Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis.

Pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, or AA-NAT) generates the large circadian rhythm in melatonin, the hormone that coordinates daily and seasonal physiology in some mammals. Complementary DNA encoding ovine AA-NAT was cloned. The abundance of AA-NAT messenger RNA (mRNA) during the day was high in the ovine pineal gland and somewhat lower in retina. AA-NAT mRNA was found unexpectedly in the pituitary gland and in some brain regions. The night-to-day ratio of ovine pineal AA-NAT mRNA is less than 2. In contrast, the ratio exceeds 150 in rats. AA-NAT represents a family within a large superfamily of acetyltransferases.

Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation.

The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins.

A family of proteins known as 14-3-3 is currently receiving increased attention by investigators studying a broad range of biological systems, including plants and invertebrates. The outstanding feature of this family is the extraordinarily high sequence conservation observed. Current thinking indicates that these proteins may function as regulators in signal transduction/phosphorylation mechanisms.

Expression and structural analysis of 14-3-3 proteins.

The 14-3-3 family of proteins plays a role in a wide variety of cellular functions including regulation of protein kinase C and exocytosis. Using antisera specific for the N termini of 14-3-3 isoforms described previously and an additional antiserum specific for the C terminus of epsilon isoform, protease digestion of intact 14-3-3 showed that the N-terminal half of 14-3-3 (a 16 kDa fragment) was an intact, dimeric domain of the protein. Two isoforms of 14-3-3, tau and epsilon, were expressed in E. coli and their secondary structure was shown by circular dichroism to be identical to wild-type protein, and expression of N-terminally-deleted epsilon 14-3-3 protein showed that the N-terminal 26 amino acids are important for dimerization. Intact 14-3-3 is a potent inhibitor of protein kinase C, but the N-terminal domain does not inhibit PKC activity. Site-specific mutagenesis of several regions in the tau isoform of 14-3-3, including the mutation of a putative pseudosubstrate site to a potential substrate sequence, did not alter its inhibitory activity. Intact 14-3-3 proteins are phosphorylated by protein kinase C with a low stoichiometry, but truncated isoforms are phosphorylated much more efficiently by this kinase. This may imply that the proteins may adopt a different structural conformation, possibly upon binding to the membrane, which could modulate their activity. 14-3-3 proteins are found at high concentration on synaptic plasma membranes and this binding is mediated through the N-terminal 12 kDa of 14-3-3.

New light is shining on the melatonin rhythm enzyme: the first postcloning view.

One of the most interesting molecules in circadian biology is serotonin N-acetyltransferase (arylalkyfamine N-acetyltransferase, AANAT), the enzyme that controls the daily rhythm in pineal melatonin production and blood melatonin. The recent cloning of AANAT cDNA has led to the characterization of the human gene; the realization that AANAT represents a unique gene family; the discovery of circadian rhythms in AANAT mRNA; the determination of the basis of transsynaptic and cellular regulation of expression of the AANAT gene; a new understanding of the relationship of AANAT mRNA and activity; and the surprising finding of strong expression of the AANAT gene in the retina and significant levels in select brain regions, the pituitary gland, and testes. The cloning of AANAT cDNA has not only made it possible to answer longstanding questions in circadian biology, but has also raised stimulating new issues.

Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland.

In vertebrates, the circadian rhythm in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] drives the daily rhythm in circulating melatonin. We have discovered that expression of the AA-NAT gene in the rat pineal gland is essentially turned off during the day and turned on at night, resulting in a more than 150-fold rhythm. Expression is regulated by a photoneural system that acts through an adrenergic-cAMP mechanism in pinealocytes, probably involving cAMP response element-binding protein phosphorylation. Turning off AA-NAT expression appears to involve de novo synthesis of a protein that attenuates transcription. A approximately 10-fold night/day rhythm in AA-NAT messenger RNA occurs in the retina, and AA-NAT messenger RNA is also detected at low levels in the brain.

Corticotropin-releasing hormone messenger RNA distribution and stress-induced activation in the thalamus.

