Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Effects of calcium on a parathyroid hormone-sensitive adenylate cyclase inhibitor

Inhibition of parathyroid hormone (PTH)-sensitive adenylate cyclase by {Nle-8, Nle-18, Tyr-34} bPTH-(3–34) amide was studied in thyroparathyroid-ectomized dogs. The inhibitory effect was shown to be markedly enhanced by the addition of calcium ions into the $\text{in}\text{,}\text{vitro}$ assay system. At 0.1 mM Ca²⁺, complete inhibition by the antagonist was obtained. Chelation of exogenous Ca²⁺ by EGTA eliminated the Ca²⁺-induced inhibition. Both the basal and hormone-stimulated activities were decreased in the presence of 0.1 mM Ca²⁺, whereas the addition of EGTA increased both activities. Our results suggest that Ca²⁺ modulates canine renal PTH-sensitive adenylate cyclase and its inhibition by substituted bPTH-(3–34).     

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.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I21

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the $\text{in}$$\text{vitro}$ effects of luteinizing hormone (LH) and prostaglandins (PG) E₁, E₂ and I₂. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE₂, PGE₂, or PGI₂ (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E₁ and E₂ had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI₂ was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE₂ upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE₂ caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E₁, E₂ and I₂ are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

Effect of ascorbic acid on neurochemical, behavioral, and physiological systems mediated by catecholamines

Ascorbic acid, at concentrations below that normally present in the brain, inhibited the dopamine-sensitive adenylate cyclase $\text{in}$$\text{vitro}$. Ascorbate had no effect on the norepinephrine-sensitive adenylate cyclase. To study the $\text{in}$$\text{vivo}$ effect of ascorbic acid on central dopaminergic systems, mice (C57 B1/6J) were injected with pharmacological doses (2 g/kg) of ascorbate, which produced a significant elevation in brain ascorbate concentration. Injecting the mice with ascorbate (2 g/kg) blocked the amphetamine-induced (15 mg/kg) increase in stereotype behavior which has been reported to be mediated by dopaminergic neural systems. Ascorbate had no effect on the amphetamine-induced locomotor activity thought to be mediated by norepinephrine systems. Ascorbate (1 g/kg) attenuated apmorphine-induced hypothermia in this same strain of mice. This demonstrates the specific neurochemical, physiological, and behavioral alterations in dopaminergic systems produced by ascorbic acid and suggests possible therapeutic uses for ascorbate in conditions involving functional dopamine excess.     

On the mechanism of tolerance to isoproterenol-induced accumulation of cAMP in rat pineal in vivo.

Less cyclic adenosine 3′:5′ monophosphate (cAMP) accumulated in rat pineal gland, $\text{in}$$\text{vivo}$, after two doses of l-isoproterenol (5mg/kg, i.p.) than after one dose. A single injection of l-isoproterenol decreased the ability of l-isoproterenol to activate adenylate cyclase and increased the activity of the low Km phosphodiesterase (PDE). Tolerance to l-isoproterenol-induced accumulation of cAMP in rat pineal $\text{in}$$\text{vivo}$ may be due to decreased responsiveness of adenylate cyclase as well as to increased activity of PDE.     

Effects of morphine on dopamine-stimulated adenylate cyclase and on cyclic GMP formation in primate brain amygdaloid nucleus.

