Opposite effects of acute and repeated administration of desmethylimipramine on adrenergic responsiveness in rat pineal gland.

The effect of acute and repeated desmethylimipramine (DMI) treatment on catecholamine-stimulated production of adenosine 3', 5'-monophosphate (cyclic AMP) in rat pineal gland was studied $\text{in}$$\text{vivo}$. In rats exposed to continuous illumination, the administration of isoproterenol (2μmol/kg) to control animals produced a marked increase in the concentration of cyclic AMP in pineal gland. In contrast, norepinephrine (2μmol/kg) failed to increase the levels of cyclic AMP. After acute treatment with DMI (single injection, 38μmol/kg, i. p.), the isoproterenol-induced rise in cyclic AMP was not significantly different from that measured in control animals. However, acute DMI treatment did allow a significant elevation in the concentration of cyclic AMP in pineal gland in response to norepinephrine. In rats given nine injections of DMI (38μmol/kg, i.p., twice daily) neither isoproterenol nor norepinephrine caused a significant increase in the concentration of cyclic AMP in pineal glands. Although acute treatment with DMI had no significant effect on [³H] dihydroalprenolol binding, chronic treatment with DMI significantly reduced [³H] dihydroalprenolol binding in the pineal gland. The results of this study suggest that while a single administration of DMI can enhance adrenergic responses elicited by norepinephrine, chronic administration of DMI leads to compensatory decreases in receptor density and adrenergic responsiveness.     

Uptake of estradiol by the rat pineal organ. Effects of cervical sympathectomy, stage of the estrous cycle and estradiol treatment

Rat pineal organs of spayed rats took up and retained estradiol $\text{in vitro}$ up to 32-fold the concentration present in the incubation media. This phenomenon was maximum at 37°C and after 2-h incubations. Most (86–91%) of [³H] radioactivity recovered from the incubated pineals was identified as estradiol by thin-layer chromatography. Treatment with dextran-coated charcoal of nuclei-free pineal homogenates incubated with [³H] estradiol of different SA uncovered a high affinity, low capacity binding of estradiol to cytosol components. Uptake of estradiol by the nuclear fraction also proceeded in a saturable fashion. Similar findings were made in uterine homogenates of spayed rats.Estradiol uptake by the pineal organ and the uterus of cycling rats varied as a function of the stage of the estrous cycle, maxima being observed in diestrus and minima in proestrus. The administration of a priming dose of estradiol benzoate to spayed rats caused high affinity binding components of the pineal cytosol to increase by about 150%. Nuclear binding of estradiol was also increased by the estradiol priming dose. Pineal denervation, i.e., by superior cervical ganglionectomy, caused pineal estradiol uptake to decrease significantly by about 20%. These data suggest that the early steps of estradiol action on the pineal organ may resemble those of the uterus.     

Circadian rhythm and uptake of taurine by the rat pineal gland

Taurine is believed to be a modulator of membrane excitability in muscle and a neuroinhibitory transmitter in the central nervous system. The retina and pineal contain relatively large quantities of taurine. Taurine levels in the retina are reported to be responsive to variations in lighting conditions. We report here a carcadian rhythm for taurine in the mature male rat pineal gland. The maximum taurine concentration occurs at the midpoint of the light period, 24 ± 1.9 nmoles/gland, and the minimum at the beginning of the dark period, 13.9 ± 1.6 nmoles/gland. Sympathectomy by bilateral superior cervical ganglionectomy lowered pineal taurine levels. Constant light and blinding had no effect. Taurine was demonstrated to be taken up by the pineal gland $\text{in}$$\text{vitro}$ in organ culture. The uptake was saturable, K_m = 2.0 mM, and sodium dependent. The close structural analogs hypotaurine and β-alanine inhibited taurine uptake but α-alanine did not. We have demonstrated a circadian rhythm for taurine content in the rat pineal gland and the presence of a sodium-dependent transport system for taurine in the pineal $\text{in}$$\text{vitro}$ in organ culture.     