Corticotropin-releasing hormone plays a critical role in mediating the stress response. Brain circuits hypothesized to mediate stress include the thalamus, which plays a pivotal role in distributing sensory information to cortical and subcortical structures. In situ hybridization revealed neurons containing corticotropin-releasing hormone messenger RNA in the posterior thalamic nuclear group and the central medial nucleus of the thalamus, which interfaces with the ventral posteromedial nucleus (parvicellular part). These regions are of interest because they process somatosensory and visceral information. In the first experiment, the effect of acute stress on thalamic corticotropin-releasing hormone messenger RNA levels was assessed. Rats restrained for 1 h and killed 1 h later were found to have increased corticotropin-releasing hormone messenger RNA in the posterior thalamic nuclear group. The time course of these changes was examined in a second experiment in which rats were killed immediately or 3 h after restraint. While no changes occurred in the thalamus immediately after restraint, 3 h after restraint, increases in corticotropin-releasing hormone messenger RNA occurred in both the posterior thalamic nuclear group and the central medial-ventral posteromedial nucleus (parvicellular part) of the thalamus. A different pattern of activation was observed in the paraventricular nucleus of the hypothalamus with increased corticotropin-releasing hormone messenger RNA immediately after restraint, but not 1 or 3 h later. In addition to the stress-induced changes, a prominent decrease in baseline thalamic corticotropin-releasing hormone messenger RNA was observed from 1000 to 1300 h. These results show that the thalamus contains corticotropin-releasing hormone messenger RNA that increases after restraint stress, indicating a role for thalamic corticotropin-releasing hormone systems in the stress response. Stress-induced changes in thalamic corticotropin-releasing hormone messenger RNA expression appears to be regulated differently than that in the paraventricular nucleus of the hypothalamus, and may be influenced by diurnal mechanisms.

Persistent corticotropin-releasing factor(1) receptor desensitization and downregulation in the human neuroblastoma cell line IMR-32.

Brain corticotropin-releasing factor (CRF) systems integrate various responses to stress. Pathological responses to stress may result from errors in CRF receptor regulation in response to changes in synaptic CRF levels. To establish an in vitro model to study brain CRF receptors, we characterized the CRF-induced modulation of CRF(1) receptors in the human neuroblastoma cell line, IMR-32. Treatment with CRF decreased CRF(1) receptor binding and desensitized CRF-induced increases in cAMP. The decrease in binding had an EC(50) of approximately 10 nM, was maximal by 30 min, and was blocked by the CRF receptor antagonist [D-Phe(12), Nle(21,38), C(alpha)-MeLeu(37)]CRF(12-41). The desensitization was homologous as vasoactive intestinal polypeptide-induced increases in cAMP were unchanged, and elevation of cAMP did not alter CRF(1) receptor binding. Treatment with CRF for up to 24 h did not alter CRF(1) receptor mRNA levels, suggesting that a posttranscriptional mechanism maintains the decrease in receptor binding. Interestingly, recovery of CRF receptor binding and CRF-stimulated cAMP production was only partial following exposure to 100 nM CRF. In contrast, receptor binding recovered to control levels following exposure to 10 nM CRF. These data suggest that exposure to high doses of CRF result in permanent changes characterized by only partial recovery. Identifying the mechanisms underlying this partial recovery may provide insights into mechanisms underlying the acute and chronic effects of stress on CRF receptor regulation.

Natural melatonin 'knockdown' in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase.

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.

Human hydroxyindole-O-methyltransferase: presence of LINE-1 fragment in a cDNA clone and pineal mRNA.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of the pineal hormone melatonin. In this study, an HIOMT clone was isolated from a human pineal cDNA library using synthetic oligonucleotide probes based on the bovine HIOMT sequence. The human sequence is unusual because it contains a 3' fragment (84 bp) of the LINE-1 sequence, a highly repetitive sequence in the human genome and the genome of some primates and rodents. Exclusive of this LINE-1 fragment, the human HIOMT clone is 75% and 63% homologous to bovine and avian HIOMT sequences, respectively. The deduced amino acid sequence of the human cDNA clone encodes a 41.6-kD protein. In addition, the sequence is 70% and 57% identical and 81% and 73% similar to bovine and avian HIOMT, respectively. In agreement with the results of earlier studies, it was found that vertebrate HIOMT amino acid sequences are not homologous to any other vertebrate proteins, including several methyltransferases. However, HIOMT exhibits homology with a plant O-methyltransferase and an internal 120-amino-acid region is approximately 35% identical to a region of four bacterial O-methyltransferases. The results of PCR and Southern blot analysis indicate that three species of HIOMT mRNA are typically present in the human pineal gland, only one of which contains the LINE-1 fragment. An antiserum was raised against a mixture of three synthetic peptides, corresponding to three regions of the deduced amino acid sequence of human HIOMT. This antiserum detected a single immunoreactive protein in Western blot analysis of human pineal glands. The size of the protein (approximately 42 kD) is identical to that predicted from the HIOMT clone, including the LINE-1 fragment. The human HIOMT sequence should be useful in further studies of this enzyme and will also be of special importance in evaluating the functional significance of the inclusion of a fragment of the LINE-1 in an mRNA.

