Control of estrogen and androgen receptors in the rat pineal gland by catecholamine transmitter

Sucrose gradient studies of rat pineal cytosol incubated with ³H-estradiol (female pineals) or ³H-5 α -dihydrotestosterone (male pineals) revealed a radioactivity peak in the 8 S region which disappeared after superior cervical ganglionectomy or incubation with excess unlabeled hormone. Ganglionectomy decreased significantly estradiol and testosterone uptake by the pineal gland $\text{in vitro}$ as well as high affinity binding to pineal cytoplasmic and nuclear components. Norepinephrine treatment counteracted all the effects of ganglionectomy but was unable to modify hormone uptake and binding by the pineal gland of sham-operated controls. Pre-treatment with actinomycin D or propranolol but not with phentolamine impaired norepinephrine effects; propranolol blockage however was only partial. Administration of isoproterenol, L-dopa or phentolamine increased hormone uptake by denervated pineals. The effects of isoproterenol were also observed $\text{in vitro}$ and were blocked by propranolol. These results indicate that sex steroid receptors in the pinealocytes are controlled by norepinephrine via beta-adrenergic receptors and that depletion of neural norepinephrine enhanced responsiveness of pineal hormone receptors to exogenous catecholamines.     

Regression of thyroid hormone induced cardiac hypertrophy: Effect on cardiac beta receptors and adenyl cyclase activity

Adrenergic mechanisms may be important in the symptomatic manifestations of hyperthyroidism. The chronic administration of thyroid hormone also results in cardiac hypertrophy and increased numbers of beta-adrenergic receptors in cardiac membranes. The roles of adrenergic mechanisms in the initiation and perpetuation of this hypertrophy has been open to speculation. Rats treated chronically with L-thyroxin were sacrificed after 7 days of treatment and 1–4 days after cessation of treatment. Hearts were removed and weighed and norepinephrine measured. In other groups of identically treated rats, membranes were prepared from the left ventricle for $\text{invitro}$ measurements of beta-adrenergic receptor characteristics and adenyl cyclase activity. Regression of cardiac hypertrophy with a decrease in receptor number to control values was seen as early as 2 days after stopping thyroxine. Cardiac norepinephrine concentrations had also returned to control values at this time. Displacement of bound [H³] dihydroalprenolol by isoproterenol was not changed from control. Basal and isoproterenol stimulated adenyl cyclase activity was not changed by thyroxine administration or its cessation.The rapid reversal of the increased beta-adrenergic receptor number and cardiac hypertrophy raises the possibility that thyroid hormone may play a regulatory role in cardiac function. Although the enhancement of myocardial contractility by thyroid hormone may be mediated through cardiac hypertrophy this effect of thyroid hormone is independent of the catecholamine sensitive adenyl cyclase system.     

Effects of neurotransmitters and other pharmacological agents on 32Pi incorporation into phospholipids of the iris muscle of the rabbit

The effects of norepinephrine, other catecholamines, α- and β- adrenergic receptor blocking agents and acetylcholine on the incorporation of ³²Pi into phospholipids of the iris muscle of the rabbit were studied $\text{in vitro}$. There was a marked stimulation of ³²Pi into phosphatidic acid (PhA), phosphatidyl inositol (PhI) and to a much lesser extent phosphatidyl choline but not into phosphatidyl ethanolamine. The increase in the ³²P labeling of PhA and PhI in the presence of norepinephrine or acetylcholine, which ranged from 2- to 6-fold, was found to be time- and concerntration-dependent. Under our experimental conditions, several adrenergic drugs, including DL-propranolol, phentolamine, isoproterenol, phenylephrine, but not sotalol, increased markedly (nearly up to 5-fold) the ³²Pi incorporation into PhA and PhI of the iris. In contrast, phenoxybenzamine, an α-receptor blocker, blocked completely the stimulatory effects of norepinephrine on phospholipid synthesis. The stimulation of phospholipid synthesis by acetylcholine was completely abolished by atropine. Incorporation of ³²Pi into PhA and PhI was significantly increased in the presence of serotonin, dopamine, epinephrine or histamine. Addition of γ-aminobutyric acid or cyclic AMP was ineffective. These observations suggest that in the iris muscle of the rabbit, which is innervated by cholinergic and adrenergic fibers, the phospholipid effect is probably a membrane effect that is not associated with synaptic transmission.     

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.     

Malaria enhances cyclic AMP production by immature erythrocytes in vitro.

Infection with a chloroquine-susceptible line of $\text{Plasmodium}$$\text{berghei}$ NYU-2 enhanced the production of cyclic AMP in response to isoproterenol only in immature mouse erythrocytes, which possess a functional hormone-receptor-adenylate cyclase complex. With 10^?7 M isoproterenol the increases in cyclic AMP concentrations (picomoles per 10⁸ erythrocytes) were 19 for a preparation of mature erythrocytes, 81 for a preparation enriched with immature erythrocytes, 25 for a preparation of infected mature erythrocytes, and 900 for a preparation enriched with infected immature erythrocytes. Beta-adrenergic receptor blockade with propranolol prevented this response to isoproterenol but had no effect on the stimulation produced by prostaglandin E₁. These findings indicate that the malaria parasite enhances the responsiveness of a fundamental regulatory system that is intrinsic to immature erythrocytes.     

