Minna Marttila

Adrenomedullin gene expression in the rat heart is stimulated by acute pressure overload: blunted effect in experimental hypertension.

The levels of adrenomedullin (ADM), a newly discovered vasodilating and natriuretic peptide, are elevated in plasma and ventricular myocardium in human congestive heart failure suggesting that cardiac synthesis may contribute to the plasma concentrations of ADM. To examine the time course of induction and mechanisms regulating cardiac ADM gene expression, we determined the effect of acute and short-term cardiac overload on ventricular ADM mRNA and immunoreactive ADM (ir-ADM) levels in conscious rats. Acute pressure overload was produced by infusion of arginine8-vasopressin (AVP, 0.05μ g/kg/min,iv) for 2 h into 12-week-old hypertensive TGR(mREN-2)27 rats and normotensive Sprague-Dawley (SD) rats. Hypertension and marked left ventricular hypertrophy were associated with 2.2-times higher ir-ADM levels in the left ventricular epicardial layer (178 ± 36 vs. 81 ± 23 fmol/g, P < 0.05) and 2.6-times higher ir-ADM levels in the left ventricular endocardial layer (213 ± 23 vs. 83 ± 22 fmol/g, P < 0.01). The infusio...Somatostatin (SRIF) acts on specific membrane receptors to inhibit exocrine and endocrine pancreatic functions. Five SRIF receptor genes have been cloned, producing six receptor proteins (sst-s). We used a recently developed antibody to localize the sst2A splice variant in the rat pancreas. Western blots identified the sst2A receptor as an 90 kDa glycosylated protein in pancreatic tissue. In tyramide-amplified immunostainings all acinar cells, and the glucagon and pancreatic polypeptide immunoreactive cells (A and PP, respectively) were intensely labeled for sst2A, while no signal was detected in SRIF producing (D) cells. A very few insulin immunoreactive (B) cells were also labeled for sst2A, but the signal in these cells was lower than in exocrine, A or PP cells. Absorption of the sst2A antibody with the receptor peptide abolished specific staining in both immunoblots and tissue sections (negative control). These studies are the first to localize any SRIF receptor subtype in the rat pancreas. The specific localization of sst2A receptor in acinar, A and PP cells if confirmed in humans, would suggest that subtype specific analogs will be useful for the therapeutic regulation of exocrine and/or endocrine pancreatic secretion.

Endothelin-1 Is Involved in Stretch-Induced Early Activation of B-Type Natriuretic Peptide Gene Expression in Atrial but Not in Ventricular Myocytes Acute Effects of Mixed ETA/ETB and AT1 Receptor Antagonists In Vivo and In Vitro

BACKGROUND The precise role of paracrine and autocrine factors in mechanical load-induced activation of cardiac gene expression is unknown. Here we report the effects of endothelin-1 (ET-1) and angiotensin II (Ang II) receptor antagonism on acute pressure overload-induced activation of cardiac B-type natriuretic peptide (BNP) gene expression in spontaneously hypertensive rats (SHRs) in vivo and on mechanical stretch-induced increase in atrial BNP gene expression in vitro. METHODS AND RESULTS Acute pressure overload produced in conscious SHRs by infusion of arginine8-vasopressin (0.05 microg x kg(-1) x min(-1)) for 2 hours resulted in an increase in BNP mRNA levels in the left ventricle as well as in the atrium. Bolus injections of bosentan (mixed ET(A)/ET(B) receptor antagonist, 10 mg/kg I.V.) but not losartan (AT1 receptor antagonist, 10 mg/kg I.V.) blocked the increase of the BNP mRNA levels produced by pressure overload in the left atria, whereas the elevation of BNP mRNA levels was similar (a 1.9-fold increase) in the left ventricles of vehicle-, losartan-, and bosentan-infused SHRs. In an isolated perfused rat heart preparation, infusion of bosentan (1 micromol/L) for 2 hours inhibited the mechanical stretch-induced increase in BNP mRNA levels in the right atria, whereas an AT1 receptor antagonist, CV-11974 (10 nmol/L), had no effect. CONCLUSIONS The findings of the present study demonstrate that Ang II and ET-1 are not obligatorily required for stretch to trigger the increased BNP gene expression in ventricular myocytes in vivo. In contrast, mechanical load on the atrial myocytes did initiate an ET-1-dependent expression of BNP gene showing that endogenous ET-1 production differentially regulates BNP gene expression in atrial and ventricular myocytes.