Cloning and characterization of the epsilon and zeta isoforms of the 14-3-3 proteins.

Two prominent proteins (30 and 33 kD) in a purified preparation of the sheep pineal gland were studied. Amino acid analysis of tryptic peptides indicated that the 33-kD protein was the epsilon isoform of the 14-3-3 family of proteins, and that the 30-kD protein was the zeta isoform. The sheep pineal gland was found to have six other 14-3-3 isoforms in addition to the epsilon and zeta, suggesting that copurification of the epsilon and zeta forms may reflect the existence of homo- or heterodimers comprised of these isoforms. To characterize 14-3-3 proteins further in the pineal gland, the full sequence of the epsilon isoform and a partial sequence of the zeta isoform were cloned from a rat pineal cDNA library and are reported here. Tissue distribution studies using Western blot analysis revealed that rat pineal and retina have levels of 14-3-3 protein similar to those found in brain, and that relatively low levels occur in other tissues. This investigation also revealed the epsilon isoform was present at high levels in the rat pineal gland early in development and decreased steadily thereafter and that 30-kD isoforms exhibited the inverse developmental pattern.

2D-PAGE analysis: adrenergically regulated pineal protein AIP 37/6 is a phosphorylated isoform of cytosolic malate dehydrogenase.

The adrenergic transmitter norepinephrine (NE) dramatically increases the prominence of only two out of the hundreds of [35S]methionine-labeled pineal proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). One of these regulated proteins is AIP 37/6 (37 kDa, pI approximately 6). The labeling of this protein is increased approximately 100-fold by NE. In the study presented here the identity of AIP 37/6 was investigated. The results of microsequencing, immunochemical analysis of 2D-PAGE blots and size exclusion chromatography indicate that AIP 37/6 is an isoform of cytosolic malate dehydrogenase (cMDH; approximately 36.3 kDa; pI approximately 6.5). Associated studies indicate that this isoform is phosphorylated whereas the bulk of cMDH is not. Cotranslational phosphorylation of cMDH is discussed.

Acute stress-induced increases in thalamic CRH mRNA are blocked by repeated stress exposure.

Corticotropin-releasing hormone (CRH) coordinates multiple aspects of the stress response. Recently, CRH mRNA has been identified in two regions of the thalamus: the posterior nuclear group (Po), and a region located at the interface of the central medial and ventral posteromedial nucleus (parvicellular part) (CM-VPMpc). Previous studies demonstrated that in both regions CRH mRNA increases following 1 h of restraint stress, suggesting involvement of thalamic CRH in processing somatosensory and visceral information related to stress. The current study was proposed to further understand the effects of repeated and acute restraint stress on levels of thalamic CRH mRNA. Adult male rats were assigned to one of four groups in a 2 (repeated stress, no repeated) x2 (acute, no acute) design. Brain sections were processed for CRH mRNA in situ hybridization. ANOVA revealed no main effects of acute or repeated stress in either thalamic region. However, significant interactions between acute and repeated stress for levels of CRH mRNA were found for both regions of the thalamus. Compared to the no stress condition, acute restraint significantly increased CRH mRNA in the Po (39%) and the CM-VPMpc (32%). Repeated restraint did not alter baseline CRH mRNA levels, but blocked the acute restraint-induced effects. Thus, while acute stress increases levels of thalamic CRH mRNA, repeated exposure to the same stressor is without effect and prevents the acute response. These findings add to data establishing a role for thalamic CRH in the stress response and suggest a mechanism that may underlie habituation to repeated stress exposure.

Effects of acute and repeated restraint stress on corticotropin-releasing hormone binding protein mRNA in rat amygdala and dorsal hippocampus.

