Phase II biomarker trial of a multimarker diagnostic for ovarian cancer.

PURPOSE: The primary hypothesis to be tested in this study was that the diagnostic performance (as assessed by the area under the receiver operator characteristic curve, AUC) of a multianalyte panel to correctly identify women with ovarian cancer was significantly greater than that for CA-125 alone. METHODS: A retrospective, case-control study (phase II biomarker trial) was conducted that involved 362 plasma samples obtained from women with ovarian cancer (n = 150) and healthy controls (n = 212). A multivariate classification model was developed that incorporated five biomarkers of ovarian cancer, CA-125; C-reactive protein (CRP); serum amyloid A (SAA); interleukin 6 (IL-6); and interleukin 8 (IL-8) from a modelling cohort (n = 179). The performance of the model was evaluated using an independent validation cohort (n = 183) and compared with of CA-125 alone. RESULTS: The AUC for the biomarker panel was significantly greater than the AUC for CA-125 alone for a validation cohort (p < 0.01) and an early stage disease cohort (i.e. Stages I and II; p < 0.01). At a threshold of 0.3, the sensitivity and specificity of the multianalyte panel were 94.1 and 91.3%, respectively, for the validation cohort and 92.3 and 91.3%, respectively for an early stage disease cohort. CONCLUSIONS: The use of a panel of plasma biomarkers for the identification of women with ovarian cancer delivers a significant increase in diagnostic performance when compared to the performance of CA-125 alone.

Increased expression of UBF is a critical determinant for rRNA synthesis and hypertrophic growth of cardiac myocytes.

Recent evidence suggests that increased translational efficiency of existing ribosomes alone is insufficient to account for the hypertrophic growth of cardiomyocytes and that synthesis of new functional ribosomes must occur. The rate-limiting step in ribosome accumulation is the transcription of the ribosomal 45S genes (rDNA) by RNA polymerase I. Our previous studies have demonstrated that increases in the expression of the rDNA transcription factor UBF correlated with hypertrophy of neonatal cardiomyocytes. These studies expand this observation to examine directly the hypothesis that increased UBF levels are an essential requirement for the initiation of cardiac hypertrophy. We demonstrate that the introduction of UBF antisense RNA into myocytes, using adenovirus approaches, efficiently inhibits UBF accumulation during induction of cardiomyocyte hypertrophy. Moreover, this approach results in a significant reduction in rDNA transcription, rRNA levels, and protein accumulation, which are all the hallmarks of cardiac growth. Furthermore, UBF antisense RNA expression did not alter re-expression of the fetal gene program, which confirmed that the effect was specific for transcription by RNA polymerase I. These findings demonstrate that an increase in rRNA synthesis is required for hypertrophy of cardiomyocytes and also implicate UBF as a major regulatory factor in this process. Approaches that target UBF activity may be of therapeutic use in the regression of pathophysiological cardiac hypertrophy.

Adrenomedullin is a regulated modulator of neonatal cardiomyocyte hypertrophy in vitro.

OBJECTIVE: Adrenomedullin is a potent hypotensive, natriuretic and diuretic peptide that is coexpressed in the heart with its receptor, suggesting that it may have localized actions as a modulator of cardiac function. Although expression of adrenomedullin is upregulated in the pathological heart, its cardiac function has not been clearly elucidated and it is not known whether this represents a common feature of cardiac hypertrophy, nor whether this is restricted to cardiac myocytes. We have determined the direct effects of hypertrophic agents on cardiomyocyte adrenomedullin gene expression and peptide secretion and have examined the effects of adrenomedullin on biochemical markers of cardiomyocyte hypertrophy. METHODS: Regulation of adrenomedullin expression and its effects on the hypertrophic response were studied in cultured rat neonatal ventricular cardiomyocytes. RESULTS: Incubation with phenylephrine or endothelin for 48 h led to a hypertrophic response with an associated fivefold stimulation of ANP gene expression. In contrast, adrenomedullin mRNA was inhibited by 30-50% in response to phenylephrine or endothelin-mediated hypertrophy, and this was associated with a 35-45% reduction in secretion of immunoreactive adrenomedullin. Phorbol ester mediated activation of protein kinase C and increasing intracellular Ca(2+) with ionomycin led to significant downregulation of adrenomedullin gene expression in cardiomyocytes. Co-incubation with 100 nM adrenomedullin for 48 h inhibited phenylephrine-induced cardiomyocyte hypertrophy as determined by protein:DNA ratio. Adrenomedullin partially blocked phenylephrine-mediated transcriptional activation of ANP and MLC-2 reporter gene expression in cardiomyocytes and this effect was mimicked by 2 microM forskolin, suggesting that this response was mediated via the activation of adenylate cyclase. CONCLUSION: These data demonstrate that the cardiomyocyte adrenomedullin gene is repressed by phenylephrine or endothelin-mediated hypertrophy. The inhibitory effects of adrenomedullin on the cardiomyocyte hypertrophic response suggests that this peptide acts as a regulated autocrine or paracrine modulator of cardiomyocyte function and that downregulation of adrenomedullin expression may play a role in induction and maintenance of cardiomyocyte hypertrophy.

