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.     

Prostaglandins Stimulate Serotonin Acetylation in Chick Pineal Cells: Involvement of Cyclic AMP-Dependent and Calcium/Calmodulin-Dependent Mechanisms

Abstract: The effects of prostaglandins (PGs) on the activity of the rate-limiting enzyme of melatonin biosynthesis, aryl-alkylamine- N -acetyltransferase (NAT) were investigated on primary cultures of dispersed chick pineal cells. In indomethacin-treated cells, PGs caused a four-fold increase in NAT activity. This response was associated with an eightfold increase in cyclic AMP (cAMP) levels. The potency order of PGs was the same for NAT and for cAMP responses (PGE¹ > PGE² > PGF^2α≫ cloprostenol). However, each PG tested was 30- to 200-fold more potent to increase NAT activity than to stimulate cAMP accumulation. As a result, half-maximal stimulation of NAT by PGs was not associated with an increase in cAMP levels. Half-maximal stimulation of NAT by PGE¹ was highly sensitive to inhibition by a calcium/calmodulin antagonist (W-7). In contrast, maximal stimulation of NAT by PGE¹ as well as stimulations evoked by either forskolin or 8-bromo-cAMP were poorly sensitive to inhibition by W-7. These results indicate that an increase in cAMP levels may be responsible for the maximal stimulation of NAT evoked by PGs, whereas half-maximal stimulation of NAT by PGs would rely principally on a calcium/calmodulin-dependent mechanism.     

Neuropeptide Y Inhibits β-Adrenergic Agonist– and Vasoactive Intestinal Peptide–Induced Cyclic AMP Accumulation in Rat Pinealocytes Through Pertussis Toxin–Sensitive G Protein

The effects of neuropeptide Y (NPY) on pineal gland cyclic AMP (cAMP) accumulation were investigated using dispersed pinealocytes from rats. NPY inhibited the intracellular cAMP accumulation stimulated by isoproterenol and norepinephrine in a dose-dependent manner during a 10-min incubation of pinealocytes. NPY (1 x 10(-7) M) also inhibited vasoactive intestinal peptide (VIP)- and cholera toxin-induced cAMP accumulation. The inhibitory effect of NPY on isoproterenol-induced cAMP accumulation was completely abolished by a 5-h pretreatment of pinealocytes with 1 microgram/ml of pertussis toxin (PT). These results suggest that NPY participates in modulation of cAMP production in the rat pineal gland through PT-sensitive G protein. Yohimbine, an alpha 2-adrenergic antagonist, blocked NPY inhibition of isoproterenol-stimulated cAMP accumulation. On the other hand, the alpha 2-adrenergic agonist clonidine by itself did not affect cAMP accumulation stimulated by isoproterenol but significantly potentiated NPY action. The present study demonstrates that NPY inhibits beta-adrenergic or VIPergic stimulation of the pineal gland cAMP accumulation. The inhibitory effect of NPY is mediated through PT-sensitive G protein. Our results also suggest that NPY exerts its action to affect alpha 2-adrenoceptor function.     

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.     

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.     

Down-Regulation of Pinealocyte Protein Kinase C: Effect on α1-Adrenergic Potentiation of β-Adrenoceptor Stimulation of Cyclic AMP Accumulation and Induction of Serotonin N-Acetyltransferase Activity

Treatment of rat pinealocytes with 4 beta-phorbol 12,13-dibutyrate down-regulated protein kinase C (PKC) activity. Loss of activity was concentration-dependent (50% loss at 8 x 10(-7) M after 18 h of treatment) and time-dependent (50% loss after 2 h with 3 x 10(-6) M). Phenylephrine, an alpha 1-adrenergic agonist, and phorbol esters unable to activate PKC did not down-regulate the enzyme. alpha 1-Adrenergic amplification of beta-adrenergic stimulation of cyclic AMP accumulation, a response previously shown to be mediated by PKC activation, was reduced by only 50% in cells in which PKC activity was down-regulated by approximately 95%. These data suggest that there is not a simple proportional relationship between the degree of activation of pinealocyte PKC and the alpha 1-adrenergic amplification of beta-adrenergic cyclic AMP synthesis. In down-regulated cells, alpha 1-adrenergic amplification of beta-adrenergic induction of serotonin N-acetyltransferase activity, a key cyclic AMP-responsive enzyme involved in the nocturnal synthesis of the pineal hormone melatonin, was unchanged. Thus, even though alpha 1-adrenergic amplification of cyclic AMP synthesis is impaired, sufficient cyclic AMP is generated to allow a full induction of serotonin N-acetyltransferase activity. This finding raises the important question of whether the alpha 1-adrenergic amplification mechanism has a physiological role in regulating melatonin synthesis in vivo.     