STIMULATION OF SEROTONIN N-ACETYLTRANSFERASE IN PINEAL ORGAN CULTURE BY DRUGS

Abstract— Drugs such as cocaine, procaine, pheniprazine (Catron) and veratridine, which have actions on sympathetic nerves and nerve terminals, were examined for their ability to increase serotonin N-acetyltransferase (EC 2.3.1.5; NAT) in pineal organ culture. The absence of potassium (0 KCl) was also examined. NAT is known to respond to β-adrenergic stimulation. It was found that these drugs and 0 KCl increased the enzyme activity 100 to 2000-fold in innervated pineals but had virtually no effect in denervated pineals. The effects on innervated pineals were blocked by the β-blocker propranolol but not by the α-blocker, phentolamine. These drugs and 0 KCl inhibited to varying degrees [³H] 1-norepinephrine uptake in pineals. It is concluded that these agents activated the β-adrenergic receptor on pineal cells by causing an accumulation of extraneuronal norepinephrine. The accumulation of norepinephrine is due, at least in part, to the blockade of norepinephrine reuptake by nerve terminals. The ability of veratridine to stimulate NAT and to inhibit norepinephrine uptake was reversed by tetrodotoxin, a blocker of sodium permeability in excitable tissue, thus veratridine acts by increasing sodium permeability in nerve terminals. This adds support to the theory that catecholamine uptake is a process that requires a sodium gradient across the nerve terminal membrane.     

Effects of naloxone and acetylcholine on medial thalamic and cortical units in naive and morphine dependent rats.

The administration of 0.5 mg of testosterone propionate (TP) for 3 days to castrated male rats caused ³H-leucine incorporation into pineal proteins to increase significantly by 79%. TP effects depended on time of administration; rats receiving TP at 06.00 h exhibited a significant 150% increase in pineal protein synthesis 24 h later whereas rats injected at 14.00 h only showed a 54% increase in ³H-leucine incorporation into proteins. Superior cervical ganglionectomy decreased pineal testosterone uptake $\text{in vitro}$ by 21% and pineal protein synthesis by 27%; in addition it blocked the stimulatory effects of TP on protein synthesis. Ganglionectomy also modified the $\text{in vitro}$ metabolism of testosterone by pineal cells; it increased the amounts of ³H-androstenedione and decreased ³H-5∝-androstanedione extracted from pineal glands incubated with ³H-testosterone. These results indicate that the sympathetic nervous input reaching the pineal via the superior cervical ganglia is important to modulate the early steps of androgen action on the pinealocytes.     

Influence of isoproterenol on net potassium uptake in whole pigeon erythrocytes in vitro

Isoproterenol increases net uptake of potassium in whole pigeon erythrocytes $\text{in vitro}$; effect of 10^?5 M isoproterenol is blocked by 10^?4 M propranolol. Pentifylline, a potent inhibitor of cAMP-phosphodiesterase, significantly amplifies effect of isoproterenol, indicating that isoproterenol-effect is mediated by cAMP. cAMP alone has no direct influence on net potassium uptake, while dibuturyl-cAMP has a very weak effect. Isoproterenol-effects are also mediated by the cell membrane protein-phosphorylation.     

Catecholamine-mediated constrictor effects of aldosterone on vascular smooth muscle

The possibility that the corticosteroid hormone, aldosterone, might possess direct vasoconstrictor properties was examined in the isolated central ear artery of the New Zealand white rabbit. Aldosterone alone produced only minimal contractile effects in the arterial segments; but following pretreatment of the tissue with desipramine (10^?7M), a blocker of neuronal uptake of norepinephrine, aldosterone concentrations of 10^?6M, 10^?5M, and 10^?4M produced stepwise contractile responses of 0.16 ± 0.03 (SE)g, 0.48 ± .04g, and 1.31 ± 0.06g. The possible involvement of norepinephrine in this action of aldosterone was tested in a series of tissues stored for 2 days at 2°C in Krebsbicarbonate medium so as to deplete endogenous catecholamine stores. Treatment with desipramine followed by aldosterone (10^?4M) now produced an average contraction of only 0.1 ± 0.06g; but if the labile neuronal tissue stores of norepinephrine in these tissues were then replenished by exposure to norepinephrine 10^?7M, contractions of 1.2 ± 0.3g now occurred when desipramine and aldosterone were added. To examine whether aldosterone's action might be due to blockade of extraneuronal norepinephrine uptake (uptake-2), ³H-norepinephrine was added to ear artery tissues exposed to desipramine with or without aldosterone: a significant (P<0.005) decrease of 25% in ³H-norepinephrine uptake occurred in the tissues treated with aldosterone. In further studies, the contractile effects of aldosterone could be prevented by pretreatment with prazosin (10^?7M) or phentolamine (10^?7M); if added after the aldosterone, each of these alpha-blockers completely reversed the contractile responses. Although the physiological relevance of these findings is yet to be fully defined, these studies indicate that the $\text{in}$$\text{vitro}$ contractile effects of aldosterone are dependent upon its inhibition of extraneuronal uptake of endogenous norepinephrine; it is likely that the resulting increase in extracellular norepinephrine concentration then produces vasoconstriction by stimulation of post-synaptic alpha-adrenergic receptors.     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

In vivo stimulation of rat pineal type II thyroxine 5'-deiodinase activity by either norepinephrine or isoproterenol

