Pituitary Adenylate Cyclase-Activating Polypeptide: Control of Rat Pineal Cyclic AMP and Melatonin but Not Cyclic GMP

Abstract: In this study, the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10⁻⁷ M ) increased MT production 2.5-fold. PACAP (10⁻⁷ M ) also increased cyclic AMP accumulation four- to fivefold; this effect was potentiated two- to three-fold by α₁-adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because α₁-adrenergic activation is known to translocate PKC and the PACAP-stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4β-phorbol 12-myristate 13-acetate (PMA). Phenylephrine and PMA also potentiated the PACAP-stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an α₁-adrenergic → PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.     

Neurotransmitter-Mediated Regulation of Brain Aromatase: Protein Kinase C- and G-Dependent Induction

Abstract: Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by α₁-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the α₁-agonist phenylephrine than in control neurons, whereas prazosin, an α₁-antagonist, suppressed this increase, and ligands for α₂- or β-adrenergic receptors did not exert any influence. The profile of α₁-adrenergic receptor subtypes during actual development in vivo suggested that the α_1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via α₁ (possibly α_1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. β-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.     

Adrenergic Stimulation of Cyclic GMP Formation Requires NO-Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α₁-adrenergic receptors. The effects of N -monomethyl- l -arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α₁-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l -arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.     

Regulation of α2A-Adrenergic Receptor Expression by Epinephrine in Cultured Astroglia from Rat Brain

Abstract: Epinephrine (Epi) mediates various physiological effects via α_2A-adrenergic receptors (α_2A-ARs). Studies in mice with a point mutation in the gene for α_2A-AR have shown that these receptors are responsible for the centrally mediated depressor effects of α₂-AR agonists. These studies underscore the importance of understanding the basic cellular mechanisms involved in the expression of α_2A-ARs, of which little is known. We use astroglia cultured from the hypothalamus and brainstem of adult Sprague-Dawley rats as a model system in which to study factors that regulate α_2A-AR expression. These cells contain α₂-ARs, which are predominately of the α_2A-AR subtype. Our studies have shown that Epi causes a dose- and time-dependent decrease in steady-state levels of α_2A-AR mRNA and number of α_2A-ARs, effects that are mediated via α₁- and β-adrenergic receptors (α₁-ARs and β-ARs). These effects of Epi on α_2A-AR mRNA and α_2A-AR number are mimicked by activation of protein kinase C or increases in cellular cyclic AMP, which are intracellular messengers activated by α₁-ARs and β-ARs, respectively. Taken together, these results indicate that expression of α_2A-ARs is regulated in a heterologous manner by Epi, via α₁-AR- and β-AR-mediated intracellular pathways.     

Presynaptic and Postsynaptic Effects of Neuropeptide Y in the Rat Pineal Gland

Abstract: Neuropeptide Y is colocalized with noradrena-line in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC₅₀ of 50 n M , the potassium-evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin-sensitive G protein-coupled neuropeptide Y-Y2 receptors and is independent from, but additive to, the α₂-adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC₅₀ of 5 n M , the β-adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y-Y1 receptors. This moderate neuropeptide Y-induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a β-adrenergic stimulation. On the contrary, in the presence of 1 m M ascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.     

Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers

Abstract. A defined cultivation system was developed for the differentiation of pluripotent embryonic stem cells of the mouse into spontaneously beating cardiomyocytes, allowing investigations of chronotropic responses, as well as electrophysiological studies of different cardioactive drugs in vitro.
The β-adrenoceptor agonists (—)isoprenaline and clenbuterol, the mediators of cAMP metabolism, forsko-lin and isobutylmethylxanthine (IBMX), the α₁-adreno-ceptor agonist (—)phenylephrine, and the heart glyco-side digitoxine induced a positive, the muscarinic cholin-oceptor agonist carbachol and L-type Ca²⁺ channel blockers nisoldipine, gallopamil and diltiazem induced a negative chronotropic response.
In early differentiated cardiomyocytes β₁-, α₁-, but not β₂-adrenoceptors, cholinoceptors, as well as L-type Ca²⁺ channels participated in the chronotropic response. In terminally differentiated cardiomyocytes β₂-adrenoceptors and digitoxine responses were also functionally expressed.
The contractions of spontaneously beating cardiomyocytes were concommitant with rhythmic action potentials very similar to those described for embryonic cardiomyocytes and sinusnode cells. We conclude that cardiomyocytes differentiating from pluripotent embryonic stem cells are able to develop adrenoceptors and cholinoceptors and signal transduction pathways as well as L-type Ca²⁺ channels as a consequence of cell-cell interactions during embryoid body formation in vitro, independent of the development in living organisms.
The cellular system described may be useful as in vitro assay for toxicological investigations of chronotropic drugs and a model system for studying commitment and cellular differentiation in vitro.     

Enhancement of the responsiveness of cortical adrenergic receptors by chronic administration of the 5-hydroxytryptamine uptake inhibitor citalopram.