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.     

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.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

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.     

α2-Adrenergic Regulation of Arylalkylamine N-Acetyltransferase in Organ-Cultured Chick Pineal Gland: Characterization with Agonists and Modulation of Experimentally Stimulated Enzyme Activity

In the chicken pineal gland, norepinephrine, released at sympathetic nerve endings, plays a role in synchronizing the circadian rhythm of melatonin synthesis. This effect appears to be exerted via an adrenergic inhibition of arylalkylamine N-acetyltransferase, the melatonin rhythm-generating enzyme. The present study indicates that the nighttime peak of N-acetyltransferase activity developed by organ-cultured chick pineal glands is inhibited by adrenergic agonists with a potency order characterizing alpha 2-adrenergic receptors: UK 14,304 greater than clonidine greater than alpha-methylnorepinephrine = epinephrine greater than cirazoline greater than phenylephrine greater than isoproterenol. The mechanism of this alpha 2-adrenergic response was further analyzed in organ cultures, by studying the ability of clonidine to block the cyclic AMP-dependent and the depolarization-dependent stimulations of N-acetyltransferase activity. Clonidine prevented the rise in N-acetyltransferase activity evoked by the adenylate cyclase activators forskolin and cholera toxin or by the phosphodiesterase inhibitor Ro 20,1724. The stimulatory effect of dibutyryl cyclic AMP was also blocked by clonidine. Activation of pineal alpha 2-adrenergic receptors effectively prevented the stimulation of N-acetyltransferase by depolarizing concentrations of KCl. The possibility that the alpha 2-adrenergic effect might be exerted at a step distal to cyclic AMP production is discussed.     

Cyclic AMP-induced release of [14C]taurine from pinealocytes.

Pinealocytes were prelabelled with [¹⁴C]taurine. Twenty-four hours later they were treated with derivatives of cyclic AMP. It was found that dibutyryl cyclic AMP and ?-chloro-phenyl-thio cyclic AMP treatment caused a large increase in the release of [¹⁴C]taurine. The effect of dibutryl cyclic AMP on [¹⁴C]taurine release was near maximal fifteen minutes after treatment started. In view of the known stimulatory effects of norepinephrine on pineal cyclic AMP and the recent discovery that norepinephrine causes the release of taurine from pinealocytes, one can conclude that norepinephrine stimulates [¹⁴C]taurine release from pinealocytes by acting through a cyclic AMP mechanism.     

Circadian Variation of Cyclic AMP in the Rat Pineal Gland

Abstract: This study was carried out to investigate circadian variation of cyclic AMP contents in the rat pineal glands, using the high-energy microwave radiation technique. The pattern of cyclic AMP concentration in the pineal gland showed a distinct circadian variation, with the maximum level at 0200 and the lowest at 1400. The administration of propranolol completely blocked the dark-induced increase in the pineal cyclic AMP level at 0200, and the administration of isoproterenol induced a threefold, rapid increase in the cyclic AMP level at 1400, although it did not change the level at 0200.     

Effects of morphine on cyclic AMP levels in the prostate glands of the mouse.

Morphine sulfate (20 mg/kg daily × 5 or 10 days) failed to produce any significant changes in the weights of the testes, liver or sex accessory organs. Injections of morphine for 1, 5 or 10 days caused significant increases in the prostate gland levels of ³H-cAMP (P < 0.01). Levels of ³H-cAMP formed from ³H-adenosine and endogenous cAMP were likewise observed to be elevated when prostate glands were incubated $\text{in vitro}$ with varying concentrations of morphine sulfate. When morphine sulfate (10, 20 or 40 mg/kg daily for 1, 5 or 10 days) was injected (s.c.), endogenous cyclic AMP levels were also significantly elevated in the prostate gland. The higher doses of morphine and the longer duration of its administration caused the greatest degree of elevation of endogenous cAMP. These dose regimens exerted little effect upon the liver's ability to metabolize tritiated testosterone.     

Effect of adrenergic agents on α-amylase release and adenosine 3′,5′-monophosphate accumulation in rat parotid tissue slices

The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20–30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both α-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a β₁-adrenergic mechanism.At the lowest concentrations tested, each of the adrenergic agonists stimulated significant α-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased α-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of α-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of α-amylase release by isoproterenol.Stimulation of α-amylase release by phenylephrine was only partially blocked by either α- or β-adrenerg blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and α-amylase release. However, phenoxybenzamine also potentiated the stimulation of α-amylase release by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using α-adrenergic blocking agents as tools for investigation of α- and β-adrenergic antagonism.     

