Serotonin 5-HT1 receptors potentiate histamine and thrombin stimulated prostaglandin synthesis in endothelial cells.

The ability of serotonin 5-HT1 receptors to increase vascular tone was previously found to be activated by vasoconstrictiors such as histamine. In this study, treatment of cultured human aortic endothelial cells (HAEC) with the 5-HT1-selective agonist 5-carboxamidotryptamine (5-CT) alone had no effect on the levels of prostaglandin F2alpha (PGF2alpha) or 6-keto-prostaglandin F1alpha (6-keto PGF1alpha). However, 5-CT potentiated the histamine and thrombin stimulated increases in prostaglandins released by HAEC. In the presence of histamine, increasing doses of 5-CT caused a steep rise in PGF2alpha levels resulting in an increase in the ratio of PGF2alpha over 6-keto PGF1alpha. The ability of 5-CT to potentiate prostaglandin production was correlated with its ability to potentiate the histamine and thrombin mediated mobilization of arachidonic acid. These results demonstrate that the ability of 5-HT1 receptors to stimulate prostaglandin production in endothelial cells is activated by histamine and thrombin.

The activation of plasminogen activator inhibitor-1 expression by IL-1beta is attenuated by estrogen in hepatoblastoma HepG2 cells expressing estrogen receptor alpha.

A low estrogen status in postmenopausal women is associated with elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1). In this study, the ability of estrogen compounds to regulate PAI-1 expression was determined in a hepatocyte HepG2 cell line made to stably express estrogen receptor alpha (ERalpha). In both the wild type and ER expressing HepG2 cells, estrogen had no effect on basal PAI-1 expression. However, in the ER expressing cells the ability of IL-1beta to increase PAI-1 mRNA and protein levels was attenuated by 17beta-estradiol, tamoxifen and twelve estrogen components of Premarin. In contrast, the mixed agonist/antagonist raloxifene had weak agonist activity and like the pure antagonist ICI 182780, it dose dependently blocked the effect of 17beta-estradiol on IL-1beta stimulated PAI-1 levels. These results suggest that estrogen agonists may lower PAI-1 levels in vivo by inhibiting cytokine activated PAI-1 expression by an ER dependent mechanism.

E1A represses apolipoprotein AI enhancer activity in liver cells through a pRb- and CBP-independent pathway.

The apolipoprotein AI (apoAI) promoter/enhancer contains multiple cis -acting elements on which a variety of hepatocyte-enriched and ubiquitous transcription factors function synergistically to regulate liver-specific transcription. Adenovirus E1A proteins repress tissue-specific gene expression and disrupt the differentiated state in a variety of cell types. In this study expression of E1A 12Sor 13S in hepatoblastoma HepG2 cells repressed apoAI enhancer activity 8-fold. Deletion mapping analysis showed that inhibition by E1A was mediated by the apoAI promoter site B. E1A selectively inhibited the ability of HNF3beta and HNF3alpha to transactivate reporter genes controlled by the apoAI site B and the HNF3 binding site from the transthyretin promoter. The E1A-mediated repression of HNF3 activity was not reversed by overexpression of HNF3beta nor did E1A alter nuclear HNF3beta protein levels or inhibit HNF3 binding to DNA in mobility shift assays. Overexpression of two cofactors known to interact with E1A, pRb and CBP failed to overcome inhibition of HNF3 activity. Similarly, mutations in E1A that disrupt its interaction with pRb or CBP did not compromise its ability to repress HNF3beta transcriptional activity. These data suggest that E1A inhibits HNF3 activity by inactivating a limiting cofactor(s) distinct from pRb or CBP.

Involvement of early growth response factor Egr-1 in apolipoprotein AI gene transcription.

Liver-specific expression of the apolipoprotein AI (apoAI) gene is mediated by transcription factors bound to three sites (A, B, and C) in the apoAI enhancer. Sites A and C bind various members of the nuclear receptor superfamily, including the orphan nuclear receptor apolipoprotein regulatory protein-1 (ARP-1); site B binds the liver-enriched factor hepatic nuclear factor-3. The immediate early growth response factor (Egr-1), which is transiently expressed in various pathophysiologic states of the liver, activates the apoAI enhancer and overcomes ARP-1-mediated repression of the enhancer in hepatoblastoma HepG2 cells. Deletion mapping analysis revealed two Egr-1 binding sites, E1 and E2, flanking site A. Erg-1 bound efficiently to both E1 and E2. Sp1 in HepG2 nuclear extracts bound to E2 but not E1. In HepG2 cells, E1 functioned as an Egr-1 response element, whereas E2 had high basal activity and was not further induced by Egr-1. Mutations that prevent Egr-1 binding to the apoAI enhancer abolished its responsiveness to Erg-1, while they had only minor effects on its constitutive activity. These mutations also diminished the ability of Egr-1 to overcome ARP-1-mediated repression. Elimination of transcription factor binding to sites A, B, or C reduced enhancer activity without affecting Egr-1-dependent activation. We argue that Egr-1 is recruited to the apoAI enhancer complex under unusual circumstances, such as those prevailing during liver regeneration, to maintain apoAI transcription levels by overriding prior transcriptional controls.