Corticotropin-releasing hormone (CRH) mediates endocrine, behavioral, and autonomic responses to stress. In addition to binding to two receptor subtypes, CRH binds to a CRH-binding protein (CRH-BP). While CRH-BP is hypothesized to play a role in regulating levels of free CRH and modulating the stress response, the effects of stressors on brain CRH-BP are relatively unexplored. The present study determined effects of acute and repeated restraint on CRH-BP mRNA in basolateral amygdala (BLA) and dorsal hippocampus (DH), brain regions involved in fear and motivation. Using in situ hybridization, we found that a single acute period of restraint significantly increased CRH-BP mRNA in BLA by 20% but had no effect in DH. Repeated restraint had no effect on basal levels of CRH-BP mRNA in BLA or DH. Importantly, repeated restraint blocked the effects of acute restraint in the BLA. These results demonstrate differential effects of acute and repeated restraint on CRH-BP mRNA.

Anxiety-related behavioral inhibition in rats: a model to examine mechanisms underlying the risk to develop stress-related psychopathology.

Behavioral inhibition (BI) is an adaptive defensive response to threat; however, children who display extreme BI as a stable trait are at risk for development of anxiety disorders and depression. The present study validates a rodent model of BI based on an ethologically relevant predator exposure paradigm. We show that individual differences in rat BI are stable and trait-like from adolescence into adulthood. Using in situ hybridization to quantify expression of the immediate early genes homer1a and fos as measures of neuronal activation, we show that individual differences in BI are correlated with the activation of various stress-responsive brain regions that include the paraventricular nucleus of the hypothalamus and CA3 region of the hippocampus. Further supporting the concept that threat-induced BI in rodents reflects levels of anxiety, we also show that BI is decreased by administration of the anxiolytic, diazepam. Finally, we developed criteria for identifying extreme BI animals that are stable in their expression of high levels of BI and also show that high BI (HBI) individuals exhibit maladaptive appetitive responses following stress exposure. These findings support the use of predator threat as a stimulus and HBI rats as a model to study mechanisms underlying extreme and stable BI in humans.

Predator stress induces behavioral inhibition and amygdala somatostatin receptor 2 gene expression.

Psychological stressors precipitate and maintain stress-induced psychopathology, and it is likely that altered amygdala function underlies some of the deleterious effects of psychological stress. To understand the mechanisms underlying the linkage between the response to psychological stressors and maladaptive or psychopathological responses, we have focused on amygdala responsivity in animal models employing species-specific psychological stressors. In the present study, we characterized the effects of a 15-min exposure to a natural predator, the ferret, on rat behavior and the expression of the somatostatin family of genes in the amygdala. We examined the somatostatin family of genes because substantial evidence shows that central somatostatin systems are altered in various neuropsychiatric illnesses. We report that rats respond to acute ferret exposure with a significant increase in fearful and anxious behaviors that is accompanied by robust amygdala activation and an increase in somatostatin receptor 2 (sst2) messenger RNA expression within the amygdala and anterior cingulate cortex. These studies are the first to show stress-induced changes in amygdala sst2 expression and may represent one mechanism by which psychological stress is linked to adaptive and maladaptive behavioral responses.

Neuropharmacology of venlafaxine.

Venlafaxine (Effexor) is an effective antidepressant and has also been approved for the treatment of generalized anxiety disorder. Venlafaxine was initially characterized as an inhibitor of both serotonin (5HT) and norepinephrine (NE) uptake and was therefore termed a "dual uptake inhibitor." This chapter reviews data from both in vitro and in vivo studies regarding its effects on 5HT and NE neurotransmission. In addition, the effects of venlafaxine on other systems that may play a role in its therapeutic efficacy effects are described. The data indicate that venlafaxine is a relatively weak inhibitor of NE transport in vitro. In vivo studies indicate that venlafaxine selectively inhibits 5HT uptake at low therapeutic doses and inhibits both 5HT and NE uptake at higher therapeutic doses. This chapter concludes with a discussion of the effects of venlafaxine on various aspects of physiology.

Acute in vivo amphetamine produces a homologous desensitization of dopamine receptor-coupled adenylate cyclase activities and decreases agonist binding to the D1 site.

