50 kDa and 22 kDa proteins, reported as substrates of cyclic GMP- and cyclic AMP-dependent protein kinases that are related to the inhibition of platelet functions. Finally, PCA-4230 also potentiated the forskolin- and sodium nitroprusside-inhibited serotonin release evoked by thrombin, probably related to the increased cyclic nucleotide level. © 1997 Elsevier Science Ltd     

Amantadine: an antiparkinsonian agent inhibits bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

The effect of amantadine (an antiparkinsonian agent) on calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes was investigated. Amantadine inhibited bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase but not the bovine brain 63 kDa, heart and lung calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes. The inhibition of bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase was overcome by increasing the concentration of calmodulin. This suggests that amantadine may be an antagonist of calmodulin or act specifically and reversibly on the action of calmodulin. The bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme is predominantly expressed in the brain and its inhibition may result in increased intracellular levels of cyclic AMP (cAMP). The increased intracellular levels of cAMP have a protective role for dopaminergic neurons. The present findings suggest that amantadine may be a valuable tool to investigate the physiological role of 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme in the progression of Parkinson's disease and gives a new insight into the action of this drug.     

Forskolin enhancement of acetylcholine-evoked cyclic AMP formation and catecholamine release in perfused dog adrenals

Summary Unstimulated efflux of cyclic AMP from perfused dog adrenal glands was not altered by 0.1M of forskolin and was slightly increased by 0.3 and 1.0M of forskolin. ACh stimulated efflux of cyclic AMP which preceded CA release and the efflux was dose-dependently enhanced by forskolin. Forskolin did not affect the spontaneous CA release but enhanced ACh-evoked catecholamine (CA) release. There was a close correlation between the dose relationship of forskolin enhancement of stimulated-cyclic AMP efflux and that of evoked-CA release. ACh-evoked CA release in the presence of forskolin was further potentiated by R020-1724, a phosphodiesterase inhibitor. CA release evoked by excess K⁺, or by caffeine in the presence or absence of external Ca²⁺ was also potentiated by forskolin. These results suggests that cyclic AMP generation may increase in response to stimulation of adrenal chromaffin cells and that the resulting increase of the nucleotide may function as a facilitating modulator of CA release.     

Chronic treatment of newborn rats with naltrexone alters astrocyte production of nerve growth factor

Newborn rats were treated with the opiate antagonist naltrexone daily for 1–2 wk in order to examine the effects of endogenous opioid peptides on astrocytes during CNS development. Nerve growth factor (NGF) and cyclic AMP were measured in astrocytes cultured from cerebellum, striatum, and hippocampus of 1 d, 1 wk, and 2 wk postnatal rats. Cerebellar and striatal, but not hippocampal, astrocytes prepared from naltrexone-treated animals produced higher levels of NGF than those from controls. The turnover rate of cyclic AMP, measured following treatment of the cells with forskolin in the presence of the phosphodiesterase inhibitor IBMX, was increased in naltrexone-derived cerebellar and striatal astrocytes. Opiate receptors could not be detected on the cultured astrocytes, either by direct binding of³H-etorphine or by modulation of cyclic AMP content. These results suggest that endogenous opioid peptides may function indirectly to alter trophic factor synthesis in astrocytes.     

Development of adenosine-dependent cyclic AMP accumulation in the avian optic tectum

The present work shows the existence of adenosine-dependent cyclic adenosine monophosphate (AMP) accumulation in the chick optic tectum. When tecta from 18-day-old embryos were incubated with the phosphodiesterase inhibitor IBMX and RO 20-1724, the cyclic AMP level increased from 39.2 to 73.3 and 285.5 pmol/mg protein, respectively. The high level obtained with RO 20-1724 could be inhibited by increasing concentrations of IBMX or by adenosine deaminase, but not by dipyridamole. 2-Chloroadenosine promoted a dose-dependent cyclic AMP accumulation in tecta incubated with RO 20-1724 and adenosine deaminase. This effect was blocked by IBMX and varied substantially during the development of the tissue. The degree of stimulation increased after day 11 of incubation, attaining maximal levels on day 14. The effect of 2-chloroadenosine remained constant until day 18, a period when both the protein content and the basal cyclic AMP levels are increasing in the developing tectum. The cyclic AMP increase elicited by 2-chloroadenosine was greatly reduced in tecta from 20-day-old embryos and 2-day-old chicks. The putative transmitters glutamate and glycine and the neurotransmitter analogs isoproterenol and carbachol had no stimulatory effect on the cyclic AMP accumulation of tecta from 10- and 17-day-old embryos.     

Effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein

We have discovered that bradykinin is effective in causing the synthesis of prostacyclin in endothelial cells cultured from calf and pig aorta and human umbilical cord vein. Maximal stimulation is attained at 10 ng bradykinin per ml with a 10 fold increase of PGI₂ as assayed by radioimmunoassay for 6-k-PGF_1α. Ionophore A23187, trypsin, and the precursor arachidonic acid are also stimulatory. We have confirmed the study of Weksler $\text{et al}$ ($\text{J. Clin. Invest., 62}$, 923, 1978) and Czervionke $\text{et al}$ ($\text{Thrombosis Research, 14}$, 781, 1979) that thrombin is effective in stimulating PGI₂ synthesis in endothelial cells from human umbilical cord vein. However, we found that cells from calf or pig aorta are not stimulated as well by thrombin. Thus, there appears to be a difference in the response to thrombin between these cells. When calf cells were grown in the presence of [³H]arachidonic acid, the radioactivity is incorporated mainly into the phospholipids. Bradykinin, ionophore A23187, and trypsin stimulated the release of radioactive materials into medium from [³H]arachidonic acid labeled calf cells. Arachidonic acid is the major radioactive substance released and PGI₂ is the major known arachidonic acid metabolite formed.     

Phorbol-12,13-dibutyrate antagonizes while forskolin potentiates the presynaptic autoreceptor-mediated inhibition of [3H]-5-hydroxytryptamine release in rat hypothalamic slices

In superfused rat hypothalamic slices prelabeled with [3H]-5-hydroxytryptamine [( 3H]-5-HT), the 5-HT autoreceptor agonists 5-methoxytryptamine and RU 24969 inhibited in a concentration-dependent manner the electrically evoked release of [3H]-5-HT. Exposure to phorbol-12,13-dibutyrate increased in a concentration-dependent manner the stimulation-evoked overflow of [3H]-5-HT and shifted to the right the 5-methoxytryptamine inhibition curve. Exposure to forskolin, a potent activator of adenylate cyclase, increased the stimulation-evoked [3H]-5-HT overflow and shifted to the left the 5-methoxytryptamine or RU 24969 inhibitory curves on transmitter release. A similar interaction was observed in the presence of 1-isobutyl,3-methylxanthine (IBMX), a phosphodiesterase inhibitor, or 8-bromo-cyclic AMP and the serotonin autoreceptor agonist 5-methoxytryptamine. In the presence of phorbol-12,13-dibutyrate or forskolin, the enhancing effect of the 5-HT autoreceptor antagonist methiothepin on the stimulation-evoked [3H]-5-HT overflow was significantly less pronounced than in the absence of these compounds. These results indicate that the presynaptic 5-HT autoreceptors that modulate the release of [3H]-5-HT in rat hypothalamic slices may be coupled to the phosphoinositide cycle and protein kinase C-dependent mechanisms. In addition, the increase in intracellular level of cyclic AMP by forskolin, IBMX, and 8-bromo-cyclic AMP potentiates the inhibitory effects of the autoreceptor agonist 5-methoxytryptamine on [3H]-5-HT release, although the mechanism of the interaction remains to be elucidated.     

Modulation of rhythmic melatonin synthesis in Xenopus retinal photoreceptors by cyclic AMP