In homogenates of $\text{Macaca}$$\text{mulatta}$ (Rhesus) or $\text{Cebus}$$\text{apella}$ amygdaloid nuclear complex, adenylate cyclase activity was approximately doubled by either 10μ$\text{M}$ dopamine or 8m$\text{M}$ NaF. In the presence of morphine, the stimulation by dopamine was reduced. A 90–100% inhibition of the dopamine stimulation was obtained with 20μ$\text{M}$, and a 50% inhibition, with 5μ$\text{M}$ morphine. The effects of 10μ$\text{M}$ morphine on dopamine stimulation were reversed by 10μ$\text{M}$ naloxone. Morphine itself did not significantly affect the basal adenylate cyclase activity, but in the presence of 10μ$\text{M}$ morphine the stimulation by 8m$\text{M}$ NaF was reduced approxiamtely 50%. The data suggest an action of morphine at a receptor site which is distinct from the dopamine receptor, but which inhibits the dopamine-stimulated adenylate cyclase. In addition, the cyclic GMP content of $\text{Cebus}$ amygdala slices was reduced by 50–75% during incubation for 5–20 minutes with morphine. Maximum effects on cyclic GMP were obtained with 10μ$\text{M}$, and half-maximum effects, with 0.1μ$\text{M}$ morphine. The effect of morphine on amygdala cyclic GMP was not reversed by naloxone. Thus, this action of morphine may not be receptor mediated, or may involve the interaction of morphine with receptors other than the opiate receptor.     

Striatal adenylate cyclase activity following reserpine and chronic chlorpromazine administration in rats.

Adenylate cyclase activity was assayed in rat striatal homogenates. Dopamine and, to a lesser extent, 1-norepinephrine added $\text{in vitro}$ produced a dose-dependent enhancement of adenylate cyclase activity. Fluphenazine did not alter basal enzyme activity, but prevented both dopamine- and 1-norepinephrine-elicited increases. No significant changes in basal- or dopamine-stimulated adenylate cyclase activity were found in homogenates from rats pretreated with chlorpromazine for 21 days or reserpine for 2 days. It is concluded that the behavioral and neurophysiologic postsynaptic supersensitivities that follow similar pretreatments are not mediated by alterations in the sensitivity of striatal adenylate cyclase to dopamine.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the β-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used.Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, $\text{n-}\text{octylamine}$ and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitrophenols, indomethacin and octanoic acid did not affect the total number of β-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected.As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.     

Extracellular magnesium is not required for beta adrenergic drug-receptor interactions in intact human lymphocytes

We have investigated the effect of extracellular magnesium ions on the function of beta adrenergic receptors in $\text{intact}$ human lymphocytes. We examined adenylate cyclase stimulation by isoproterenol displacement of (-) (³H) dihydroalprenolol from beta receptor sites, and down regulation (desensitization) of beta receptors by prolonged exposure of the cells to isoproterenol. Contrary to results obtained using broken cell preparation, in none of these situations did the presence or absence of extracellular magnesium ions make any difference. The importance of selecting the most nearly physiological preparations for conducting $\text{in vitro}$ studies that may be extrapolated to the whole organism is stressed.     

Stimulation of guinea-pig brain adenylate cyclase by adenosine analogues with potent pharmacological activity in vivo

1-Methylisoguanosine, a marine natural product with potent muscle-relaxant and cardiovascular actions $\text{in vivo}$, interacts directly with adenosine receptors in guinea-pig brain slices to stimulate adenylate cyclase. These effects are blocked by theophylline. Comparison of the $\text{in vivo}$ pharmacological activity of a number of synthetic analogues of 1-methylisoguanosine with $\text{in vitro}$ adenylate cyclase-stimulating ability indicates that compounds lacking the latter biochemical activity have little muscle-relaxant activity. Adenosine is a potent stimulator of adenylate cyclase but is inactive $\text{in vivo}$ because of rapid removal from the extracellular environment by uptake and deamination. Unlike adenosine, 1-methylisoguanosine is resistant to deamination and is only poorly accumulated by brain tissue slices or homogenates containing synaptosomes. Since it is an extremely weak competitive inhibitor of adenosine deaminase and only a weak inhibitor of adenosine uptake, it is unlikely to act by potentiating the effects of adenosine itself at extracellular receptors. Thus, the pharmacological effects of 1-methylisoguanosine are apparently due to its actions as a long-lasting adenosine analogue.     

