Ovine arylalkylamine N-acetyltransferase in the pineal and pituitary glands: differences in function and regulation

The enzyme arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) has been conventionally linked with the biosynthesis of melatonin within the pineal gland and retina. This study establishes that AANAT messenger RNA (mRNA) and functional enzyme occurs within the pars tuberalis (PT) and to a lesser degree within the pars distalis (PD) of the sheep pituitary gland; expression in these tissues is approximately 1/15th (PT) and 1/300th (PD) of that in the ovine pineal gland. AANAT mRNA in the PT appears to be expressed in the same cells as the Mel1a receptor. No evidence was obtained to indicate that either PT or PD cells have the ability to synthesize melatonin, suggesting that this enzyme plays a different functional role in the pituitary. We also found that cAMP regulation of the abundance of AANAT mRNA differs between the PT and pineal gland. Forskolin (10 microM) has no effect on pineal AANAT mRNA levels, yet represses expression in the PT. This suppressive influence could be mediated by ICER (inducible cAMP response early repressor), which is induced by forskolin in both tissues. Although it appears that the specific function and regulation of AANAT in the pituitary gland differ from that in the pineal gland, it seems likely that AANAT may play a role in the broader area of signal transduction through the biotransformation of amines.     

Regulation of K+ channel mRNA expression by stimulation of adenosine A2a-receptors in cultured rat microglia

Previous investigations suggest that the expression of K+ channels in cultured rat microglia is related to the activation status of these cells. Both, lipopolysaccharide (LPS) and agents that raise intracellular cyclic AMP have been shown to inhibit microglial proliferation. LPS also regulates the mRNA expression levels of K+ channels in cultured microglia, which led us to investigate possible regulatory interactions between K+ channels and adenosine A2a-receptors, which are coupled to the cAMP-signal transduction pathway. The selective adenosine A2a-receptor agonist CGS 21680 induced enhanced mRNA expression of both Kv1.3 and ROMK1, as well as an elevation of Kv1.3 protein. The selective adenosine A2a-receptor antagonist aminophenol (ZM 241385) and the nonselective antagonist 8-phenyltheophylline (8-PT) inhibited these effects. Elevations of cyclic AMP by use of dibutyryl cyclic AMP (dbcAMP), phosphodiesterase-inhibitor (RO 20-1724), forskolin, or cholera toxin (CTX), strongly enhanced Kv1.3-mRNA expression, but decreased ROMK1-mRNA levels. Results from experiments with actinomycin D suggest that K+ channel mRNA levels in cultured microglia were regulated by altered mRNA synthesis. Evidently, the CGS 21680-induced effects upon Kv1.3 were mediated via an increase in intracellular cyclic AMP, whereas ROMK1-mRNA expression appeared to be regulated by coupling of adenosine A2a-receptors to an alternative pathway, which involves activation of protein kinase C (PKC). It is concluded that the cyclic AMP second messenger system in microglia is not only involved in regulation of K+ channel activity, but also in regulation of de novo K+ channel synthesis.     

Phase II study of irinotecan and etoposide in patients with metastatic non-small-cell lung cancer

Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; EC 2.3.1.87), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.     

Human hydroxyindole-O-methyltransferase in pineal gland, retina and Y79 retinoblastoma cells

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) was studied in extracts of human pineal gland, retina and Y79 retinoblastoma cells. HIOMT enzyme activity and immunoreactive protein (approximately 42 kDa) were undetectable in the human retina; very low levels of HIOMT mRNA were detected using a highly sensitive RT-PCR/Southern blot method, as has been reported. Analysis of extracts of Y79 cells indicated that HIOMT enzyme activity, immunoreactivity (approximately 42 kDa) and mRNA (approximately 1.3 kb) were detectable at approximately 1/5-1/40 the levels found in the pineal gland. This unambiguously establishes that the HIOMT gene is expressed in Y79 cells. Kinetic analysis of Y79- and pineal-derived HIOMT indicates that the enzyme is generally similar in both tissues; one difference, however, is that substrate inhibition by N-acetylserotonin is greater with the Y79-derived enzyme. These studies show that Y79 cells represent a valid model to study the regulation of human HIOMT protein and mRNA; the differences detected may reflect the existence of tissue-specific regulatory mechanisms or differential patterns of expression of HIOMT isoforms.     

