Identification of the Subunit Structure of Rat Pineal Adrenergic Receptors by Photoaffinity Labeling

The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I-CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (+/- 5) pM and 30 (+/- 5) pM, respectively. Binding site maxima were 481 (+/- 63) and 1,020 (+/- 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of alpha 1-adrenergic receptors. 125I-CYP-labeled beta-adrenergic receptors were characterized as a homogeneous population of beta 1-adrenergic receptors. The alpha 1- and beta 1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-(4-[5-(4-azido-3-[125I]iodophenyl) pentanoyl]-1-piperazinyl) quinazoline (125I-APDQ) and 125I-p-azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an alpha 1-adrenergic receptor peptide of Mr = 74,000 (+/- 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single beta 1-adrenergic receptor peptide with a Mr = 42,000 (+/- 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed.     

Identification of α2-Adrenergic Receptors in Chicken Pineal Gland Using [3H]Rauwolscine

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.     

Evidence Against Dopaminergic and Further Support For α-Adrenergic Receptor Involvement in the Pineal Pho sphatidy lino sitol Effect

Abstract: Several α-adrenergic receptor agonists and antagonists were used to strengthen the earlier findings that the stimulation by (-)-norepinephrine of ³²P₁ incorporation into acidic phospholipids, especially phosphatidylinositol, in the rat pineal gland is mediated through α-adrenergic receptors. Dopamine was able to induce similar stimulation, although always to a smaller extent than equimolar concentrations of norepinephrine. The dopaminergic agonists apomorphine and piribedil did not increase phosphatidylinositol labeling. A number of antagonists considered to act primarily at dopaminergic or α-adrenergic receptors respectively completely prevented dopamine from exerting its effect. Both types of antagonists also were able to inhibit in varying degree the elevation of phospholipid labeling induced by norepinephrine. Dopamine increased phosphatidylinositol turnover without first being converted to norepinephrine, inasmuch as dopamine β-hydroxylase inhibitors had no influence on dopamine activity. Dopamine and α-agonists competitively activated the receptors involved in the phospholipid effect. The conclusion drawn from the several lines of evidence is that only α-adrenergic receptors are concerned with the changes in pineal phospholipid metabolism brought about by the various agonists used and that the action of dopamine occurs through these receptors rather than through discrete dopaminergic receptors.     

Vasoactive Intestinal Peptide Stimulates Chick Pineal Melatonin Production and Interacts with Other Stimulatory and Inhibitory Agents but Does Not Show α1-Adrenergic Potentiation

Vasoactive intestinal peptide (VIP) is known to mimic the effects of beta-adrenergic receptor stimulation in the rat pineal, including marked potentiation by alpha 1-adrenergic receptor stimulation, and to cause increased melatonin synthesis. In contrast, the chick pineal does not respond to beta-adrenergic stimulation, and melatonin synthesis is inhibited by norepinephrine via an alpha 2-adrenergic receptor. The present experiments show that chick pineal cells in primary culture do, however, respond to VIP with increased melatonin production. The effect of VIP was inhibited by addition of norepinephrine or of nitrendipine or by exposing the cells to "unexpected" white light. Stimulation by VIP was enhanced by addition of forskolin or Bay K 8644 but not by alpha 1-adrenergic receptor stimulations. Although stimulation by VIP appears similar in the chick pineal to that seen in the rat pineal and other systems, "dual-receptor regulation," at least with alpha 1-adrenergic receptors, appears to be absent.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

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.     

