Prolyl 4-hydroxylase (EC 1.14.11.2), an a2132 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important a subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The 13 subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the a and 13 subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodopterafrugiperda insect cells by baculovirus vectors. When the 13 subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the a subunit was expressed alone, only traces of the a subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These a subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both a-and 1subunit-producing viruses, an enzyme tetramer was formed, but signifcant amounts of a and 13 subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the a subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.Prolyl 4-hydroxylase (EC 1.14.11.2), an enzyme residing in the lumen of the endoplasmic reticulum, catalyzes the formation of 4-hydroxyproline in collagens and related proteins by the hydroxylation of proline residues in peptide linkages. This cotranslational and posttranslational modification plays a crucial role in collagen synthesis, as the 4-hydroxyproline residues formed are essential for the folding of the newly synthesized procollagen polypeptide chains into triple-helical molecules. The active prolyl 4-hydroxylase is an a2P32 tetramer consisting of two types of inactive monomer with mo-

To evaluate the mechanisms of brain natriuretic peptide (BNP) gene expression, we determined the effect of acute cardiac overload (from 30 min to 4 h) on atrial and ventricular BNP mRNA levels in normal and hypertrophied myocardium. Arginine8 vasopressin (AVP; 0.05 microgram/kg.min) and l-phenylephrine (PHE; 20 micrograms/kg.min) were infused iv to increase cardiac workload in conscious spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. At the age of 10-22 months, during the established phase of ventricular hypertrophy, baseline BNP synthesis was increased in the hypertrophic ventricular cells of SHR, as reflected by about 2-fold (P < 0.05-0.001) elevation of levels of immunoreactive BNP (IR-BNP) and BNP mRNA. Intravenous infusions of AVP and PHE increased mean arterial pressure, plasma IR-BNP levels, and ventricular BNP mRNA levels within 1 h of pressure overload; peak levels of BNP mRNA were reached at 4 h. The increase in BNP mRNA levels was slightly greater in the epicardial (2.0- to 2.6-fold; P < 0.01) than in the endocardial layer (1.9- to 2.0-fold; P < 0.01) of the left ventricle. The rapid stimulation of ventricular BNP mRNA synthesis induced by AVP and PHE was accompanied by the simultaneous activation of left atrial BNP gene expression. Left atrial BNP mRNA levels were increased significantly in response to 1-h infusions, and values peaked in both the AVP- and PHE-infused SHR at 2 h, i.e. a 3.6-fold increase in BNP mRNA levels in left atria in AVP-infused SHR, and a 2.5-fold increase in PHE-infused SHR. Right atrial BNP mRNA levels remained unchanged during drug infusion, except for a transient increase in the WKY after 30 min of infusion. The induction of BNP synthesis was also reflected by increased ventricular IR-BNP levels, whereas AVP and PHE did not affect atrial IR-BNP concentrations or contents. In conclusion, the present study shows that pressure overload rapidly stimulates BNP gene expression in the hearts of normal and hypertensive rats. Thus, locally generated BNP in the heart muscle may play a significant role in cardiac adaptation to acute changes in mechanical load.

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.

␣1-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 ␣1-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 ␣1-adrenergic stimulation. Interestingly, Zfp260 transcripts are induced by ␣1-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 ␣1-adrenergic signaling.The endogenous catecholamines epinephrine and norepinephrine are key regulators of numerous physiologic functions, including learning, memory, and cardiovascular and endocrine homeostasis. Their dysregulation has been implicated in human conditions such as depression and addiction and in cardiovascular and metabolic diseases. Their effects are mediated by three classes of adrenergic receptors (ARs), ␤, ␣1, and ␣2, each comprised of three distinct gene products. They all belong to the superfamily of seven transmembrane G-protein-coupled receptors. ␣1-ARs are critical for a variety of catecholamine actions such as the control of blood pressure, smooth muscle contraction, myocardial function, and glycogenolysis. The importance of ␣1-ARs in physiology and pathophysiology is evidenced by the wide clinical use of ␣1-AR agonists and antagonists for the treatment of cardiovascular disease, flu and allergy symptoms, and benign prostate hyperplasia (40, 43). Paradoxically, the molecular mechanisms underlying ␣1-AR action remain undefined.Historically, the role of ␣1-ARs in different biologic systems was largely inferred from pharmacologic studies, but the development of transgenic mice with targeted deletion or overexpression of specific ␣1-AR subtypes has further confirmed the essential role of specific ␣1-ARs in regulation of physiologic processes (reviewed in references 40 and 44). For example, ␣1-null mice rapidly develop hyperinsulinemia, insulin resistance, and obesity in response to high-fat feeding, confirming the important role of ␣1-AR in the regulation of glucose homeostasis (7). The use of genetically altered mice also confirmed the essential role of ␣1-ARs in mediating the effects of some psychostimulants and opiates and, more generally, their involvement in the regulation of vario...

Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide are the known members of the mammalian natriuretic peptide system. Like ANP, BNP is a natriuretic and diuretic hormone that also causes peripheral vasodilation and inhibition of the sympathetic and renin-angiotensin systems. Although originally isolated from porcine brain, the BNP gene is expressed in a specific manner in cardiac myocytes in both the atria and the ventricles, but it is mainly released from the ventricles. The major determinant of BNP secretion is wall stretch, and the levels of BNP mRNA increase substantially in response to cardiac overload. In the clinical setting, BNP appears to be the most powerful neurohumoral predictor of left-ventricular function and prognosis. An acute increase in BNP gene expression occurs within 1 h and mimics the rapid induction of proto-oncogenes in response to hemodynamic stress. BNP can be used as a myocyte-specific marker to identify mechanisms that couple acute mechanical overload to alterations in cardiac gene expression. This paper is focused on the mechanisms that regulate BNP gene expression in cardiac overload. Particularly, autocrine-paracrine factors as well as cytoplasmic signaling pathways and transcription factors involved in mechanical stretch-induced BNP gene expression are discussed.