Immortalization of neuroendocrine pinealocytes from transgenic mice by targeted tumorigenesis using the tryptophan hydroxylase promoter

Tryptophan hydroxylase (TPH) is the first enzyme in both serotonin and melatonin biosynthesis in neuroendocrine cells of the pineal gland. The lack of immortalized neuroendocrine pineal cell lines has been a major obstacle to the study of the tissue-specific and circadian regulation of TPH gene expression in the pineal gland. Previously, we demonstrated that a 6.1 kb 5' upstream region of the mouse TPH gene directs the restricted expression of a lacZ reporter gene to the pineal gland and the raphe nuclei of transgenic mice. Therefore, to develop TPH-expressing pineal cell lines we first established transgenic mice carrying a construct consisting of 6.1 kb of 5' flanking region fused to the SV40 T-antigen. These animals developed highly invasive pineal tumors and died at 12-15 weeks of age. The pineal tumors obtained from the transgenic mice were utilized to establish the immortalized pinealocyte-derived cell lines. These cells express two marker enzymes, TPH and serotonin N-acetyltransferase (NAT). In pineal gland TPH and NAT expressions have been known to be regulated during circadian cycle. The two established cell lines therefore promise to be a valuable in vitro model system for the study of the rhythmic nature of the pineal function at molecular level in mammal.     

Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis through nuclear factor-kappa B activation and monocyte chemoattractant protein-1 synthesis

Angiotensin-converting enzyme (ACE) inhibitors reduce macrophage infiltration in several models of renal injury. We approached the hypothesis that angiotensin II (AngII) could be involved in inflammatory cell recruitment during renal damage through the synthesis of monocyte chemoattractant protein-1 (MCP-1). In a model of immune complex nephritis, we observed an up-regulation of renal MCP-1 (mRNA and protein) coincidentally with mononuclear cell infiltration that were markedly reduced by treatment with the ACE inhibitor quinapril. Exposure of cultured rat mesangial cells to AngII increased MCP-1 mRNA expression (2.7-fold) and synthesis (3-fold), similar to that observed with TNF-alpha. Since NF-kappaB is involved in the regulation of MCP-1 gene, we explored whether the effects of AngII were mediated through NF-kappaB activation. Untreated nephritic rats showed increased renal NF-kappaB activity (3.5-fold) that decreased in response to ACE inhibition. In mesangial cells, AngII activated NF-kappaB (4.3-fold), and the NF-kappaB inhibitor pyrrolidine dithiocarbamate abolished the AngII-induced NF-kappaB activation and MCP-1 gene expression. Our results suggest that AngII could participate in the recruitment of mononuclear cells through NF-kappaB activation and MCP-1 expression by renal cells. This could be a novel mechanism that might further explain the beneficial effects of ACE inhibitors in progressive renal diseases.     

Apposition-dependent induction of plasminogen activator inhibitor type 1 expression: a mechanism for balancing pericellular proteolysis during angiogenesis