Adrenomedullin signalling in cardiomyocytes is dependent upon CRLR and RAMP2 expression.

Adrenomedullin (AM) is a multifunctional peptide with a range of cardiovascular functions including direct effects on cardiomyocytes. Despite the demonstration of high numbers of AM binding sites in rat heart, the receptor responsible for cardiac AM action has not been characterized. We show here that the hearts of adult and neonatal rats, and neonatal cardiomyocyte cultures coexpress mRNA transcripts for the calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs). RAMP2 mRNA predominated over RAMP1 in adult (20:1 ratio) and neonatal heart (10:1 ratio), and in cardiomyocyte cultures (1.7:1 ratio), though the relative abundance of these transcripts appears to be developmentally regulated. Transient transfection of cardiomyocytes using a cAMP-responsive element (CRE)-luciferase reporter gene as an indirect measure of cAMP activation, demonstrated that overexpression of either CRLR or RAMP2 potentiated the AM signalling response. Overexpression of CRLR and RAMP2 together led to an additive effect on AM signalling that was approximately 4-fold greater than control cardiomyocytes stimulated with AM. Furthermore, the AM-mediated induction of CRE-luciferase activity was abolished by coincubation with the receptor antagonist CGRP(8-37) or by overexpression of a CRLR antisense construct. These data demonstrate that AM action in the rat cardiomyocyte requires CRLR and RAMP2 to constitute functional AM receptors.

In vivo and in vitro evidence for impaired arginine transport in human heart failure.

BACKGROUND: The clinical features of congestive heart failure (CHF) result from a complex interaction between reduced ventricular function, neurohormonal activation, and impaired endothelial function. Although endothelial dysfunction has been well documented, the mechanisms that contribute to this abnormality remain unknown. Recent studies, however, indicate a potential therapeutic role for supplemental L-arginine, suggesting the presence of an underlying disorder of L-arginine metabolism. METHODS AND RESULTS: We used 2 complementary approaches to assess L-arginine transport in control subjects and patients with CHF. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), forearm clearance of [(3)H]L-arginine was significantly reduced in CHF patients compared with forearm kinetics in control subjects (64+/-2 versus 133+/-14 mL/min, P=0.002). In conjunction with this, [(3)H]L-arginine uptake by peripheral blood mononuclear cells (PBMCs) was also substantially reduced in heart failure patients compared with controls (V(max) 10. 1+/-1.3 versus 49.8+/-7.1 pmol/10(5) cells per 5 minutes, P<0.001). In association with this finding, we observed a 76% (P<0.01) reduction in mRNA expression for the cationic amino acid transporter CAT-1, as assessed by ribonuclease protection assay. CONCLUSIONS: These data document both in vivo and in vitro evidence for a marked depression of L-arginine transport in human CHF and therefore provide an explanation for the restorative actions of supplemental L-arginine on vascular function in CHF.

Adverse effects of constitutively active alpha(1B)-adrenergic receptors after pressure overload in mouse hearts.

Cardiac hypertrophy and function were studied 6 wk after constriction of the thoracic aorta (TAC) in transgenic (TG) mice expressing constitutively active mutant alpha(1B)-adrenergic receptors (ARs) in the heart. Hearts from sham-operated TG animals and nontransgenic littermates (WT) were similar in size, but hearts from TAC/TG mice were larger than those from TAC/WT mice, and atrial natriuretic peptide mRNA expression was also higher. Lung weight was markedly increased in TAC/TG animals, and the incidence of left atrial thrombus formation was significantly higher. Ventricular contractility in anesthetized animals, although it was increased in TAC/WT hearts, was unchanged in TAC/TG hearts, implying cardiac decompensation and progression to failure in TG mice. There was no increase in alpha(1A)-AR mRNA expression in TAC/WT hearts, and expression was significantly reduced in TAC/TG hearts. These findings show that cardiac expression of constitutively actively mutant alpha(1B)-ARs is detrimental in terms of hypertrophy and cardiac function after pressure overload and that increased alpha(1A)-AR mRNA expression is not a feature of the hypertrophic response in this murine model.