Abstract: The aim of this study was to evaluate the effect of citalopram, a second generation antidepressant agent producing no β-down-regulation, on the receptors and second messenger systems related to noradrenergic transmission in the cerebral cortex of the rat. We confirmed that citalopram does not bind to α₁-, α₂-, and β₂-adrenoceptors, but we found that it attenuates the inhibitory action of the protein kinase C activator, 12- O -tetradecanoylphorbol 13-acetate, on the noradrenergic response from α₁-adrenoceptor. In contrast to most antidepressants, chronic treatment with citalopram does not produce β-down-regulation, but increases the responses to noradrenaline from β-adrenoceptors without increasing the β₁,-adrenoceptor density. Chronic treatment with citalopram also increases the maximal response from α₁-adrenoceptor. The results indicate that β-down-regulation is not a necessary characteristic of an efficient antidepressant drug.     

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.     

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Presynaptic Inhibition by Concanavalin A: Are α-Latrotoxin Receptors Involved in Action Potential-Dependent Transmitter Release?

Abstract: Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [³H]noradrenaline release by up to 30% with half-maximal inhibition at 0.16 µ M . Concanavalin A also reduced the release triggered by extracellular Ca²⁺ in neurons depolarized by 25 m M K⁺ or rendered Ca²⁺-permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the α₂-adrenergic agonist UK 14,304. Inactivation of G_s and G_i/G_o type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage-dependent K⁺ and Ca²⁺ currents. In contrast, the lectin efficiently blocked both the Ca²⁺-dependent and -independent α-latrotoxin-induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as α-latrotoxin-evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl α- d -mannopyranoside. The dimeric derivative, succinyl-concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to α-latrotoxin, concanavalin A failed to alter K⁺-evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of α-latrotoxin receptors may reduce action potential-dependent transmitter release.     

The δ-Opioid Receptor in SK-N-BE Human Neuroblastoma Cell Line Undergoes Heterologous Desensitization

Abstract: The human neuroblastoma cell line SK-N-BE expresses δ-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 n M etorphine leads to an almost complete desensitization (8.2 ± 5.9 vs. 45.8 ± 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, α₂-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 µ M 2-bromo-α-ergocryptine or 1 µ M arterenol resulted in a marked desensitization of D2-dopaminergic and α₂-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the δ-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-bromo-α-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the α₂-adrenergic receptor response, whereas pretreatment with 1 µ M arterenol partly desensitized the δ-opioid receptor response. It is concluded that the δ-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterologous desensitization, and it is suggested that δ-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G_i2 protein, whereas α₂-adrenergic receptor could be coupled to the enzyme via two G proteins, G_i2 and another member of the G_i/G_o family.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Alterations in beta 1- and beta 2-adrenergic receptor density in the cerebellum of aging rats

Abstract: The densities of β¹, and β₂-adrenergic receptors were determined in homogenates of cerebral cortex and cerebellum of rats between 3 and 14 mo of age. No change in either receptor population occurred in the cortex during this period. In the cerebellum, a 20–25% decrease in the density of β₂, receptors and a 3509% increase in the density of β₁, receptors occurred. The increase in β₁ receptors in the cerebellum may be the result of a decrease in the function of the noradrenergic projections from the locus coeruleus which synapse on cerebellar Purkinje cells.     

In Situ Hybridization Evidence of Differential Modulation by Pentobarbital of GABAA Receptor α1- and β3-Subunit mRNAs

Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABA_A receptor α₁- and β₃-subunit mRNA was conducted using synthetic 3'- end ³⁵S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α₁-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β₃-subunit mRNA were not affected. Dramatically increased levels of GABA_A receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α₁ subunit and in cerebral cortex only for the β₃-subunit. These data provide further support to the structural and pharmacological GABA_A receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.     

Functional Rescue of β1-Adrenoceptor Dimerization and Trafficking by Pharmacological Chaperones

Site-directed mutagenesis guided by evolutionary trace analysis revealed that substitution of V179 and W183 within a cluster of evolutionarily important residues on the surface of the fourth transmembrane domain of the β₁-adrenergic receptor (β₁AR) significantly reduced the propensity of the receptor to self-assemble into homodimers as assessed by bioluminescence resonance energy transfer in living cells. These results suggest that mutation of V179 and W183 result in conformational changes that reduce homodimerization either directly by interfering with the dimerization interface or indirectly by causing local misfolding that result in reduced self-assembly. However, the mutations did not cause a general misfolding of the β₁AR as they did not prevent heterodimerization with the β₂AR. The homodimerization-compromised mutants were significantly retained in the endoplasmic reticulum (ER) and could not be properly matured and trafficked to the cell surface. Lipophilic β-adrenergic ligands acted as pharmacological chaperones by restoring both dimerization and plasma membrane trafficking of the ER-retained dimerization-compromised β₁AR mutants. These results clearly indicate that homodimerization occurs early in the biosynthetic process in the ER and that pharmacological chaperones can promote both dimerization and cell surface targeting, most likely by stabilizing receptor conformations compatible with the two processes.