Cyclic AMP regulates melatonin synthesis in vertebrate photoreceptor cells. In the present study, we investigated whether the circadian rhythm of melatonin synthesis in Xenopus retinal photoreceptor layers is driven by rhythmic changes in cyclic AMP. When the photoreceptor layers were continuously treated with 8-(4-chlorophenylthio)-cyclic AMP (8-CPT-cAMP) at a saturating concentration (1 mM), melatonin release was increased at all times of the day, but robust melatonin rhythms were maintained for 2 days in constant darkness (DD). We also measured cyclic AMP efflux and melatonin release simultaneously from photoreceptor layers that were continuously treated with forskolin and/or 3-isobutyl-1-methylxanthine (IBMX) in light–dark (LD) and DD. Circadian rhythmicity was observed in melatonin release, but not in cyclic AMP efflux, suggesting that changes of melatonin levels are not always caused by the changes of the cyclic AMP levels. In addition, the simultaneous treatment of forskolin and IBMX appeared to saturate sensitivity of melatonin synthesis to cyclic AMP, but this treatment did not abolish melatonin rhythms. These results suggest that circadian rhythms of melatonin can be driven without rhythmic changes of cyclic AMP, and that cyclic AMP regulates melatonin in parallel with the output pathways from the circadian oscillator.     

Low-dose aspirin inhibits platelet and venous cyclo-oxygenase in man

In a group of normal subjects we have compared rates of recovery of venous prostacyclin (PGI₂) and platelet thromboxane B₂ (TXB₂) production after a single 300 mg dose of aspirin. Marked inhibition of both venous PGI₂ synthesis and platelet TXB₂ synthesis persisted for the 72 hours duration of the study. We also examined the effect of four daily doses of 40 mg aspirin on platelet and vessel wall prostaglandin biosynthesis. This produced a cumulative inhibitory effect on TXB₂ production with 98.6 per cent mean inhibition at 72 hours. At this time 81 – 100 per cent inhibition of PGI₂ was demonstrated in the four subjects studied. We conclude that the cyclo-oxygenase enzyme in human vein is sensitive to inhibition by very low doses of aspirin. No evidence was found to substantiate the view that rapid recovery of vessel wall cyclo-oxygenase activity occurs after inhibition by a single dose of aspirin.     

Cyclic AMP-mediated potentiation of muscarinic hyperpolarization in neuroblastoma cells

Adenosine, 2-chloroadenosine and prostaglandin E₁ which are known to increase cyclic AMP in neuroblastoma cells potentiated the acetylcholine-induced muscarinic hyperpolarization of the cells without changing the resting membrane potential. The potentiation caused by 2-chloroadenosine was further augmented by Ro 20-1724, a phosphodiesterase inhibitor. A direct intracellular pressure application of cyclic AMP potentiated the muscarinic hyperpolarization without changing the resting membrane potential. Morphine which inhibits adenylate cyclase antagonized 2-chloroadenosine-induced potentiation of the muscarinic hyperpolarization. These results suggest that changes in cyclic AMP level modulate the muscarinic response of neuroblastoma cells.     

Splitomicin suppresses human platelet aggregation via inhibition of cyclic AMP phosphodiesterase and intracellular Ca release

Splitomicin is derived from β-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 μg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 μM) or ADP (10 μM). Splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 μM) was combined with PGE1 (0.5 μM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition, splitomicin (300 μM) attenuated intracellular Ca⁺⁺ mobilization, and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin, collagen, AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca⁺⁺ mobilization, TXB2 formation and ATP release.     

Regulation of adipokinetic hormone release from locust neuroendocrine tissue: participation of calcium and cyclic AMP