Beta(2)-adrenergic receptor overexpression driven by alpha-MHC promoter is downregulated in hypertrophied and failing myocardium.

OBJECTIVE: The alpha-myosin heavy chain (alpha-MHC) promoter is frequently used to direct cardiac specific transgene expression. We studied whether transgene expression controlled by this promoter was altered under conditions of cardiac hypertrophy and failure. METHODS: Transgenic (TG) mice overexpressing human beta(2)-adrenergic receptors (beta(2)AR) and wild type (WT) controls were subjected to thoracic aortic constriction (TAC) or sham operation and studied at 1, 3 and 8 weeks after surgery. RESULTS: Sham operated TG mice had higher heart rates and left ventricular (LV) contractility than WT (all P<0.01), demonstrating enhanced betaAR activation. TAC at 1, 3 and 8 weeks produced progressive LV hypertrophy which was similar between WT and TG mice. Evidence of heart failure was more marked in TG mice with a greater increase in weights of the right ventricle and lungs and a higher prevalence of atrial thrombus (P<0.05 in each case). In hypertrophied TG hearts, endogenous alpha-MHC mRNA transcripts in LV were maintained at 1 and 3 weeks, but were reduced by approximately 40% relative to the sham-operated group at 8 weeks after TAC. Transgene expression, measured as human beta(2)AR mRNA, was reduced by 45% at 1 and 3 weeks and by 70% at 8 weeks after TAC. beta(2)AR binding sites were reduced by 35, 47 and 65%, respectively, at 1, 3 and 8 weeks. CONCLUSION: Cardiac hypertrophy and failure cause downregulation of the endogenous alpha-MHC as well as cardiac specific overexpression of the transgene directed by an alpha-MHC promoter.

beta(2)-adrenergic receptor overexpression exacerbates development of heart failure after aortic stenosis.

BACKGROUND: Beta-adrenergic signaling is downregulated in the failing heart, and the significance of such change remains unclear. METHODS AND RESULTS: To address the role of beta-adrenergic dysfunction in heart failure (HF), aortic stenosis (AS) was induced in wild-type (WT) and transgenic (TG) mice with cardiac targeted overexpression of beta(2)-adrenergic receptors (ARs), and animals were studied 9 weeks later. The extents of increase in systolic arterial pressure (P<0.01 versus controls), left ventricular (LV) hypertrophy (TG, 94+/-6 to 175+/-7 mg; WT, 110+/-6 to 168+/-10 mg; both P<0.01), and expression of ANP mRNA were similar between TG and WT mice with AS. TG mice had higher incidences of premature death and critical illness due to heart failure (75% versus 23%), pleural effusion (81% versus 45%), and left atrial thrombosis (81% versus 36%, all P<0.05). A more extensive focal fibrosis was found in the hypertrophied LV of TG mice (P<0.05). These findings indicate a more severe LV dysfunction in TG mice. In sham-operated mice, LV dP/dt(max) and heart rate were markedly higher in TG than WT mice (both P<0.01). dP/dt(max) was lower in both AS groups than in sham-operated controls, and this tended to be more pronounced in TG than WT mice (-32+/-5% versus -16+/-6%, P=0.059), although dP/dt(max) remained higher in TG than WT groups (P<0.05). CONCLUSIONS: Elevated cardiac beta-adrenergic activity by beta(2)-AR overexpression leads to functional deterioration after pressure overload.

Corticosteroid receptors and 11beta-hydroxysteroid dehydrogenase isoforms in rat intestinal epithelia.