Cellular and molecular mechanisms controlling melatonin release by mammalian pineal glands

1. The pineal gland is regulated primarily by photoperiodic information attaining the organ through a multisynaptic pathway initiated in the retina and the retinohypothalamic tract. 2. Norepinephrine (NE) released from superior cervical ganglion (SCG) neurons that provide sympathetic innervation to the pineal acts through alpha1- and beta 1- adrenoceptors to stimulate melatonin synthesis and release. 3. The increase in cyclic AMP mediated by beta 1-adrenergic activation is potentiated by the increase in Ca2+ flux, inositol phospholipid turnover, and prostaglandin and leukotriene synthesis produced by alpha 1-adrenergic activation. 4. Central pinealopetal connections may also participate in pineal control mechanisms; transmitters and modulators in these pathways include several neuropeptides, amino acids such as gamma-aminobutyric acid (GABA) and glutamate, and biogenic amines such as serotonin, acetylcholine, and dopamine. 5. Secondary regulatory signals for pineal secretory activity are several hormones that act on receptors sites on pineal cells or at any stage of the neuronal pinealopetal pathway.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Effects of Fluoronorepinephrines on Phosphatidylinositol Turnover in Rat Pinealocytes

(+/-)-Norepinephrines (NAs), substituted with fluorine at positions 2, 5, and 6 of the ring, were compared with the unsubstituted compound with respect to their capacity for eliciting increased incorporation of [32P]orthophosphate into phosphatidylinositol of pinealocytes. 5F-NA and 6F-NA were approximately equipotent with NA, whereas 2F-NA required a higher concentration and gave lower maximum stimulation. Inhibition of these effects by prazosin confirmed the participation of alpha 1-adrenergic receptors. The results are comparable with those reported for alpha 1-adrenergic receptor-mediated events in other systems and different from the beta-adrenergic receptor-mediated elevation of cyclic AMP in pinealocytes. These and earlier results emphasize the importance of the hydroxyl group at position 3 of the ring and at the beta-position of the side chain in catecholamine activation of the pineal alpha-adrenergic receptors, which are involved in alterations of phospholipid metabolism.     

Stimulation of Distinct D2 Dopaminergic and α2-Adrenergic Receptors Induces Light-Adaptive Pigment Dispersion in Teleost Retinal Pigment Epithelium

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4-dihydoxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half-maximal effect at approximately 2 nM; clonidine, at 1 microM. Dopamine-induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by D1 or alpha-adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a D1 dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine-induced dispersion was inhibited by the alpha 2-adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and alpha 2-adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark-adaptive pigment aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Dopamine and β-Adrenergic Receptors Linked to Cyclic AMP Formation in Intact Cells of the Clone D384 Derived from a Human Astrocytoma

3,4-Dihydroxyphenylethylamine (dopamine) and beta-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKF 38393, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 microM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 microM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isoprenaline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 microM. The beta 2-selective antagonist ICI 118,551 (Ki,0.8 nM) was almost 8,000-fold more potent than the beta 1-selective antagonist practolol (Ki, 5.9 microM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and beta 2-adrenergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-receptor and therefore the use of this clone represents another approach to studying central D1-receptors.     

In Vivo Partial Inactivation of Dopamine D1 Receptors Induces Hypersensitivity of Cortical Dopamine-Sensitive Adenylate Cyclase: Permissive Role of α1-Adrenergic Receptors

As shown by autoradiography, peripheral injections of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a dose-dependent decrease of [3H]SCH 23390 and [3H]prazosin high-affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin-sensitive alpha 1-adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA-sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA-sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA-sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (-20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ-induced supersensitivity of DA-sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenergic Control of Rat Pineal NO Synthase

Abstract: We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We present here the evidence that chronic (5 days) norepinephrine (NE) or isoproterenol treatment prevents the effect of LL and enhances pineal NOS activity in LL animals. This effect of NE appears to be mediated by β-adrenoceptors, because it was not mimicked by the α-agonist phenylephrine. Pineal NOS activity was reduced in 16-h light/8-h dark animals treated for 4 days with the β-adrenergic antagonist propranolol but not with the α₁-antagonist prazosin, indicating again an involvement of β-adrenergic receptor in the control of NOS. Treatment with adrenergic antagonists did not affect cortical NOS activity, suggesting that the control of NOS is different in these two tissues or that the pineal expresses a specific isoform of the enzyme. Taken together, these data suggest that NE controls NOS in the pineal gland through β-adrenergic receptors. To our knowledge, this represent the first demonstration of a regulation of NOS by a neurotransmitter in the CNS, as assayed under V _max conditions.     