Involvement of Transcriptional and Posttranscriptional Mechanisms in Cardiac Overload–Induced Increase of B-Type Natriuretic Peptide Gene Expression

The induction of atrial and ventricular B-type natriuretic peptide (BNP) gene expression is one of the earliest events occurring during hemodynamic overload. To examine the molecular mechanisms for increased BNP gene expression during cardiac overload, we studied the induction of the BNP gene expression compared with that of atrial natriuretic peptide (ANP) in a modified perfused rat heart preparation. An increase in right atrial pressure of 5 mm Hg resulted in a 1.4-fold (P < .05) and 2.2-fold (P < .01) increase in BNP mRNA levels after 1 and 2 hours, respectively, whereas ANP mRNA levels remained unchanged. Stretching for up to 2 hours also significantly increased right atrial immunoreactive BNP (ir-BNP) levels (from 15.8 +/- 2.2 to 20.1 +/- 1.2 ng/mg, P < .05). Actinomycin D (10 micrograms/mL), a transcriptional inhibitor, completely inhibited the stretch-induced increase in atrial BNP mRNA levels at 1 hour (P < .05) and 2 hours (P < .001), whereas a protein synthesis inhibitor, cycloheximide (90 micrograms/mL), had no effect on basal or direct mechanical stretch-induced increase in right atrial BNP mRNA levels. Furthermore, we examined the role of tyrosine kinase and protein kinase C activities in acute mechanical stretch-induced increase in BNP synthesis. Tyrosine kinase inhibitors lavendustin A (1 mumol/L) and tyrphostin A25 (3 mumol/L) and protein kinase C inhibitors staurosporine (30 nmol/L) and chelerythrine (1 mumol/L) prevented the stretch-induced increase in right atrial ir-BNP concentrations at 2 hours. In addition, chelerythrine inhibited the increase of right atrial BNP mRNA levels stimulated by cardiac overload. These resuls demonstrate that the early increase of BNP mRNA levels by mechanical stretch results from increased transcriptional activation and is independent of protein synthesis. Our results also suggest that protein kinase C and tyrosine kinases activities may be involved in coupling cardiac overload to alterations in atrial BNP synthesis.

Synthesis and secretion of natriuretic peptides in the hypertensive TGR(mREN-2)27 transgenic rat

To examine the pathophysiological mechanisms in transgenic rats carrying the murine Ren-2d renin gene, we studied atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) gene expression and secretion in 12-week-old hypertensive TGR(mREN-2)27 and normotensive Sprague-Dawley rats. Hypertension and marked left ventricular hypertrophy in TGR(mREN-2)27 rats were associated with high baseline plasma levels of immunoreactive ANP (148 +/- 18 versus 34 +/- 3 pmol/L, hypertensive versus normotensive rats; P < .001), whereas plasma immunoreactive BNP levels did not differ significantly between the strains (19 +/- 4 versus 12 +/- 3 pmol/L, P = .06). ANP mRNA and immunoreactive ANP levels in the left ventricular endocardial and epicardial layers in TGR(mREN-2)27 rats were about 20 to 40 times higher (P < .001) than those in normotensive rats. There were no statistically significant differences between atrial and ventricular BNP mRNA levels, but left ventricular immunoreactive BNP concentrations were twofold higher in hypertensive TGR(mREN-2)27 than in normotensive rats. Infusion of [Arg8]-vasopressin (0.05 microgram/kg per minute IV, for 2 hours) in normotensive rats produced rapid increases (twofold, P < .05 to .01) in left ventricular BNP mRNA and immunoreactive BNP levels, whereas ventricular BNP mRNA and peptide levels did not change significantly in hypertensive rats. The increase in left atrial BNP mRNA levels in response to acute pressure overload was also significantly smaller in the hypertensive than normotensive rats (3.5-fold versus 5.2-fold, P < .01). Furthermore, the proportional but not absolute (in picomoles per liter) increase in plasma immunoreactive ANP was smaller in transgenic rats in response to acute saline and [Arg8]-vasopressin infusions (0.9% NaCl: 1.9-fold increase versus 4.4-fold increase in normotensive rats, P < .001; [Arg8]-vasopressin: 2.2-fold versus 4.8-fold increase, P < .001). These results show that baseline and cardiac overload-induced increases in BNP synthesis are markedly attenuated in transgenic rats carrying the murine Ren-2d renin gene. In addition, acute volume and pressure overload produced a smaller proportional increase in ANP secretion in hypertensive rats than normotensive rats. These alterations in the natriuretic peptide system may contribute to the pathogenesis of hypertension and cardiovascular complications in the TGR(mREN-2)27 rat.