To evaluate the potential roles that both receptors and enzymes play in corticosteroid regulation of intestinal function, we have determined glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) expression in intestinal epithelial cells. GR and MR mRNA and receptor binding were ubiquitously expressed in epithelial cells, with receptor levels higher in ileum and colon than jejunum and duodenum. RNase protection analysis showed that 11beta-HSD1 was not expressed in intestinal epithelial cells, and enzyme activity studies detected no 11-reductase activity. 11beta-HSD2 mRNA and protein were demonstrated in ileal and colonic epithelia; both MR and GR binding increased when enzyme activity was inhibited with carbenoxolone. Duodenal and jejunal epithelial cells showed very little 11beta-HSD2 mRNA and undetectable 11beta-HSD2 protein; despite minor (<7%) dehydrogenase activity in these cells, enzyme activity did not alter binding of corticosterone to either MR or GR. These findings demonstrate the ubiquitous but differential expression of MR and GR in intestinal epithelia and that 11beta-HSD2 modulates corticosteroid binding to both MR and GR in ileum and proximal and distal colon but not in duodenum or jejunum.

Co-expression of prepro-adrenomedullin with a putative adrenomedullin receptor gene in vascular smooth muscle.

The prepro-adrenomedullin (prepro-AM) gene encodes several biologically active peptides, including the potent vasodilator AM. At least part of the vasodilator action of AM appears to be mediated via interactions with receptors on vascular smooth muscle cells (VSMC); however, the specific receptors involved are not known. The aim of the present study was to identify putative AM receptor genes that are co-expressed with AM in cultured rat aortic VSMC that may mediate the actions of AM. AM mRNA was shown to be expressed in rat aortic VSMC cultures. In acute (4 h) secretion studies, only 20% of the total immunoreactive AM was intracellular, with the majority (80%) found in the medium, indicating active release of AM peptide from VSMC. Using highly specific ribonuclease protection analysis, mRNAs encoding three putative AM receptors [L1, calcitonin-receptor-like receptor (CRLR) and RDC1] were shown to be present at high concentrations in RNA extracts from lung. In cultured VSMC, however, whereas RDC1 mRNA was expressed at relatively high concentrations, transcripts encoding CRLR and L1 were not detected. The co-expression of prepro-AM mRNA with the RDC1 receptor implies that AM may act in a localized manner via this receptor to modulate VSMC function.

Rapid regulation of adrenomedullin in metabolically compromised vascular smooth muscle cells.

OBJECTIVE: The prepro-adrenomedullin gene encodes the biologically active peptide adrenomedullin, which acts as a potent vasodilator as well as a modulator of vascular smooth muscle cell growth. We investigated the question of whether adrenomedullin is regulated in response to metabolic perturbations in vascular smooth muscle. MATERIALS AND METHODS: Acute inhibition of glycolysis, leading to partial depletion of cellular ATP, was produced in cultured rat aortic vascular smooth muscle cells by replacing glucose with 2-deoxyglucose. Solution hybridization/RNase protection analysis was used to quantitate changes in expression of the prepro-adreno-medullin messenger RNA and a specific radioimmunoassay was used to assess levels of secreted adrenomedullin. RESULTS: Acute incubation of rat aortic vascular smooth muscle cells with 2-deoxyglucose caused a rapid and sustained induction of low basal levels of adrenomedullin messenger RNA, which reached twice the control levels by 1 h and four times control levels by 6 h. The induction of adrenomedullin messenger RNA expression was dependent upon de-novo gene transcription and was reversed by the re-introduction of glucose. Despite the sustained increase in adrenomedullin messenger RNA, secretion of immunoreactive-adrenomedullin from vascular smooth muscle cells was reduced by as much as 75% and paralleled the inhibition of radiolabeled amino acid incorporation into protein during glycolytic inhibition; both parameters recovered towards control levels following re-introduction of glucose. CONCLUSIONS: The rapid and reversible activation of the adrenomedullin gene and inhibition of adrenomedullin peptide release in response to metabolic inhibition suggest that adrenomedullin represents a novel localized mechanism that may modulate regional blood flow and vascular smooth muscle cell proliferation in response to perturbations of normal metabolism.

Reduced reperfusion-induced Ins(1,4,5)P3 generation and arrhythmias in hearts expressing constitutively active alpha1B-adrenergic receptors.