Herein we show, for the first time, a very marked increase in thyroxine 5'-deiodinase (5'-D) activity in rats injected with norepinephrine (NE) and desmethylimipramine, a drug which inhibits NE uptake by nerve terminals. The response to NE was greater in pineals collected from hypothyroid animals than in glands from euthyroid animals. NE was more effective in stimulating pineal 5'-D than was isoproterenol, suggesting that, in addition to beta-adrenergic receptors, alpha-adrenergic receptors might be involved in the 5'-D activation. However, phenylephrine, an alpha-adrenergic agonist, did not potentiate the effect of isoproterenol on pineal 5'-D activity. The nocturnal increase in pineal 5'-D activity was completely abolished by propranolol, a beta-adrenergic receptor blocker, while prazosin, an alpha-adrenergic receptor blocker, had minimal effect. These results show that the role of alpha-receptors in promoting the NE-mediated rise in rat pineal 5'-D activity is minor in contrast to the role of beta-adrenergic receptors.     

Studies on the hyperglycemia and hepatic glycogenolysis produced by alpha and beta adrenergic agonists in the cat

Previous studies in this laboratory showed that both alpha and beta receptors can mediate adrenergically-induced hyperglycemia in the cat. In the present study, the results of experiments on the isolated perfused cat liver provide affirmation that hepatic glycogenolysis in this species can be subserved by both types of receptors. Thus, the acute hepatic release of glucose induced by isoproterenol was found to be antagonized by propranolol but not by phentolamine or phenoxybenzamine. The opposite was found for the glycogenolytic action of phenylephrine. Experiments $\text{in}$$\text{vivo}$ showed that the hyperglycemic response to the beta agonist was associated with activation of hepatic phosphorylase and increased intracellular cAMP content while the hyperglycemia induced by the alpha agonist was associated with an activation of phosphorylase which was independent of cAMP.     

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.     

Stimulation by local anesthetics of the metabolism of acidic phospholipids in the rat pineal gland

The efficacy of five local anesthetics in causing stimulation of phospholipid metabolism in rat pineal gland $\text{in vitro}$ paralleled their anesthetic potency and decreased in the order: dibucaine, tetracaine, cocaine, procaine, lidocaine. When stimulation occurred, the patterns of labeling resembled that produced by propranolol, a β-adrenergic receptor blocking agent with local anesthetic activity. Isotope incorporation into phosphatidylglycerol and CDP-diglyceride was markedly enhanced and increases of labeling of phosphatidic acid and phosphatidylinositol were also seen. At concentrations of 1–10 mM, propranolol and local anesthetics inhibited labeling of phosphatidylcholine and phosphatidylethanolamine by more than 90% and incorporation of ³²P_i into other phospholipids to a smaller extent.     

Dopamine and norepinephrine stimulate somatostatin release by median eminence fragments invitro

The effect of catecholamines on somatostatin release by median eminence (ME) fragments was evaluated using an $\text{in}$$\text{vitro}$ incubation system. Adult male rats were used as tissue donors. Somatostatin release was readily detected during short-term incubations (10 and 30 minutes). Dopamine (DA) significantly stimulated somatostatin release during a 30 minute incubation period at the two doses tested (0.6 and 6 μM). Under similar conditions, norepinephrine (NE) stimulated somatostatin release only at the 6 μM dose. Using a shorter incubation period (10 min) and a 6 μM dose, only DA stimulated somatostatin release. The effects of DA and NE were specifically blocked by the $\text{in}$$\text{vitro}$ addition of pimozide or phentolamine, respectively, suggesting that dopaminergic and noradrenergic receptors may be present in the somatostatinergic terminals of the ME. The results indicate that both DA and NE may be involved in the regulation of somatostatin secretion.     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

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.     

Uptake of (-) 3H-norepinephrine by storage vesicles prepared from whole rat brain: properties of the uptake system and its inhibition by drugs.

Neurotransmitter storage vesicles were isolated from rat brain by differential centrifugation and the uptake of (?) ³H-norepinephrine was determined $\text{in vitro}$. Uptake showed a marked temperature dependence, an absolute requirement for ATP-Mg²⁺, and was inhibited $\text{in vitro}$ by reserpine. Uptake was linear for 5 min at 30°, but not at 37°. The uptake was saturable and displayed a single Km value of 4 × 10^?7 M. Other phenylamines and indoleamines displayed competitive inhibition of norepinephrine uptake; the affinities followed the rank order: reserpine>harmaline>serotonin>epinephrine> dopamine>norepinephrine>metaraminol. Uptake was reduced in vesicles isolated from rats treated intracisternally with 6-hydroxydopamine but not from rats treated with 5,6-dihydroxytryptamine, suggesting that most of the uptake occurs in catecholaminergic, and not serotonergic, vesicles. This method provides a ready characterization of pharmacologic effects on rat brain storage vesicle properties, as demonstrated by the prompt and complete inhibition of uptake $\text{in vitro}$ after administration of reserpine $\text{in vivo}$.     