Noradrenergic Inhibition and α2-Adrenergic Stimulation of Melatonin Secretion in the Pigeon

Adrenergic regulatory mechanisms of melatonin synthesis and secretion were studied in the pigeon in vivo. Late-afternoon intraperitoneal injection of noradrenaline (NA; 1 mg/kg) resulted in a significant decrease in plasma melatonin levels in 3 h. The same effect was seen after phenylephrine treatment (1 mg/kg i.p.), indicating that an alpha 1-adrenergic mechanism may mediate the inhibition. Propranolol treatment had no effect on plasma melatonin levels, supporting this concept. Detomidine (1 mg/kg i.p.), an alpha 2-adrenergic agonist, increased melatonin levels. This stimulatory effect was blocked by yohimbine, an alpha 2-adrenergic antagonist. However, yohimbine alone had no effect on the plasma melatonin levels, suggesting that alpha 2-adrenergic transmission is not primarily responsible for the nocturnal stimulation of melatonin synthesis and secretion in the pigeon.     

Effect of Immobilization Stress on Rat Pineal β-Adrenergic Receptor-Mediated Function

The results of this investigation indicate that stress produced by immobilization alters rat pineal function. Chronic stress reduced the density of pineal beta-adrenergic receptors and the activities of the intracellular enzyme serotonin N-acetyltransferase (NAT), its product N-acetylserotonin (NAS), and the pineal hormone melatonin, which was measured during the dark phase of the diurnal lighting cycle. Removal of the adrenal medulla did not prevent the reduction of pineal beta-adrenergic receptor binding sites that is observed after chronic stress. Acute immobilization stress suppressed the dark-induced elevations of pineal NAT activity and NAS levels 10 h after the stress session without altering pineal beta-adrenergic receptor binding. Although the precise mechanisms responsible for these effects are not completely clear, the results indicate that they are related to changes in sympathetic neuronal activity and not mediated by stress-induced elevations in plasma catecholamines.     

Selective inhibition of β2-adrenergic receptor-mediated cAMP generation by activation of the P2Y2 receptor in mouse pineal gland tumor cells

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.     

Effects of adrenergic agonists and antagonists on the numbers of synaptic ribbons in the rat pineal gland

In the pineal gland numbers of synaptic ribbons (SR) undergo day/night changes which parallel the rhythm of melatonin synthesis. Since pineal biosynthetic activity is controlled by activation of adrenoreceptors, we investigated the effects of adrenergic agonists and antagonists on pineal synaptic ribbon numbers and N-acetyltransferase (NAT) activity, the key enzyme of melatonin synthesis in rats. In vivo application of the beta-adrenergic antagonist propranolol decreased melatonin synthesis when given during the dark phase but did not affect SR numbers. Treatment during daytime with the beta-adrenergic agonist isoproterenol increased pineal NAT activity whereas SR numbers did not change. Norepinephrine stimulated NAT activity in vitro in a dose-dependent manner, but did not elevate SR numbers. Incubation with an analog of the second messenger cyclic adenosine monophosphate increased both NAT activity and SR numbers. These results suggest that the beta-adrenergic system does not play a decisive role in the regulation of the nocturnal increase in SR numbers observed in the rat pineal gland.     

Differential Involvement of the Arachidonic Acid Cascade on the α1-Adrenergic Potentiation of Vasoactive Intestinal Peptide-Versus β-Adrenergic-Stimulated Cyclic AMP and Cyclic GMP Accumulation in Rat Pinealocytes