Reperfusion of globally ischemic rat hearts causes the generation of inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and the initiation of arrhythmias. These responses are mediated by alpha1-adrenergic receptors (ARs), but the subtype of receptor involved has not been identified. Under normoxic conditions, hearts from transgenic animals expressing constitutively active alpha1B-ARs in heart (alpha1B-constitutively active mutant [CAM]) showed higher [3H] inositol phosphate responses to norepinephrine (2.3-fold) than hearts from nontransgenic animals (alpha1B-WT) (1.6-fold). alpha1B-WT hearts responded to 2 minutes of reperfusion after 20 minutes of global ischemia by generation of Ins(1,4,5)P3 (5301+/-1310 to 11 413+/-1597 CPM/g tissue; mean+/-SEM; n=6; P<0.01 in [3H] labeling studies and 3.8+/-0.2 to 6.3+/-0.6 nmol/g by mass analysis, n=6; P<0.05). In contrast to findings in normoxia, hearts from alpha1B-CAM animals showed no Ins(1,4,5)P3 response in early reperfusion. In parallel studies, alpha1B-WT hearts developed ventricular tachycardia and ventricular premature beats (VPB) during 5 minutes of reperfusion after 20 minutes of ischemia. The incidence of these arrhythmias was reduced in the alpha1B-CAM hearts (95% to 62% for VPB and 47% to 12% for ventricular tachycardia; both P<0.05). The resistance of the alpha1B-CAM hearts was not due to alpha1B-AR-mediated preconditioning, as the Ins(1,4,5)P3 response to thrombin receptor activation during reperfusion was not different between the 2 groups. To investigate the possibility of reduced alpha1A-receptor activity in the alpha1B-CAM hearts, expression of the mRNA for alpha1A- and alpha1B-receptors was measured. alpha1B-WT hearts contained mRNA for both receptor subtypes, but the levels of alpha1B-receptor mRNA were 5-fold higher than alpha1A-receptor mRNA. alpha1B-CAM hearts contained very high levels of alpha1B-receptor mRNA (26-fold increase), but the expression of mRNA for the alpha1A-receptors (0.141+/-0.035 amol/ microg RNA; mean+/-SEM; n=6) was reduced by 50% relative to alpha1B-WT controls (0.276+/-0.046 amol/ microg RNA; n=6; P<0.01). The reduction in arrhythmogenic and Ins(1,4,5)P3 responses in alpha1B-CAM hearts provides evidence that these response are not mediated by alpha1B-receptors.

Cardiac expression of genes encoding putative adrenomedullin/calcitonin gene-related peptide receptors.

Recent studies have suggested that adrenomedullin (AM) may play a role in the pathophysiology of heart disease, though the specific cardiac receptors involved have not been defined. RT-PCR cloned fragments of three putative AM/calcitonin gene-related peptide (CGRP) receptors were used to established a quantitative RNase protection assay to identify and quantitate expression of receptor mRNAs in heart and in cardiac myocytes. Intact rat heart expressed mRNA encoding the putative AM/CGRP receptors RDC1 and CRLR at 37- and 15-fold higher levels, respectively, than the AM-selective receptor L1, with a qualitatively similar profile in cultured neonatal cardiac myocytes. The high level of expression of RDC1 and CRLR suggests that both AM and CGRP may have direct actions on the cardiac myocyte via common receptors that can interact with either ligand.

Selective activation of alpha1A-adrenergic receptors in neonatal cardiac myocytes is sufficient to cause hypertrophy and differential regulation of alpha1-adrenergic receptor subtype mRNAs.

Prolonged stimulation of cardiac alpha1-adrenergic receptors causes myocyte hypertrophy, although the receptor subtypes involved remain controversial. We have used a potent and selective alpha1A agonist, A-61603, to test whether activation of the alpha1A-adrenergic receptor subtype is sufficient to mediate the morphological, biochemical and molecular alterations associated with cardiomyocyte hypertrophy. In neonatal rat cardiomyocyte cultures, 48 h incubation with 50 nm A-61603 led to a marked increase in myocardial cell size that was associated with a significant elevation in the rate of protein synthesis. The increased rate of incorporation of radiolabelled amino acids into protein stimulated by A-61603 was totally abolished by the selective alpha1A antagonist KMD-3213. A-61603 increased ANF secretion three-fold, and ANF mRNA 12-fold above control levels in cardiomyocyte cultures. RNase protection analysis demonstrated a A-61603-mediated two to three-fold increase in alpha1A-adrenergic receptor mRNA with a concomitant 50% decrease in alpha1B mRNA levels by 48 h. Identical responses of differential regulation of alpha1A- and alpha1B-adrenergic receptor mRNA were observed with phenylephrine. Both the stimulation of alpha1A- and repression alpha1B-adrenergic receptor mRNA caused by A-61603 could be abolished by 10-20 nm KMD-3213. The present data provide evidence that selective activation of alpha 1A-adrenergic receptors on cardiomyocytes is sufficient to mediate the phenotypic changes associated with cardiac hypertrophy. In addition, the differential regulation of alpha1A and alpha1B mRNA in response to selective alpha1A-adrenergic receptor stimulation suggests that cross-talk between receptor subtypes may be involved in regulating receptor populations during chronic agonist exposure.