Adentylate cyclase activity in myocardium of spontaneously hypertensive rat: effect of endogenous factors and solubilization

The isoproterenol- and glucagon-stimulated adenylate cyclase activities in the myocardial membranes of hypertensive rat were consistantly lower as compared with normal controls. Addition of cytosolic fraction (100,000 x$\text{g}$ supernatant) to the particulate preparation had an additive effect for glucagon and Gpp(NH)p stimulated enzyme activity and a synergistic effect for isoproterenol stimulation. Cytosolic fraction of normal control animals did not bring the adenylate cyclase activity in SHR equivalent to the control values. The basal and F^?-stimulated enzyme activity of solubilized adenylate cyclase was reduced by about 30% in SHR as compared with WKY, which could be due to a decrease in the actual amount of adenylate cyclase in the myocardium of SHR.     

Effect of prostaglandin E2 on vascular responses of the rabbit kidney to nerve stimulation and noradrenaline, in vitro and in situ

The effect of prostaglandin E₂ on vascular responses of the rabbit kidney to renal nerve stimulation and noradrenaline was examined $\text{in}$$\text{vitro}$ and $\text{in}$$\text{situ}$ as a test of the hypthesis that prostaglandins of the E series may be involved in the regulation of adrenergic neuroeffector transmission. Intraarterial administration of prostaglandin E₂ to the $\text{in}$$\text{vitro}$ kidney caused marked inhibition of vascular responses to nerve stimulation whereas the responses to noradrenaline were not significantly altered. In the $\text{in}$$\text{situ}$ preparation, vascular responses to both nerve stimulation and noradrenaline were inhibited by prostaglandin E₂ infusion, although its effect on responses to nerve stimulation was approximately twice that observed on responses to noradrenaline.It is concluded that prostaglandin E₂ acts primarily at a prejunctional level of adrenergic neuroeffector transmission in the kidney, although a postjunctional effect has also been observed.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

Comparative biological activities of synthetic leukotriene b4 and its ω-oxidation products

Synthetic leukotriene B₄ (LTB₄) and its ω-oxidation products, 20 OH-LT₄ and 20 COOH-LTB₄, were tested for their ability to induce the aggregation of rat neutrophils $\text{in}$$\text{vitro}$, to contract the guinea pig parenchymal strip $\text{in}$$\text{vitro}$ and to cause vascular permeability changes in rabbit skin $\text{in}$$\text{vivo}$. 20 OH-LTB₄ had 10, 100 and 20% of the activity of LTB₄ in the neutrophil aggregation, parenchymal strip and vascular permeability assays respectively. 20 C00H-LTB₄ was inactive $\text{in}$$\text{vivo}$ and showed <1% of the activity of LTB₄$\text{in}$$\text{vitro}$. These results show that while ω-oxidation is a route for biological inactivation of LTB₄, 20 OH-LTB₄ still retains significant biological activity.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

Influence of lithium on dopamine-stimulated adenylate cyclase activity in rat brain

Dopamine-stimulated adenylate cyclase activity from various rat brain areas was inhibited $\text{in vitro}$ by lithium. The inhibition was dose-dependent and non-competitive. In lithium-treated rats no changes in enzyme activity could be demonstrated.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

Effects of ergots on adenylate cyclase activity in the corpus striatum and pituitary

Adenylate cyclase activity was determined in homogenates of the corpus striatum and pituitary gland. Dopamine and several ergots stimulated cyclic AMP synthesis in the striatum, but no stimulation was seen in the pituitary gland. None of the ergots tested were as active as dopamine itself, and all were able to partially inhibit the dopamine-induced activation of adenylate cyclase. Lergotrile, a simple ergoline derivative which displays dopamine agonist activities in the pituitary gland and striatum, did not stimulate adenylate cyclase in either tissue. These findings show that the $\text{in vivo}$ dopaminergic activity of ergots is not reflected in the dopamine-dependent adenylate cyclase assay using either the corpus striatum or the pituitary gland. It is suggested that those dopamine receptors in the pituitary gland which mediate prolactin release are not associated with adenylate cyclase.