The failure of aminophylline to modulate glucagon release in man

There are conflicting results regarding the impact of cyclic AMP on pancreatic glucagon release. The effect of aminophylline, a phosphodiesterase inhibitor, on glucagon secretion was studied in four non-obese, non-diabetic, healthy young male volunteers. The subjects received separate infusions of: 1) aminophylline; 2) aminophylline and propranolol; 3) arginine; 4) aminophylline and arginine; 5) insulin; 6) aminophylline and insulin; and 7) aminophylline and isoproterenol. Aminophylline not only failed to alter glucagon levels but also did not affect the glucagon responses observed after arginine and insulin-induced hypoglycemia. The concurrent infusion of isoproterenol and aminophylline also failed to cause a glucagon response. Although glucagon release has been evoked by cyclic AMP in some $\text{in vitro}$ systems, administration of aminophylline to human subjects does not enhance secretion. These results indirectly suggest that cyclic AMP is of little importance in the control of glucagon secretion in man, though the effects of aminophylline at the cellular level may be complex.     

LOW CONCENTRATIONS OF LITHIUM INHIBIT THE SYNTHESIS OF CYCLIC AMP AND CYCLIC GMP IN THE RAT PINEAL GLAND

Abstract— Lithium chloride (2 m m ) significantly inhibited the increases in cyclic AMP and in cyclic GMP caused by norepinephrine or high concentrations of potassium in intact rat pineal glands. Adenylyl cyclase activity in homogenates and its stimulation by isoproterenol, a β-adrenergic agonist, were also inhibited. Lithium reduced the apparent V _max of isoproterenol-stimulated adenylyl cyclase activity without significantly affecting the apparent affinity for isoproterenol. There was no effect on the binding of the antagonist [³H]dihydroalprenolol to the β-adrenergic receptors, nor on the competition for binding sites by isoproterenol. Inhibition of adenylyl cyclase activity by lithium was inversely related to the magnesium concentration in the reaction mixture. There was no differential effect of lithium on adenylyl cyclase activity from supersensitive vs subsensitive glands. Lithium may inhibit cyclic nucleotide synthesis by interfering with the role of divalent cations.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

Apparent absence of alpha-2 adrenergic receptors from hamster brown adipocytes

The possible presence of α adrenergic control of lipolysis and cyclic AMP production in brown adipocytes of hamsters was studied in adipocytes isolated from interscapular, subscapular, cervical and axillary regions of normal male hamsters maintained at 25°C. Lipolysis activated by either 3-isobutyl-1-methyl xanthine or isoproterenol was unaffected by the presence of the α adrenergic selective agonists clonidine and methoxamine. Similarly, accumulation of cyclic AMP in response to β-receptor stimulation, alone or in combination with a methyl xanthine, was unaffected by clonidine or methoxamine. In contrast, both lipolysis and cyclic AMP accumulation in brown fat cells were effectively suppressed in the presence of nicotinic acid, prostaglandin E₁ or N⁶-phenylisopropyl adenosine. Accumulation of cyclic AMP in response to the mixed agonist norepinephrine was not influenced when cells were exposed to the alpha adrenergic blocking drugs yohimbine or tolazoline. These observations suggest that alpha-2 adrenergic receptors which are present on hamster white fat cells and control production of cyclic AMP and lipolysis are absent from hamster brown adipocytes. On the other hand, brown fat cells of this species appear to respond to a number of other inhibitory compounds in a manner not markedly different from that of white adipocytes.     

Uterine cyclic AMP formation: biphasic effect of estrogen on catecholamine sensitivity.

Female, ovariectomized rats were treated with estradiol and then, after various time periods, given an intravenous injection of isoproterenol or epinephrine. 30 seconds later uteri were frozen $\text{in}$$\text{situ}$ and assayed for cyclic AMP and glycogen phosphorylase. The cyclic AMP response to catecholamines was significantly depressed as early as 30 minutes after estrogen and at 6, 12 and 24 hours was 50% of that in non-estrogen-treated controls. Catecholamine-induced glycogen phosphorylase activation was unchanged until 24 hours after estrogen when it was significantly increased over controls. At 48 hours of estrogen both the cyclic AMP and phosphorylase responses to catecholamines were greater than controls. Estrogen regulates uterine β-adrenergic sensitivity but the time courses of estrogen effects on the cyclic AMP and glycogen phosphorylase response changes are different. Catecholamine-induced uterine cyclic AMP formation is biphasic: suppression during the first 24 hours of estrogen followed by recovery and finally augmentation by 48 hours. Catecholamine-induced glycogen phosphorylase activation shows only augmentation after 24–48 hours of estrogen. It is concluded that estrogen has independent effects on the β-adrenergic-glycogen phosphorylase activation pathway at two different points; one prior to cyclic AMP formation and another after cyclic AMP formation.     

Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.