We have previously reported that, 30 min after a single injection of 7.5 mg/kg d-amphetamine sulfate, there was a significant 25% decrease in the apparent Vmax for stimulation of adenylate cyclase activity by the D1 receptor-selective partial agonist SKF 38393 in rat striatal membranes, as compared with saline-injected controls. This desensitization was seen in the striatal but not the mesolimbic forebrain. In the present study this desensitization was further characterized by using various ligands that interact with the three components of the D1 receptor-coupled adenylate cyclase complex to determine the site of modification that resulted in the desensitization. The desensitization was not associated with a change in the stimulation of adenylate cyclase at the level of the catalytic subunit or the guanyl nucleotide-regulatory protein Ns. Receptor number, as assessed by the binding of the D1 selective antagonist [3H]SCH 23390, was unaltered in the desensitized state. In contrast, the number of high affinity binding sites, as measured with the agonist [3H]dopamine was decreased 30% by acute amphetamine exposure. This suggests that the amphetamine-induced desensitization may be the result of an uncoupling of the receptor from Ns. In order to further assess the effects of amphetamine on receptor/Ns coupling, we measured the ability of the guanyl nucleotide guanosine-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] to decrease high affinity [3H]dopamine binding to striatal membranes. The inclusion of 100 microM Gpp(NH)p in the assay decreased the number of receptors in the high affinity state by 40% and 52% in membranes from saline- and amphetamine-pretreated rats, respectively. These results imply that amphetamine treatment does not modify the ability of Gpp(NH)p to decrease high affinity agonist binding. It is possible that amphetamine treatment decreases the number of receptors that can couple to Ns but the remaining receptors can still form a high affinity complex and are sensitive to the effects of Gpp(NH)p. We also report that maximal D2 dopamine receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity was decreased in striatal membranes prepared from amphetamine-treated rats as compared with saline-injected controls, implying that the D2 pathway was desensitized by amphetamine treatment. Conversely, acute amphetamine injection did not alter the ability of either the adenosine A2 receptor to stimulate or the muscarinic cholinergic receptor to inhibit adenylate cyclase activity in the rat striatum. These results suggest that acute amphetamine treatment produces a dopamine receptor-specific or homologous desensitization.

Norepinephrine stimulation of pineal cyclic AMP response element-binding protein phosphorylation: primary role of a beta-adrenergic receptor/cyclic AMP mechanism.

Norepinephrine (NE) regulates melatonin production and many other aspects of pineal function through actions involving cAMP. In the present study the effects of NE on the phosphorylation of the cAMP response element-binding protein (CREB) were studied to determine whether CREB phosphorylation might be involved in cAMP signal transduction in this tissue. CREB was detected using gel mobility-shift analysis with the radiolabeled Ca2+/cAMP response element of the c-fos promoter. CREB phosphorylation was estimated in the gel mobility-shift assay using an antiserum specific for phosphorylated CREB. This antiserum generates a supershifted CREB signal with protein extracts obtained from glands treated with NE (EC50 approximately equal to 10 nM) in organ culture, demonstrating that NE stimulates CREB phosphorylation. CREB phosphorylation peaks 30-45 min after NE treatment is initiated and then gradually returns to base-line values. Pharmacological studies show that NE-stimulated CREB phosphorylation is mediated primarily through beta 1-adrenergic receptor-stimulated increases in cAMP. Activation of alpha 1-adrenergic receptors, which is known to elevate the intracellular free Ca2+ concentration, does not cause CREB phosphorylation. However, it is possible to produce CREB phosphorylation with certain pharmacological agents that elevate the intracellular free Ca2+ concentration. In vivo studies show that CREB phosphorylation can be induced by treatment with isoproterenol (1 mg/kg), demonstrating that phosphorylation of pineal CREB occurs in intact animals. These studies indicate that cAMP-dependent CREB phosphorylation could play a role in the adrenergic regulation of gene expression in pinealocytes.

Bovine hydroxyindole-O-methyltransferase. Significant sequence revision.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step in melatonin synthesis. The nucleotide and deduced amino acid sequences of bovine HIOMT have been reported. Our laboratory recently isolated a cDNA clone encoding human HIOMT. Comparison of the human and bovine nucleotide sequences revealed several discrepancies which prevented perfect alignment and produced defined regions of virtually no homology in the deduced amino acid sequence. Consequently, we repeated sequence analysis of the original bovine HIOMT cDNA clone, the results of which are reported here. The revised nucleotide sequence includes 23 differences from the published sequence. This completely changes the deduced amino acid sequence in two regions, encompassing a total of 96 residues, or 28% of the protein. The revised deduced amino acid sequence predicts different post-translational modifications as compared to that of the original deduced sequence. This information will make it possible in future investigations of HIOMT to design improved polymerase chain reaction primers, peptides for the generation of antisera, and probes for various types of analysis and screening of libraries.