Stress-induced stimulation of pituitary POMC gene expression is associated with activation of transcription factor AP-1 in hypothalamus and pituitary.

The response to environmental stimuli such as stress involves changes in gene transcription in both brain and pituitary, which in turn, facilitate adaptive phenotypic alterations favoring survival. In the present study we have examined the expression of the inducible immediate-early genes of the fos and jun families, and the activity of transcription factor AP-1 in the hypothalamus and anterior pituitary gland of rats, after a single restraint challenge. Restraint led to a rapid transient increase in c-fos but not c-jun expression in hypothalamus and pituitary. Changes in jun-B expression in hypothalamus were qualitatively similar to c-fos, though not statistically significant at 30 min. Furthermore, a single episode of restraint stress led to significant increases (50-100%) in nuclear AP-1 DNA binding activity in both hypothalamus and pituitary, while DNA binding of an unrelated transcription factor (Sp1) was unchanged. Associated with the stress-induced activation of pituitary AP-1 was a parallel three- to fourfold transcriptional stimulation of pituitary POMC gene expression. These data demonstrate that the rapidly inducible members of the fos and jun gene families contribute to increased activity of transcription factor AP-1 in both hypothalamus and pituitary following stress, and suggest that AP-1 may be a crucial factor involved in rapid transcriptional responses during stress.

The involvement of nitric oxide in the secretion of beta-endorphin from the pituitary intermediate lobe of the rat.

Nitric oxide (NO) generated by the enzyme nitric oxide synthase (NOS) has been implicated in the regulation of a variety of endocrine functions. A number of biochemical and anatomical studies have demonstrated the presence of neuronal NOS (nNOS) in the neuroendocrine axis and have shown significant effects of NO on the release of hypothalamic and pituitary hormones. Using a C-terminal directed peptide antibody that is specific for nNOS we have found a predominance of nNOS in the neural lobe of the pituitary and in a single layer of epithelial cells, possibly a remnant of Rathke's pouch that form a border between the intermediate lobe and the anterior lobe. Furthermore, we have examined the effect of sodium nitroprusside (SNP), a donor of NO on the secretion of beta-endorphin (beta-EP) from the isolated neuro-intermediate lobe (NIL) of the rat and cultured rat melanotrophs. It was shown that in explant cultures of intact neuro-intermediate lobes, SNP (100 microM) was able to cause an inhibition of beta-EP secretion. In the presence of sulpiride (10 microM), a dopamine D2-receptor antagonist, there was a partial reversal of the SNP effect. On the other hand SNP did not affect beta-EP secretion in primary cultures of melanotrophs that no longer possessed any innervation. Taken together these data suggest that NO has an indirect inhibitory effect on the secretion of beta-EP by the intermediate lobe via the release of dopamine.

Concomitant up-regulation of proopiomelanocortin and dopamine D2-receptor gene expression in the pituitary intermediate lobe of the spontaneously hypertensive rat.

Alterations in central dopaminergic mechanisms in the Spontaneously Hypertensive Rat (SHR) have been previously implicated in the development of the hypertensive phenotype in this rat strain. We have examined the expression and regulation of the dopamine-responsive gene proopiomelanocortin (POMC) in the neurointermediate lobe (NIL) of the pituitary in both SHR and normotensive Wista Kyoto (WKY) rats. Solution hybridization/nuclease protection analysis showed that adult SHR express POMC mRNA in the NIL at approximately 2-4 times the level seen in normotensive WKY controls, associated with a concomitant 2-fold increase in dopamine D2-receptor (D2-R) mRNA expression. Despite the obvious difference in D2-R gene expression, NIL POMC mRNA in both rat strains was regulated in an identical manner following 4 d in vivo bromocriptine or haloperidol treatment. In contrast, though D2-R mRNA expression in the WKY NIL was significantly up-regulated by D2-R blockade with haloperidol, the elevated levels of D2-R mRNA in the NIL of the hypertensive strain were not altered by D2-R antagonism. Following isolation from all hypothalamic input by 5 d in vitro culture, SHR melanotrophs exhibited a 2-3 fold higher rate of beta EP secretion and POMC mRNA expression than melanotrophs derived from normotensive WKY rats, though beta EP secretion was inhibited in a similar fashion by the D2-R agonist quinpirole in both cultures. The current data demonstrate changes in expression of both POMC and D2-R mRNA in the SHR NIL which may be a consequence of altered dopaminergic input and/or alterations in D2-R regulation in this tissue, possibly enabling other factors in addition to dopamine to maintain the NIL of the SHR in a relatively hyperactive state. Whether or not POMC-derived peptides or other factors secreted from the melanotroph cell play any role in the development or maintenance of hypertension in this strain is yet to be established.