Dopamine increases L-type calcium current more in newborn than adult rabbit cardiomyocytes via D1 and beta2 receptors

Dopamine is used to treat heart failure, particularly after cardiac surgery in infants, but the mechanisms of action are unclear. We investigated differences in the effect of dopamine on L-type calcium current (I(Ca)) between newborn (NB, 1-4 days) and adult (AD, 3-4 mo) rabbit ventricular myocytes. Myocytes were enzymatically dissociated from NB and AD rabbit hearts. I(Ca) was recorded by using the whole cell patch-clamp technique. mRNA levels of cardiac dopamine receptor type 1 (D1), type 2 (D2), and beta-adrenergic receptors (beta-ARs) were measured by real-time RT-PCR. Dopamine (100 microM) increased I(Ca) more in NB (E(max) 87 +/- 10%) than in AD ventricular cells (E(max) 21 +/- 3%). Further investigation of this difference showed that mRNA levels of the D1 receptor were significantly higher in NB, and, with beta-AR blockade, dopamine increased I(Ca) more in NB than AD cells. Additionally, SKF-38393 (selective D1 receptor agonist) significantly increased I(Ca) by 55 +/- 4% in NB (P < 0.05, n = 4) and by 11 +/- 1% in AD (P < 0.05, n = 6). Dopamine in the presence of SCH-23390 (D1 receptor antagonist) increased I(Ca) in NB cells by 67 +/- 5% and by 22 +/- 2% in AD cells, suggesting a role for beta-AR stimulation. Selective blockade of beta(1)- or beta(2)-receptors (with block of D1 receptors) showed that the beta-AR action of dopamine in the NB was largely mediated via beta(2)-AR activation. Dopamine produces a larger increase in I(Ca) in NB cardiomyocytes compared with ADs. The mechanism of action is not only through beta(2)-ARs but also due to higher expression of cardiac D1 receptor in NB.     

Ontogeny of Dopamine D1 Receptors in Rat Striatum

The development of dopamine D1 receptors in rat striatum during the early postnatal period is described, using [3H]piflutixol as ligand. Dopamine D1 receptors increase in number from day of birth until about 21 days of age, when they reach adult levels. This increase in number parallels the increase in several other dopamine markers in striatum during the same time period. The increase is reflected in an increase in Bmax of ligand binding to D1 receptors. All other properties of D1 receptors that were examined do not change throughout this developmental period and are essentially the same as those found in adult tissue. These include association and dissociation rates, affinity for piflutixol as determined by kinetic and saturation studies, and pharmacology. These studies provide a biochemical and pharmacological basis for further studies on the ontogeny of dopamine receptors and of striatum and on factors regulating development of this region.     

α1-Adrenergic Receptors in Neuronal Cultures from Rat Brain: Increased Expression in the Spontaneously Hypertensive Rat

Neuronal cells in primary culture from 1-day-old brains of normotensive, Wistar-Kyoto strain (WKY) and spontaneously hypertensive (SH) rats have been utilized to study the expression of alpha 1-adrenergic receptors. Binding of a selective alpha 1 antagonist, [125I]2-[beta-(4-hydroxy-3-iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) to neuronal membranes prepared from primary brain cultures of WKY and SH rats was 75-80% specific, rapid, and time-dependent although the binding was 1.5-2 times higher in neuronal membranes from SH rat brain cultures. Kinetic analysis of the association and dissociation data demonstrated no significant differences between rat strains. Competition-inhibition experiments provided IC50 values for various antagonists and agonists in the following order: prazosin less than phentolamine less than yohimbine less than phenylephrine less than norepinephrine less than propranolol, suggesting that [125I]HEAT bound selectively to alpha 1-adrenergic receptors. Scatchard analysis of the binding data provided straight lines for both strains of rats, indicating the presence of a homogeneous population of binding sites. It also showed that the increase in the binding in neuronal cells from SH rat brains over those from normotensive WKY controls was a result of an increase in the number of alpha 1-adrenergic receptors. Incubation of neuronal cultures from both strains of rats with phenylephrine, an alpha 1-adrenergic agonist, caused a time- and dose-dependent decrease in the binding of [125I]HEAT. This decrease was due to a decrease in the number of alpha 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal transduction and regulation of melatonin synthesis in bovine pinealocytes: impact of adrenergic,peptidergic and cholinergic stimuli