In the locust, cyclic adenosine monophosphate (cAMP) mediates at least part of the effects of octopamine, the neurotransmitter which regulates the release of two adipokinetic hormones (AKHs) from the glandular lobe of the corpus cardiacum (CC). We have examined the requirement for extracellular Ca2+ in the process of AKH release mediated by octopamine and by agents which artificially elevate intracellular cAMP levels. Octopamine and the adenylate cyclase activator forskolin elevate the cAMP content of the glandular lobe in normal saline, in normal saline with the Ca2+ channel blocker, methoxyverapamil, and in Ca2+-free saline during 10-min exposure periods. Octopamine, forskolin, and 8-bromo cAMP mediate release of AKHs in vitro in normal saline, but release is prevented in the absence of extracellular Ca2+. When glands are exposed to these agents in normal saline in the presence of methoxyverapamil, AKH release is curtailed in a similar manner. Lanthanum and EGTA dramatically reduce cAMP production elicited by octopamine and forskolin, and lanthanum prevents octopamine-mediated release of AKHs. The phosphodiesterase inhibitor, IBMX, elevates cAMP content in the presence and absence of extracellular Ca2+, and stimulates normal release of AKHs both in the presence and absence of extracellular Ca2+. However, following extensive washing in Ca2+-free saline, IBMX fails to evoke AKH release. Methoxyverapamil has no effect on IBMX-mediated secretion. These results suggest that IBMX may mobilize intracellular stores of Ca2+ to induce release. Extracellular Ca2+ is apparently required for the process of neurotransmitter-evoked release, as has been shown for release of other peptide hormones. Cyclic AMP is intimately associated with Ca2+ in mediating this process. The release of AKHs is more dependent upon extracellular Ca2+ than is cAMP production under the conditions examined in this study. Ca2+ may provide the signal which initiates the secretory response, although cAMP may modulate this signal or the cells' responsiveness to this signal in some way. Support for this hypothesis is provided by experiments with the Ca2+ ionophore, A23187. This agent provokes release of AKHs in a Ca2+-dependent manner, probably by elevating intracellular Ca2+ levels. A23187 does not elevate cAMP levels in the glandular lobe, indicating that cAMP elevation is not a prerequisite for secretion.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

Orexins/Hypocretins Acting at G<Subscript>i</Subscript> Protein-Coupled OX<Subscript>2</Subscript> Receptors Inhibit Cyclic AMP Synthesis in the Primary Neuronal Cultures

Orexins A and B are newly discovered neuropeptides with pleiotropic activity. They signal through two G protein-coupled receptors: OX₁ and OX₂. In this study, we examined the expression of orexin receptors and effects of the receptors’ activation on cyclic AMP formation in the primary neuronal cell cultures from rat cerebral cortex. Both types of orexin receptors were expressed in rat cortical neurons; the level of OX₂R was markedly higher compared to OX₁R. Orexin A (an agonist of OX₁R and OX₂R) and [Ala¹¹-D-Leu¹⁵]orexin B (a selective agonist of OX₂R) did not affect basal cyclic AMP formation in the primary neuronal cell cultures. Both peptides (0.001–1 μM) inhibited, in a concentration-dependent manner and IC₅₀ values in low nanomolar range, the increase in the nucleotide production evoked by forskolin (1 μM; a direct activator of adenylyl cyclase), pituitary adenylate cyclase-activating polypeptide (PACAP27; 0.1 μM), and vasoactive intestinal peptide (VIP; 3 μM). Effects of orexin A on forskolin-, PACAP27-, and VIP-stimulated cyclic AMP synthesis were blocked by TCS OX2 29 (a selective antagonist of OX₂R), and unaffected by SB 408124 (a selective antagonist of OX₁R). Pretreatment of neuronal cell cultures with pertussis toxin (PTX) abolished the inhibitory action of orexin A on forskolin- and PACAP-stimulated cyclic AMP accumulation. It is suggested that in cultured rat cortical neurons orexins, acting at OX₂ receptors coupled to PTX-sensitive G_i protein, inhibit cyclic AMP synthesis.     

Chronic infusion of agents that increase cyclic AMP concentration enhances the regeneration of mammalian peripheral nerves in vivo

Our previous investigation indicates that forskolin, a robust activator of adenylate cyclase, promotes sensory nerve regeneration in amphibians. The present study was designed to determine if forskolin had a similar effect in mammals. We also wished to test the hypothesis that cyclic AMP modulates nerve regeneration by comparing the effects of chronically infused forskolin with the effects of infused dibutyryl cyclic AMP, 8-bromo cyclic AMP, and the phosphodiesterase inhibitor, theophylline. Our results indicated that all agents promoted some aspect of regeneration. The two which presumably generated the largest increase in cyclic AMP concentration, forskolin and 8-bromo cyclic AMP, had the most profound effect on axonal elongation. All agents decreased the time to sprout initiation, but theophylline produced the largest decrease and its effect was mimicked by caffeine, a methylxanthine with limited ability to inhibit phosphodiesterase. This suggests that sprout formation may be triggered by an increase in intraaxonal free Ca2+, possibly modulated by cyclic AMP. The role of cyclic AMP in axonal elongation remains to be determined, but may be associated with stimulation of protein synthesis in the nerve cell body.