Adrenomedullin stimulates cAMP accumulation and inhibits atrial natriuretic peptide gene expression in cardiomyocytes.

Adrenomedullin (ADM) is a novel vasodilating and natriuretic peptide which may play an important role in cardiovascular regulation. In neonatal cardiomyocyte cultures we have shown that ADM leads to dose-dependent inceases in cAMP accumulation and subsequent inhibition of atrial natriuretic peptide (ANP) gene expression and secretion. Forskolin-mediated elevation of intracellular cAMP levels led to a qualitatively similar inhibitory effect on both ANP gene expression and secretion. These data show that ADM has direct effects on expression of ANP in the cardiomyocyte by a mechanism that may involve the activation of adenylate cyclase, lending further support to the hypothesis that ADM may act in vivo as an important endocrine or paracrine modulator of cardiovascular function.

CRF stimulates expression of multiple fos and jun related genes in the AtT-20 corticotroph cell.

Recent studies have shown that corticotropin releasing factor (CRF) stimulates c-fos gene expression in the AtT-20 corticotroph cell line, and that overexpression of c-Fos results in activation of POMC gene transcription. Since transactivation by c-Fos requires dimerization with a Jun family member to form the active transcription factor AP-1, we have examined the expression of multiple fos and jun related genes and have correlated their expression with AP-1 DNA binding activity in AtT-20 nuclear extracts after stimulation with CRF. Although basal expression of c-fos mRNA was extremely low, it was rapidly and transiently stimulated in AtT-20 cells following administration of either constant or a single pulse of CRF. In contrast, basal expression of c-jun mRNA was slightly higher and underwent little or no change in response to CRF. Specific ribonuclease protection analysis showed that in addition to c-fos, mRNA transcripts encoding fos B and jun B were rapidly stimulated in response to CRF, though levels of induced fos B mRNA were 20-40 times lower than c-fos or jun B, respectively. Gel shift analysis demonstrated that CRF caused a sustained increase in AP-1 DNA binding to both a canonical AP-1 element as well as to the POMC exon-1 AP-1 site. Studies with specific antisera directed against c-Fos revealed that although no c-Fos could be detected in AP-1 complexes in basal cell extracts, c-Fos became a prominent component of AP-1 following CRF stimulation, reaching maximal levels by 4 h. Despite the fact that AP-1 DNA binding activity remained elevated for at least 24 h after CRF, c-Fos was most prominent during the early phase of the response. Similarly, JunB was shown to be a major component of AP-1 DNA binding activity in CRF-stimulated AtT-20 nuclear extracts that persisted for at least 24h after stimulation. Despite the obvious induction of fos B mRNA in response to CRF, FosB protein was not detected in DNA bound AP-1 complexes. These data demonstrate that CRF is a potent stimulus for corticotroph expression of c-fos, jun B and fos B, and suggest that the subsequent increase in AP-1 may play a role in activation of gene expression and/or as a modulator of glucocorticoid receptor function.

Adrenomedullin induces expression of c-fos and AP-1 activity in rat vascular smooth muscle cells and cardiomyocytes.

The recently described hypotensive peptide adrenomedullin has been shown to activate various second messenger pathways in cells of the cardiovascular system though no genomic actions have yet been described. In cultured rat vascular smooth muscle cells, cardiomyocytes, and cardiac fibroblasts, adrenomedullin caused a rapid, but transient induction of c-fos mRNA expression in all three cell types that varied in magnitude. Adrenomedullin increased AP-1 DNA binding activities in vascular smooth muscle cells and cardiomyocytes, but not in cardiac fibroblasts. These data suggest that cardiomyocytes and vascular smooth muscle cells may be important genomic targets for this novel hormone.