Limited studies of the regulation of pineal melatonin biosynthesis in ungulates indicate that it differs considerably from that in rodents. Here we have investigated several signal transduction cascades and their impact on melatonin synthesis in bovine pinealocytes. Norepinephrine increased the intracellular calcium ion concentration ([Ca2+]i) via alpha(1)-adrenergic receptors. Activation of beta-adrenergic receptors enhanced cAMP accumulation and rapidly elevated arylalkylamine N-acetyltransferase (AANAT) activity and melatonin secretion. The beta-adrenergically evoked increases in AANAT activity were potentiated by alpha(1)-adrenergic stimulation, but this was not seen with cAMP or melatonin production. PACAP treatment caused small increases in cAMP, AANAT activity and melatonin biosynthesis, apparently in a subpopulation of cells. VIP and glutamate did not influence any of these parameters. Activation of nicotinic and muscarinic acetylcholine receptors increased [Ca2+]i, but did not alter cAMP levels, AANAT activity or melatonin production. Our study reveals that discrete differences in pineal signal transduction exist between the cow and rodent, and emphasizes the potential importance that the analysis of ungulate pinealocytes may play in understanding regulation of pineal melatonin biosynthesis in primates and man, whose melatonin-generating system appears to be more similar to that in ungulates than to that in rodents.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

D2 Dopamine Receptors on Bovine Chromaffin Cell Membranes: Identification and Characterization by [3H]N-Methylspiperone Binding

Although dopamine-containing cells are known to be present in sympathetic ganglia, the site of action and the role of dopamine in ganglion function remain obscure. In the present work, we evaluated the interaction of dopamine receptor ligands with particulate membrane fractions from bovine chromaffin cells and adrenal medullary homogenates using the D2 dopamine receptor radioligand [3H]N-methylspiperone ([3H]NMSP). Scatchard analysis of [3H]NMSP saturation experiments revealed a Bmax of 24.1 +/- 1.6 fmol/mg of protein and a KD of 0.23 +/- 0.03 nM in the particulate fraction from adrenal medulla homogenates and a Bmax of 26.5 +/- 2.7 fmol/mg of membrane protein and a KD of 0.25 +/- 0.02 nM in the particulate fraction prepared from isolated adrenal chromaffin cells. There were approximately 1,000 receptors/cell. There were no detectable levels of specific [3H]NMSP binding in the particulates prepared from adrenal cortical or capsular homogenates. Competition studies with the nonradioactive D2 receptor antagonists spiperone, chlorpromazine, and (-)-sulpiride revealed KI values of 0.28, 21, and 196 nM, respectively. The (+) isomer of butaclamol displayed a 604-fold higher affinity than the (-) isomer. Competition studies with the dopamine receptor agonists dopamine and apomorphine revealed affinities of 3,960 and 417 nM, respectively. A correlation coefficient of 0.96 was obtained in studies comparing the potencies of drugs in inhibiting specific [3H]NMSP binding in bovine adrenal medullary homogenates and in inhibiting specific [3H]NMSP binding to brain D2 dopamine receptors. In summary, radiolabeling studies using [3H]NMSP have revealed the presence of D2 dopamine receptors on bovine adrenal chromaffin cells.(ABSTRACT TRUNCATED AT 250 WORDS)