The Zinc Finger-Only Protein Zfp260 Is a Novel Cardiac Regulator and a Nuclear Effector of α1-Adrenergic Signaling

ABSTRACT α1-Adrenergic receptors mediate several biological effects of catecholamines, including the regulation of myocyte growth and contractility and transcriptional regulation of the atrial natriuretic factor (ANF) gene whose promoter contains an α1-adrenergic response element. The nuclear pathways and effectors that link receptor activation to genetic changes remain poorly understood. Here, we describe the isolation by the yeast one-hybrid system of a cardiac cDNA encoding a novel nuclear zinc finger protein, Zfp260, belonging to the Krüppel family of transcriptional regulators. Zfp260 is highly expressed in the embryonic heart but is downregulated during postnatal development. Functional studies indicate that Zfp260 is a transcriptional activator of ANF and a cofactor for GATA-4, a key cardiac regulator. Knockdown of Zfp260 in cardiac cells decreases endogenous ANF gene expression and abrogates its response to α1-adrenergic stimulation. Interestingly, Zfp260 transcripts are induced by α1-adrenergic agonists and are elevated in genetic models of hypertension and cardiac hypertrophy. The data identify Zfp260 as a novel transcriptional regulator in normal and pathological heart development and a nuclear effector of α1-adrenergic signaling.

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132+/-6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA (P < 0.001) and IR-BNP (P < 0.05) levels in 2-month old rats. There was also a 1.5-fold (P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% (P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% (P < 0.05) and 39% (P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% (P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.

Mechanisms regulating adrenomedullin gene expression in the left ventricle: role of mechanical load

Adrenomedullin (AM) may function as an autocrine and/or paracrine factor in the heart, but the exact mechanisms regulating cardiac AM gene expression are unknown. The aim of the present study was to characterize the role of mechanical load in regulating gene expression of AM by using two hypertensive rat strains as experimental models. Acute pressure overload was produced by arginine(8)-vasopressin (AVP, 0.05 microg/kg/min, i.v.) infusion in conscious spontaneously hypertensive rats (SHR) and double transgenic rats (dTGR) harboring both the human renin and angiotensinogen genes and in their respective normotensive strains. A significant increase in left ventricular AM mRNA levels was seen in the left ventricles of all rat strains, the increase being augmented in hypertensive strains. Direct left ventricular wall stretch in isolated, perfused rat heart preparation also activated AM gene expression. However, stretching of cultured neonatal ventricular myocytes resulted in inhibition of AM gene expression, and stretch also blocked hypoxia-induced increase in AM gene expression. The present study shows that cardiac AM gene expression is upregulated in response to pressure overload and that this upregulation may be mediated via cell types other than cardiac myocytes.

Augmentation of BNP gene expression in atria by pressure overload in transgenic rats harbouring human renin and angiotensinogen genes

We studied the role of angiotensin II in pressure overload-induced B-type natriuretic peptide (BNP) gene expression by using a double transgenic rat (dTGR) model, in which transgenic rats for the human angiotensinogen and renin genes are crossed. Pressure overload produced by [Arg8]-vasopressin (AVP) infusion (i.v., 0.05 microg/kg/min for 2 h) in conscious, chronically instrumented rats, resulted in a significantly greater increase in BNP mRNA levels in the left atrium of the dTGR rats than in Sprague-Dawley (SD) control rats (3.6- vs 1.6-fold, p < 0.05), while in the left ventricle there was no significant difference between the strains. In dTGR rats, the early activation of the BNP gene expression was associated with a decrease in immunoreactive BNP levels in the atrium (27.5%, p < 0.05), but not in the ventricle. In SD rats, ir-BNP levels did not change significantly in either atria or ventricles in response to AVP infusion. These results show that the pressure overload-induced activation of BNP gene expression differs between atrial and ventricular myocytes in the dTGR model of experimental hypertension.