Low density lipoproteins induce parasympathetic responsiveness in embryonic chick ventricular myocytes in parallel with a coordinate increase in expression of genes coding for the M2 muscarinic receptor, G alpha i2, and the acetylcholine-sensitive K+ channel.

Growth of chick atrial cells in medium supplemented with lipoprotein-depleted serum has been shown to result in an increase in total cell cholesterol, and an increase in the negative chronotropic response to muscarinic stimulation in parallel with an increase in levels of muscarinic receptors and the G-protein alpha-subunits alpha i and alpha o (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In this study we determined whether growth of chick ventricular cells in medium supplemented with lipoprotein depleted serum could alter levels of muscarinic receptors and G-protein alpha-subunits and induce a negative chronotropic response to muscarinic stimulation. We further determined whether levels of mRNA coding for muscarinic receptors, G-proteins, and the acetylcholine-sensitive K+ channel were coordinately regulated. Growth of embryonic chick ventricular cells from hearts 14 days in ovo in medium supplemented with lipoprotein depleted serum resulted in a 21 +/- 5% (n = 3, +/- S.E.) increase in muscarinic receptor number as demonstrated by [3H]quinuclidinyl benzilate binding and a 4.7 +/- 1.0 (+/- S.E., n = 4)-fold increase in G alpha i2 as demonstrated by Western blot analysis. These changes in receptor and G-protein were associated with a coordinate increase in levels of mRNA coding for the M2 muscarinic receptor, G alpha i2 and the acetylcholine sensitive K+ channel as determined by RNase protection. These increases were reversed by addition of 30 microM mevinolin, an inhibitor of HMG-CoA reductase activity. Carbamylcholine (0.1 mM) had no effect on beat rate in ventricular cells grown in medium supplemented with fetal calf serum. Cells grown in medium supplemented with lipoprotein depleted serum demonstrated a 40 +/- 8% (+/- S.E., n = 10, p < 0.0001) decrease in beat rate in response to 0.1 mM carbamylcholine which was reversed by the addition of 30 microM mevinolin. These data suggest that, during growth in medium supplemented with lipoprotein depleted serum, a component of the cholesterol biosynthetic pathway plays a role in the coordinate induction of mRNAs coding for receptors, G-proteins, and an effector (ion channel) that results in the induction of a parasympathetic response in the ventricular cell characteristic of the atrial phenotype.

Cloning of cDNAs coding for the G alpha i1 and G alpha i2 G-proteins from chick brain.

We have cloned and characterized several cDNAs coding for G-protein inhibitory alpha subunits (G alpha i) from a chick brain cDNA library. Based on homology to G alpha subunits from other eukaryotes, these clones were designated chick G alpha i1 and G alpha i2. On the deduced amino-acid level, G alpha i1 and G alpha i2 were found to be 98 and 95% identical to rat G alpha i1 and G alpha i2, respectively. Using RNase protection analysis, the G alpha i1 and G alpha i2 mRNAs were found to be expressed in chick atria, ventricle, lung, liver, brain and kidney.

Isolation and expression of a novel chick G-protein cDNA coding for a G alpha i3 protein with a G alpha 0 N-terminus.

We have cloned cDNAs coding for G-protein alpha subunits from a chick brain cDNA library. Based on sequence similarity to G-protein alpha subunits from other eukaryotes, one clone was designated G alpha i3. A second clone, G alpha i3-o, was identical to the G alpha i3 clone over 932 bases on the 3' end. The 5' end of G alpha i3-o, however, contained an alternative sequence in which the first 45 amino acids coded for are 100% identical to the conserved N-terminus of G alpha o from species such as rat, mouse, human, bovine and hamster. Both clones were found to be expressed in all tissues studied. The unusual alpha o-alpha i3-like G-protein chimera, G alpha i3-o, was found to be expressed at significantly lower levels than G alpha i3. In vitro transcription and translation of the G alpha i3-o cDNA clone gave a protein of approx. 41 kDa which stably bound guanosine 5'-[gamma-thio]triphosphate. G alpha i3-o appears to be the first G-protein alpha subunit cloned which contains ends that are homologous to two different alpha subunit isoforms, G alpha o and G alpha i3.

Bradykinin elevates tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels in PC12 cells.

Bradykinin is known to rapidly elevate intracellular calcium leading to secretion of neurotransmitters and short term activation of tyrosine hydroxylase (TH). In this study we examined the effect of bradykinin on mRNA levels of two catecholamine biosynthetic enzymes. Treatment of PC12 cells with 1 microM bradykinin for 3 h markedly elevated both TH and dopamine beta-hydroxylase (DBH) mRNA levels.

Regulation of carboxypeptidase E by membrane depolarization in PC12 pheochromocytoma cells: comparison with mRNAs encoding other peptide- and catecholamine-biosynthetic enzymes.

PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine alpha-amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mM KCl. Short-term (1- to 3-h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]-Met into CPE. Also, CPE mRNA is not altered by 2-h treatments with KCl. In contrast, prolonged treatment (24-48 h) of PC12 cells with 50 mM KCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long-term treatment of the cells with KCl, with increases of 35% after 5 h and 55-75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine beta-hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long-term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin or DBH.