In the rat pineal gland, alpha 1-adrenergic agonists, which stimulate arachidonic acid release, also potentiate vasoactive intestinal peptide (VIP)- or beta-adrenergic-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation. In this study, the possible involvement of the arachidonic acid pathway in the potentiation mechanism was examined in dispersed rat pinealocytes using two inhibitors of the arachidonic acid cascade, indomethacin and nordihydroguaiaretic acid. These two inhibitors appeared to have differential effects on the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP responses. Whereas nordihydroguaiaretic acid was effective in suppressing both the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP responses, indomethacin inhibited selectively the VIP-mediated cAMP and cGMP responses. The role of arachidonic acid metabolites was further determined using several prostaglandins--A2, I2, E2, and F2 alpha--and leukotrienes--B4, C4, and D4. Of the seven compounds tested, prostaglandins E2 and F2 alpha stimulated basal cAMP but not cGMP accumulation. The prostaglandin E2- and F2 alpha-stimulated cAMP responses were additive to those stimulated by VIP or beta-adrenergic receptors. The other five compounds had no effects on basal or VIP- or beta-adrenergic-stimulated cAMP or cGMP accumulation. Taken together, these findings indicate that the arachidonic acid cascade is likely involved in the alpha 1-adrenergic potentiation of VIP- or beta-adrenergic-stimulated cAMP and cGMP accumulation. However, the specific arachidonic acid metabolite involved in the potentiation mechanisms of VIP- versus beta-adrenergic-stimulated cyclic nucleotide responses may be different.     

Ethanol Reduces Norepinephrine-Stimulated Melatonin Synthesis in Rat Pinealocytes

In this study, the in vitro effects of ethanol on norepinephrine-stimulated cyclic AMP (cAMP), N-acetyltransferase (NAT), and melatonin (MT) production were examined in dispersed rat pinealocytes. Cellular cAMP content was determined 15 min after treatment; whereas NAT activity and MT release in the medium were determined 4.5 h after treatment. It was found that ethanol less than or equal to 200 mM had no effect on norepinephrine-stimulated cAMP response, whereas 25 mM ethanol resulted in a significant inhibition of norepinephrine-stimulated NAT and MT levels. Furthermore, ethanol was equally effective in inhibiting the dibutyryl cAMP-stimulated NAT and MT levels. The inhibitory action of ethanol was not due to a direct effect or a delay in the onset of NAT activity. When alcohols with different chain lengths were used, it was found that their inhibitory potencies were related to their chain lengths with butanol greater than propanol greater than ethanol greater than methanol. Taken together, these findings indicate that (1) ethanol has an inhibitory action on norepinephrine-stimulated MT synthesis, (2) one site of ethanol action is distal to cAMP elevation, and (3) the inhibitory effect of ethanol on pineal MT synthesis appears to be secondary to its hydrophobic action.     

Effect of brefeldin A on melatonin secretion of chick pineal cells

Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.     

Cannabinoids attenuate norepinephrine-induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N-acetyltransferase activity without involvement of cannabinoid receptors

Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.     

Delta Sleep-Inducing Peptide Modulates the Stimulation of Rat Pineal N-Acetyltransferase Activity by Involving the α1 -Adrenergic Receptor

Delta sleep-inducing peptide (DSIP) has been isolated and characterized by its capacity to enhance delta sleep in rabbits. Up to now, sleep was the main target of DSIP research, but different extra-sleep effects of the peptide have been reported as well. Several mechanisms of action have been proposed, though no convincing evidence for any of them has been obtained so far. We recently detected that DSIP reduced the nocturnal increase of N-acetyltransferase (NAT) activity in rat pineal in a dose-dependent manner. The activity of this enzyme is known to be induced by adrenergic agonists and several studies have suggested that stimulation of alpha 1-adrenergic receptors potentiates the "basic" effect of beta-receptors. DSIP in the range between 20 and 300 nM significantly enhanced NAT activity induced by 10(-6) M norepinephrine in vitro, and a similar effect was observed with 2nMP-DSIP, a phosphorylated analog. Incubation with prazosin eliminated the enhancement, whereas propranolol reduced norepinephrine stimulation that was still increased by P-DSIP and probably DSIP. It was concluded that the sleep-peptide and its analog modulate the alpha 1-adrenergic receptor of rat pineal in its response to adrenergic agonists. The same mechanism may also be responsible for other biological activities of DSIP such as sleep-induction and stress-tolerance.     