Plasminogen-activator inhibitor type I (PAI-1), the primary inhibitor of urinary-type plasminogen activator, is thought to play an important role in the control of stroma invasion by both endothelial and tumor cells. Using an in vitro angiogenesis model of capillary extension through a preformed monolayer, in conjunction with in situ hybridization analysis, we showed that PAI-1 mRNA is specifically induced in cells juxtaposed next to elongating sprouts. To further establish that PAI-1 expression is induced as a consequence of a direct contact with endothelial cells, coculture experiments were performed. PAI-1 mRNA was induced exclusively in fibroblasts (L-cells) contacting endothelial cell (LE-II) colonies. Reporter gene constructs driven by a PAI-1 promoter and stably transfected into L-cells were used to establish that both mouse and rat PAI-1 promoters mediate apposition-dependent regulation. This mode of PAI-1 regulation is not mediated by plasmin, as an identical spatial pattern of expression was detected in cocultures treated with plasmin inhibitors. Because endothelial cells may establish direct contacts with fibroblasts only during angiogenesis, we propose that focal induction of PAI-1 at the site of heterotypic cell contacts provides a mechanism to negate excessive pericellular proteolysis associated with endothelial cell invasion.     

Expression of T3 receptor genes in peripheral lymphocytes from patients with thyroid dysfunction

In order to explore the expression and regulation of T3 receptor genes, the relative amount of peripheral lymphocytes c-erbA alpha and c-erbA beta mRNA from patients with Graves disease and Hashimoto thyroiditis with hypothyroidism (HTH) was determined with hybridization by using human c-erbA alpha and c-erbA beta probes. The results showed that there were two types of c-erbA alpha mRNA (6.0 kb, 3.2 kb) and three types of c-erbA beta mRNA (5.0 kb, 3.0 kb, 2.0 kb). The levels of c-erbA alpha and c-erbA beta mRNA were found to increase in HTH patients, but unaltered in Graves disease patients. This result suggests the presence of up-regulation of nuclear T3 receptor at the gene level in the lymphocytes of hypothyroid patients. The high level of c-erbA alpha and c-erbA beta mRNA may contribute in part to the increase in T3 receptor.     

Research on the mechanism of endothelin inflammatory effects on human mesangial cells

OBJECTIVE: To investigate the mechanism of endothelin (ET) inflammatory effects on human mesangial cells (HMC). METHODS: The following experiments were performed on cultured HMC after ET-1 stimulation: (1) the expression of tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) itself messenger ribonucleic acid (mRNA) was determined by Northern Blot analysis; (2) the TNF alpha concentration was tested with radioimmunoassay; the IL-1 activity was assayed by the enhancement of thymocyte proliferation in response to mitogen; the surface expression of ICAM-1 and VCAM-1 was measured with cell enzyme linked immunoadsorbent assay (ELISA) analysis. RESULTS: ET-1 (10(-7) mol/L) induced the following changes on HMC: (1) up-regulation of the expression of TNF alpha mRNA and protein; (2) up-regulation of the expression of ICAM-1 and VCAM-1 mRNA and protein; (3) up-regulation of the expression of ET-1 itself mRNA. However, the expression of IL-1 mRNA and protein was not changed. CONCLUSIONS: ET-1 can stimulate HMC to produce TNF alpha, ICAM-1 and VCAM-1, and thereby induce inflammatory effects. ET-1 can also stimulate HMC to up-regulate the expression of ET-1 itself, so as to amplify inflammatory effects. So, ET-1 is actually an inflammatory mediator and may play an important role in the pathogenesis of glomerulonephritis.     

Effect of prostate biopsy on the results of the PSA RT-PCR test

In this investigation, the presence of NKA-immunoreactive substances was determined in pineal glands from intact, castrated and castrated, testosterone-treated male rats. The effect of environmental light, melatonin treatment and superior cervical ganglionectomy on pineal NKA-immunoreactive substances was also investigated. The results obtained show that NKA is present in measurable amounts in the rat pineal, and NPK is probably also present, Orchidectomy was followed by an increase in the content of NKA-immunoreactive substances in the pineal gland. The replacement treatment with testosterone propionate in castrated rats blocked this effect. NKA-immunoreactive substances were not significantly different quantitatively in pineals from rats killed under light or under darkness. The removal of the superior cervical ganglia was followed by a significant increase in the NKA-immunoreactive substance content in the pineal gland of male rats. These results indicate that NKA and other tachykinins are present in the pineal gland of the male rat, and they seem to be regulated by gonadal hormones and the innervation originated from the superior cervical ganglia.     

