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.     

Sensitivity of rat brain adenylate cyclase to activation by calcium and ethanol after chronic exposure to ethanol

Rats were fed ethanol (Lieber-DeCarli diet) for three weeks. Stimulation of cerebellar adenylate cyclase by calcium was measured in control (pair-fed), chronic-alcohol and alcohol-withdrawn animals. No differences in the sensitivity or maximal stimulation of this enzyme were observed among these groups. Ethanol $\text{in}$,$\text{vitro}$ (1%) stimulated brain adenylate cyclase approximately 50% in the presence or absence of calcium. Chronic alcohol exposure $\text{in}$,$\text{vivo}$ did not alter the sensitivity of adenylate cyclase to stimulation by alcohol $\text{in}$,$\text{vitro}$.     

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.     

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.     

Ascorbic acid modulation of splenic cell cyclic GMP metabolism.

Chronic ascorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80%); ascorbate (1 mM) addition to these cells $\text{in vitro}$ restored the cellular concentration to control levels. Splenic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to exogenous ascorbate whereas thiol reducing agents diminished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of ascorbate while agents known to interfere with or promote H₂O₂ production had no effect. Guanylate cyclase activity in cell lysates increased after treatment of intact cells with ascorbate; dithiothreitol reversed this effect. Ascorbate also enhanced guanylate cyclase activity in cell lysates. The results suggest that oxidizing equivalents in the form of the monoanionic free radical of ascorbate alter cyclic GMP metabolism in these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component.     

Inhibition of ascorbate autoxidation by a dialyzed,heat-denatured extract of plant tissues

Preparations obtained from various plant sources were analyzed for their effect on the autoxidation of ascorbic acid and norepinephrine. The former reaction was followed by spectro-photometric detection of ascorbic acid at 265 nm, the latter one by measuring the formation of noradrenochrome at 480 nm. Extracts were prepared from $\text{Philodendron}$ leaves and the edible portion and seeds of green peppers ($\text{Capsicum Annuum}$). The tissues were minced, homogenized in 10 volumes of 16 mM Na-phosphate buffer pH 7.4 and centrifuged at 35,000g for 30 min. The supernatant was dialyzed in 12,000 m.w. cut-off tubing, denatured in boiling water and centrifuged at 10,000g for 10min. Aliquots (5–50 ul) of the supernatant were assayed in 5 ml 16 mM Na-phosphate buffer pH 7.4 containing 100 uM ascorbate or norepinephrine. The denatured extracts had marked dose-dependent inhibitory effect on the autoxidation of ascorbic acid, with negligible influence on the formation of noradrenochrome. EDTA inhibited both reactions. The selectiveness of the extract toward the autoxidation of ascorbic acid makes it unlikely that the inhibitory effect is based on sequestering metal-ions.     

Dietary ascorbic acid deficiency in guinea pigs: No effect on ethanol preference,spiroperidol binding,or monoamine oxidase activity

Ascorbic acid levels are commonly reported to be decreased in alcoholics. Although this deficiency could be due to dietary factors, there is evidence that ascorbic acid may be involved in the metabolism and acute effects of ethanol, possibly related to the pathogenesis of alcoholism. Therefore, we examined ethanol preference in guinea pigs receiving an ascorbate deficient $\text{vs}$ a normal diet. Brain and spleen ascorbic acid levels were dramatically decreased, but ethanol preference was not altered by the acute dietary deficiency of this vitamin. In addition, an acute stressor (cold water swim), alone or in combination with ascorbate deficiency, had no effect on ethanol preference. At termination of the experiment, two measures of brain aminergic function (MAO activity and ³H-spiroperidol binding), purportedly altered by ethanol or ascorbic acid or both, were not associated with tissue ascorbate levels.     

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.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

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.     

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.     

Adenylate cyclase and cyclic AMP through the cell cycle of Tetrahymena.

Adenylate cyclase activity and 3′, 5′ cyclic adenosinemonophosphate (cAMP) have been followed through the heat-synchronized cell cycle of Tetrahymena pyriformis. While the specific activity of adenylate cyclase remained essentially constant throughout the cycle, cAMP oscillated (between 10 and 50 pmoles/mg protein) through two cycles. Minima were observed at each division ($\text{D}\text{S}$ border) and maxima at each $\text{S}\text{G}\text{2}$ border. Each heat shock caused slight temporary reduction in cyclase activity. Further observations suggest to us that adenylate cyclase shows conformational changes in response to temperature-induced alterations and to changes in lipid composition of membranes.     

Membrane fluidity and adenylate cyclase activity in genetically obese mice

Adipocyte plasma membranes of genetically obese $\text{ob}\text{ob}$ mice are more fluid than their lean littermates but the fluidity was normalised in mice maintained at high environmental temperatures. The defective response of adenylate cyclase to isoproterenol was improved after normalisation of membrane fluidity. No major changes in the phospholipid composition of $\text{ob}\text{ob}$ membranes were detected.     