Regulated expression of the tyrosine hydroxylase gene by membrane depolarization. Identification of the responsive element and possible second messengers.

Prolonged depolarization has been used as a model of adaptive changes in the expression of various proteins, such as ion channels and neurotransmitter biosynthetic enzymes, in response to increased trans-synaptic activity in the nervous system. In depolarized PC12 cells, tyrosine hydroxylase (TH) mRNA levels increased severalfold (Kilbourne, E. J., and Sabban, E. L. (1990) Mol. Brain Res. 8, 121-127). In this study, membrane depolarization caused an increase in the expression of the reporter gene chloramphenicol acetyltransferase (CAT), under transcriptional control of the 5' region of the rat TH gene. These results indicate that membrane depolarization leads to increased transcription of the TH gene. Protein kinase C inhibitors had no effect on the induction of TH mRNA by depolarization, as well as the increase in formation of CAT under control of the upstream region of the TH gene. The depolarization responsive element in the TH gene was mapped to the region containing the cAMP responsive element. This region of the TH gene also increased CAT activity in response to the calcium ionophore, ionomycin. Interestingly, combined treatment with cAMP analogs and membrane depolarization had a greater effect than either alone on TH mRNA levels, as well as on CAT activity in PC12 cells transfected with the plasmid containing the cAMP responsive element.

Membrane depolarization by isotonic or hypertonic KCl: differential effects on mRNA levels of tyrosine hydroxylase and dopamine beta-hydroxylase mRNA in PC12 cells.

Membrane depolarization is an important and common manipulation used to study the result of enhanced neuronal activity on adaptive changes, including alterations in gene expression. In this study, the effect of elevated KCl, under isotonic and hypertonic conditions, on the changes in mRNA levels of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was compared. Treatment of PC12 pheochromocytoma cells for several hours with 50 mM KCl, under conditions where osmolarity was maintained, induced TH mRNA levels several fold, without changing DBH mRNA levels (Kilbourne and Sabban, 1990). In contrast, 50 mM KCl added to culture media without adjusting the osmolarity did not alter TH mRNA levels for up to 24 h. Longer continuous exposure to this hypertonic depolarization condition reduced TH mRNA levels to about 10% of control levels. DBH mRNA levels also declined when PC12 cells were treated from 12 h to 5 days with hypertonic 50 mM KCl. The effect appeared to be specific, since actin mRNA levels were elevated about 2-fold with these same hypertonic treatments. As a control for osmotic changes, 50 mM NaCl was used and did not alter TH or DBH mRNA levels. Viability of the cells was maintained and total protein synthesis was reduced somewhat after 12 h of exposure to hypertonic 50 mM KCl, and remained relatively constant for as long as 4 days. Thus, there appears to be an interaction between osmolarity and elevated KCl since very different results of the effects of membrane depolarization on the mRNA levels for the catecholamine biosynthetic enzymes were obtained depending on the osmolarity of the cultures. The extent of elevation of TH mRNA with isotonic KCl was also dependent on cell density. At high cell densities, membrane depolarization no longer induced TH mRNA levels. The results of this study indicate the experimental parameters which can be crucial in studies of membrane depolarization.

Hypomethylation of the rat tyrosine hydroxylase gene correlates with its expression in several cell types.

Changes in the methylation patterns of genes have been implicated in controlling tissue-specific gene expression. Since the mechanism which causes the developmental appearance of tyrosine hydroxylase (TH) is unknown, we examined the extent of DNA methylation in the rat TH gene is PC12, F4 and liver cells, which differ in their expression of TH. Using methylation-sensitive restriction endonucleases, we found that hypomethylation of the TH gene correlated with its expression in these cells.

Differential effect of membrane depolarization on levels of tyrosine hydroxylase and dopamine beta-hydroxylase mRNAs in PC12 pheochromocytoma cells.

Membrane depolarization has been widely used to elucidate the response of the nervous system to prolonged neuronal activity or stress. We studied the effect of treating PC12 cells with membrane depolarizing stimuli, 50 mM KCl, or 150 microM veratridine, and the subsequent changes in the mRNA levels of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). TH mRNA levels were found to increase 2- to 5-fold after continuous treatment for 1-12 h with 50 mM KCl. Depolarization with 150 microM veratridine had a similar effect on TH mRNA. In contrast, DBH mRNA levels were unchanged by either KCl or veratridine treatment. The role of calcium in the increase of TH mRNA levels elicited by depolarization was examined. The increase in TH mRNA was inhibited by the chelation of calcium with 3 mM EGTA. However, in contrast to their effect on phosphorylation of TH elicited by acute depolarization, the calcium channel blockers, nitrendipine and verapamil, and the calmodulin antagonists, W7 and trifluoperazine, did not prevent the increase in TH mRNA levels subsequent to several hours exposure to depolarizing stimuli. The calcium ionophore, A23187, alone was unable to induce TH mRNA levels. Thus, the increase in TH mRNA elicited by depolarization is mediated differently than the acute phosphorylation of the enzyme.