Roles of alpha and beta adrenergic receptors in control of glucose oxidation in hamster epididymal adipocytes

Oxidation of [¹⁴C]glucose in isolated epididymal adipocytes from Golden hamsters was stimulated by isoproterenol and norepinephrine, which all interact with β-adrenergic receptors and by adrenorticotrophic hormone. In contrast α-receptor agonists, such as phenylephrine, methoxamine or clonidine did not increase basal glucose oxidation. The β-adrenergic blocking drug propranolol inhibited both lipolysis and glucose oxidation when these had been stimulated by isoproterenol, ephinephrine and phenoxybenzamine did not the α-adrenergic blocking drugs phentolamine and phenoxybenzamine did not influence lipolysis or glucose oxidation when isoproterenol provided the stimulus and increased both liposlysis and glucose metabolism in the presence of either epinephrine or norepinephrine. All α-adrenergic agonists tested (phenylephrine, methoxamine and clonidine) lowered liposlysis and glucose oxidation in isolated adipocytes exposed to isoproterenol. However, when adrenorcortropin provided the stimulus for glucose oxidation and lipolysis, only clonidine produced a significant reduction in lipolysis and glucose oxidation. None of the α-agonists influenced glucose metabolism which had been increased by insulin. These data confirm the presence of both α and β adrenergic receptors on hamster epididymal adipocytes and suggests that they exert antagonistic influences on lipolysis and glucose oxidation. These data are also consistent with the view that adrenergic stimulation of glucose oxidation and lipolysis in adipocytes are both mediated through β receptors.     

Steroid hormone receptors in human malignancy.

Steroid hormones induce responses in target tissues by a mechanism involving the specific initial interaction of hormone with cytoplasmic receptor molecules. These receptors, usually localized in target tissues have high binding affinities and limited binding specificities for biologically active steroids. Examination of human leukemic lymphoblasts has revealed these receptors in some tumor samples. Their presence is well correlated with hormone responsiveness of the tumor $\text{in vitro}$. Similar studies on human breast cancer tumor homogenates has indicated that about $\text{2}\text{3}$ of primary tumors contain estrogen receptor. The absence of receptor predicts a lack of response to hormone therapy almost invariably, while the presence of receptor increases but does not assure that the tumor will be hormone responsive. Recently $\text{in vitro}$ tissue culture systems which mimic the hormone responses observed $\text{in vivo}$ have been developed which should significantly aid in the clarification of the mechanisms whereby steroid hormones stimulate and inhibit growth in target tissues.     

Metabolism of propranolol in rat: the fate of the N-isopropyl group

The metabolic fate of the side-chain of propranolol has been studied in the rat $\text{in vivo}$ and with liver preparations $\text{in vitro}$ using drug labelled with ¹⁴C in the isopropyl group. Oxidative N-dealkylation to yield acetone was the major route of metabolism $\text{in vitro}$. Direct deamination to form isopropylamine was a very minor pathway of metabolism of propranolol by rat liver. In the intact rat only 1.4% of an intra-peritoneal dose of propranolol was excreted as isopropylamine in urine. In addition, isopropylamine administered to a rat was rapidly excreted unchanged in urine. Thus, if a similar metabolic pattern obtains in man, it seems unlikely that part of the pharmacological effects of propranolol can be attributed to this active metabolite.     

Muscarinic receptors in pineal

The presence of muscarinic receptors in sheep and rat pineals was detected by binding of [³H]quinuclidinyl benzilate ([³H]QNB), a potent and specific muscarinic antagonist. [³H]QNB binding to sheep pineal membrane resuspensions was saturable and reversible, with a rate constant for association at 37°C of 6×10⁸M^?1min^?1 and a rate constant for dissociation of 1×10^?2min^?1. Kinetic and saturation experiments yielded an equilibrium dissociation constant of 13–18 pM and a concentration of binding sites equivalent to 1.1 pmol/g of original wet weight. This is only about 5% of the level of β-adrenergic receptors. Competition by a variety of cholinergic drugs confirmed the muscarinic nature of the binding sites. Experiments in rats failed to detect a significant decrease in pineal [³H]QNB binding following bilateral superior cervical ganglionectomy, suggesting that the binding sites are not localized exclusively on sympathetic terminals.