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 in vivo of a sub-hypnotic dose of ethanol on nerve conduction velocity in mice

Relative nerve conduction time (reciprocal of velocity), before and after administration of ethanol, was studied in the caudal nerves of 58 mice. Doses of 1.4, 2.0, 2.5, 3.0, and 4.24 g ethanol/kg body wt., given i.p., were used. The tail was maintained at 37.0° and a control (saline) injection preceeded the ethanol injection. Doses of 1.4 and 2.0 g/kg did not produce significant effects on conduction time but doses of 2.5, 3.0, and 4.24 g/kg did, the relative conduction times twelve minutes after ethanol increasing by 1.2, 1.8, and 3.1 percent, respectively. This appears to be the first controlled demonstration of an acute $\text{in}$$\text{vivo}$ effect of ethanol on conduction velocity.     

Chromium and chronic ascorbic acid depletion effects on tissue ascorbate,manganese, and14C retention from14C-ascorbate in guinea pigs

Chromium (Cr) potentiates the effects of insulin and a role for insulin in ascorbic acid transport has been reported. Therefore, the effects of Cr and ascorbate depletion on tissue ascorbic acid and¹⁴C distribution and excretion after a¹⁴C ascorbate dose were investigated in guinea pigs. As utilization of dietary Cr is affected by interaction with other minerals, tissue manganese (Mn), zinc (Zn), copper (Cu), and iron (Fe) were examined. For 20 wk, 40 weanling animals were fed either a Cr-deficient (<0.06 μg Cr/g diet, ?Cr) or a Cr-adequate (2 μg Cr from CrCl₃/g diet, +Cr) casein-based diet and were given 1 mg ascorbate/d (?C) or 10 mg ascorbate/d (+C) for 20 wk. Animals fed the Cr-depleted diet had decreased weight at 20 wk (p<0.01). Six hours before necropsy, animals were dosed by micropipette with 1.8 μCi ofl-[carboxyl-¹⁴C] ascorbic acid and placed in metabolic cages. Ascorbate supplementation increased Fe concentrations in most analyzed tissues, hepatic¹⁴C, tissue ascorbate and Mn concentration in the adrenal and testes, but decreased the concentrations of Cu in the kidney and Mn in the spleen. Liver Mn concentration was higher and kidney Mn concentration was lower in +Cr animals. Interactions between Cr and ascorbic acid affected Mn concentrations in bone and brain. These results indicate that ascorbate and Cr may affect Mn distribution. Chromium supplementation decreased plasma cortisol, brain¹⁴C and the amount of¹⁴C expired as carbon dioxide. These findings suggest that dietary Cr may affect ascorbic acid metabolism and the metabolic response to stress.     

Effects of LSD and 2-bromo LSD on striatal DOPAC levels.

Administration of d-lysergic acid diethylamide (LSD) and its analogue 2-bromo lysergic acid diethylamide (BOL) resulted in a shortlasting increase of 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the rat striatum. BOL was more potent than LSD in the dose range 0.5–4.0 mg/kg. Since there was a concomitant increase in the striatal $\text{in}$$\text{vivo}$ tyrosine hydroxylation as measured by DOPA accumulation after decarboxylase inhibition, our findings suggest that LSD and BOL increase the impulse flow in the nigro-neostriatal pathway probably by central dopamine (DA) receptor antagonism. However, 4 hrs after LSD the DOPAC level was decreased, while the DOPA accumulation was not. Thus the effect of LSD on the dopaminergic system appears not to be limited to a pure receptor antagonism. The possibility also exists that the effect of LSD on the nigro-neostriatal DA pathway is secondary to its effect on the central 5-hydroxytryptaminergic system.     

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.     

Further studies on the pharmacology of a false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine).

The pharmacology of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine, DEC) was studied with various preparations. It was found to inhibit the neuromuscular transmission of frog sciatic nerve-gastrocnemius muscle $\text{in}$$\text{vitro}$ with ED₅₀ of 1.93 (0.66 - 5.79) × 10⁻⁴ M. DEC was also found to inhibit dog chorda tympani-Wharton's duct (postganglionic parasympathetic neuro-effector junction) and cat superior cervical ganglionnictitating membrane (sympathetic ganglion) preparations $\text{in}$$\text{vivo}$ with ED₅₀'s of 6.2 (1.8 – 21.1) mg/kg and 12.0 (5.7 - 25.2) mg/kg, respectively. After blockade of these preparations with DEC, the former was still responsive to intravenous injection of pilocarpine (1 mg/kg) and choline (10 mg/kg) and the latter to close arterial injection of acetylcholine (100 μg/injection) and choline (3 mg/min infusion). These results support the idea that DEC paralyzes cholinergic neurons possibly through false cholinergic transmission without blocking the cholinergic receptor at the post-junctional membrane.     

A new type of enzyme electrode: The ascorbic acid eliminator electrode

A new type of enzyme electrode has been developed for $\text{in}$$\text{vivo}$ electrochemical measurements which allows discrimination between ascorbic acid and catecholamines and their metabolites. The electrode employs the enzyme ascorbic acid oxidase held between the voltammetric electrode and solution by a dialysis membrane or immobilized on the outer surface of serous membrane from rat small intestine. The electrode gives linear calibration curves for all catecholamines and metabolites independent of any ascorbic acid concentrations significant in physiological measurements. The electrode has been tested in brain slice measurements and shown to respond to releases of catecholamines initiated by potassium ion stimulation.