Twenty-four hour oscillation of cAMP in chick pineal cells: role of cAMP in the acute and circadian regulation of melatonin production

Chick pineal cells contain circadian oscillators that regulate a rhythm of melatonin biosynthesis. We explored the role of cAMP in regulating this melatonin rhythm. Chick pineal cells expressed a 24 hr oscillation of cAMP efflux with a waveform similar to that of melatonin. Elevation of cAMP in chick pineal cells stimulated melatonin. These results suggest that an oscillation of cAMP regulates the rhythm of melatonin. We investigated whether cAMP was a component of the circadian oscillator by determining the effects of 8-Br cAMP pulses on the phase of the circadian melatonin rhythm. Six hour pulses of 8-Br cAMP did not cause steady-state phase shifts of the rhythm. The acute regulation of melatonin by cAMP, the 24 hr oscillation of cAMP, and the inability of cAMP to phase-shift the melatonin rhythm strongly suggest that cAMP acts as an output signal of the circadian oscillator.     

σ Receptor Modulation of Noradrenergic-Stimulated Pineal Melatonin Biosynthesis in Rats

Abstract: Because σ receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the σ-receptor ligands 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine potentiated the enhancement of N -acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine enhanced significantly the physiological increases in both pineal N -acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific σ-receptor antagonist. These findings suggest that, in rats, the activation of pineal σ-receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it pontentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.     

Alpha 2-adrenergic receptors accelerate Na+/H+ exchange in neuroblastoma X glioma cells

The regulation of cytoplasmic pH (pHi) was examined in neuroblastoma X glioma hybrid cell-line cells (NG108-15 cells) using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. The pHi of NG108-15 cells suspended in nominally HCO-3-free, Na+-containing buffer could be reduced by the external application of acetate. The recovery of pHi to its resting value was blocked by the removal of extracellular Na+, by the addition of extra-cellular H+, and by the addition of analogs of amiloride selective for inhibition of Na+/H+ exchange. The rate of recovery of pHi from acid load exhibited an ionic selectivity of Na+ greater than Li+ much greater than K+, and no recovery was observed in N-methyl-D-glucamine+. Tetrodotoxin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on early pHi recovery. These data suggest that Na+/H+ exchange accounts primarily for the recovery of pHi in NG108-15 cells under our experimental conditions. Na+/H+ exchange in NG108-15 cells was accelerated by alpha 2-adrenergic receptors. Thus, (-)epinephrine, but not (+)epinephrine, elicited an intracellular alkalinization which was blocked by the alpha 2-adrenergic receptor selective antagonist yohimbine but not by the alpha 1-adrenergic receptor antagonist, prazosin, nor the beta-adrenergic antagonist, propranolol. Norepinephrine, clonidine, and the clonidine analog, UK-14304, also caused alkalinization of NG108-15 cells, whereas isoproterenol, a beta-adrenergic receptor agonist, and phenylephrine, a selective alpha 1-adrenergic receptor agonist, did not. Manipulations that blocked Na+/H+ exchange blocked the ability of alpha 2-adrenergic agonists to alkalinize the interior of NG108-15 cells without blocking the ability of these agonists to attenuate cAMP accumulation. These findings provide the first direct evidence of modulation of Na+/H+ exchange activity by a receptor linked to inhibition of adenylate cyclase and offer a possible mechanism whereby alpha 2-adrenergic receptors might influence cellular activity apart from changes in cyclic nucleotide metabolism.     

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.     

Day-night differences in the response of the pineal gland to swimming stress

The effect of swimming stress on pineal N-acetyltransferase activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin content was studied during the day and night in adult male rats. At night, elevated pineal activity was suppressed by light exposure before the animals swam. During the day, swimming for 2 hr did not stimulate NAT activity unless the animals were pretreated with desmethylimipramine (DMI), a norepinephrine uptake blocker. Pineal melatonin content after daytime swimming exhibited a weak rise, unless DMI was injected, in which case melatonin levels showed a highly significant increase. Swimming at night caused a greater (compared to daytime levels) increase in NAT activity in both noninjected and DMI-injected rats. Melatonin levels at night were highly significantly stimulated (compared to daytime values) even without pretreatment of the rats with DMI. The greater response of the rat pineal to swimming stress at night may relate either to an increase in the number of beta-adrenergic receptors in the pinealocyte membrane at night or to a reduced capacity of the sympathetic neurons in the pineal to take up excess circulating catecholamines. Pineal HIOMT activity was not influenced by swimming (with or without DMI) either during the day or at night.