Alteration of heart uncoupling protein-2 mRNA regulated by sympathetic nerve and triiodothyronine during postnatal period in rats

To provide tissue-specific and developmental characteristics of gene expression of rat heart uncoupling protein-2 (UCP2), we investigated developmental alterations of UCPs mRNA expression in the heart and brown adipose tissue (BAT), and examined possible up-regulators of heart UCP2 expression using in vitro studies. Heart UCP2 mRNA expression was low during the early postnatal days followed by a rapid and significant increase in the 2nd postnatal week. Heart UCP3 mRNA remained undetectable until the 2nd postnatal week when the expression reached a small but significant peak. BAT UCP1 mRNA was abundantly expressed in the neonate, but the expression rapidly decreased to the adult level. The studies using cultured cardiomyocytes demonstrated that both 10(-8) M triiodothyronine and 10(-7) M isoproterenol, but not phenylephrine, increased UCP2 mRNA expression. These results indicate that the sympathetic nervous system and/or thyroid hormones may be involved in the up-regulation of heart UCP2 gene expression during postnatal development. The increase in postnatal heart UCP2 may provide a key link between the postnatal energy shift and adaptation of rat pups to their novel environment.     

A novel pineal night-specific ATPase encoded by the Wilson disease gene

We have identified a pineal night-specific ATPase (PINA), a novel splice variant of the ATP7B gene disrupted in Wilson disease (WD). PINA expression exhibits a dramatic diurnal rhythm in both pineal gland and retina with 100-fold greater expression at night than at day. PINA is expressed in pinealocytes and a subset of photoreceptors in adult rats and is transiently expressed in the retinal pigment epithelium and the ciliary body during retinal development. Nocturnal pineal expression of PINA is under the control of a suprachiasmatic nucleus clock mediated by superior cervical ganglion innervation of the pineal. In vitro, PINA expression in pineal cells can be stimulated by agents activating the cAMP signal transduction pathway. PINA is able to restore copper transport activity in Saccharomyces cerevisiae deficient in the homologous copper-transporting ATPase CCC2, suggesting that this protein may function as a copper transporter in rat pinealocytes. These studies suggest a potential role of rhythmic copper metabolism in pineal and/or retina circadian function.     

Thiol-mediated regulation of ICAM-1 expression in endotoxin-induced acute lung injury

The intracellular redox state regulates several aspects of cell function, suggesting that strategies directed toward altering the cellular redox state may modulate cell activation in inflammatory states. As the most abundant intracellular thiol, glutathione plays a critical role as an intracellular redox buffer. Using diethylmaleate (DEM) as a glutathione-depleting agent, we evaluated the effects of GSH depletion in a rodent model of polymorphonuclear neutrophil (PMN)-dependent acute lung injury. Rats received 500 microg of LPS by intratracheal challenge, inducing a 5.5-fold increase in lung permeability and sixfold increase in lung PMN content. Pretreatment with DEM prevented the LPS-induced increase in lung PMN influx and lung permeability. Northern analysis and immunohistochemical studies suggest that this effect may be mediated by preventing up-regulation of lung ICAM-1 mRNA and protein expression. This effect is specific to ICAM-1, because lung cytokine-induced neutrophil chemoattractant and TNF-alpha mRNA levels are unaffected. This finding is not unique to the lung, because a similar effect on PMN influx was recapitulated in a rodent model of chemical peritonitis. Further, in vitro studies demonstrated that pretreatment of HUVEC monolayers with DEM prevented both ICAM-1 up-regulation and PMN transendothelial migration. These data indicate the presence of a thiol-sensitive mechanism for modulating ICAM-1 gene expression and suggest a potential novel therapeutic strategy for diseases characterized by PMN-mediated tissue injury.