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The angiotensin-converting enzyme (ACE) is a membrane-bound peptidyl dipeptidase known to act on a variety of peptide substrates in the extracellular space. Its most notable functions are the formation of angiotensin II and the degradation of bradykinin. In the current experiments, we found that exogenous ACE added to vascular smooth muscle cell culture strongly induces and upregulates the genes of bradykinin receptors [B.sub.1] and [B.sub.2]. This transcriptional regulatory property of ACE was shown to be unrelated to its known enzymatic properties. Indeed, ACE at 3.75 [micro]g/ml added in the culture medium of vascular smooth muscle cells was found to cause marked upregulation of the mRNA expression of the genes for the [B.sub.1] and [B.sub.2] receptors of bradykinin by 22- and 11-fold, respectively. This phenomenon was not altered by the addition of specific angiotensin II antagonists for the A[T.sub.1] or A[T.sub.2] receptors. Moreover, the ACE inhibitor captopril, which inhibited ACE enzymatic activity, did not block its effect at the bradykinin receptor gene transcription level. Expression of both receptor genes was completely abolished by actinomycin D. Furthermore, transcriptional upregulation was inhibited by curcumin, suggesting involvement of different transcriptional factors in this phenomenon. Electro-phoretic mobility shift assay revealed increase in NF-[kappa]B and activator protein-1 protein binding for consensus sequences, between ACE-treated cells versus untreated cells. The data indicate a novel biological function of the ACE unrelated to its well-known enzymatic function as a peptidyl dipeptidase. vascular smooth muscle cells; peptidyl dipeptidase; nuclear factor-[kappa]B; activator protein-1

Immortalized human bladder cell line exhibits typical characteristics of a mammalian urinary bladder, including stratification and amiloride-sensitive Na+ absorption. The cells are suitable for analyzing bladder pathobiology. Bladder cells are isolated through the retroviral transduction of simian virus 40 large T antigen into epithelial bladder cells. The cells are grown in a hormonally supplemented medium containing lethally irradiated NIH-3T3 fibroblast cocultures.

A study was conducted to examine the role of the C/EBP proteins of the binding site of human growth factor-activatable sodium-ion/hydrogen-ion exchanger (NHE1) in the gene transcriptional regulation of NHE1 activity. NHE1 is involved in initiating cell proliferation and growth in vascular smooth muscles and hepatic tissues. The results show that C/EBP belongs to a family of transcription factors that regulate the transcription of human NHE1 genes in hepatic and vascular smooth muscle cells.

The renal cortex of rabbits have basolateral membrane vesicles which are used to study the dependence of the basolateral Na(+)-HCO3(-) cotransporter on the physiological concentrations of angiotensin (ANG) II. The activity of the basolateral Na(+)-HCO3(-) cotransporter is induced by potent physiological accumulations of ANG II. Adenylate cyclase, phospholipase C, phospholipase A2 and receptor-operated ion channels represent various signal transduction pathways. The coupling of the pathways and the ANG II receptors in epithelial cells is observed.

Bradykinin has vasodilatory and tissue-protective effects exerted via its [B.sub.2] type receptor, whereas the [B.sub.1] receptor is constitutively absent but inducible by inflammation and toxins. In previous studies, we found that [B.sub.2] receptor gene knockout mice exhibit overexpression of the [B.sub.1] receptor, which assumes a vasodilatory function and is further upgraded in renovascular hypertension. The present study was designed to explore the effects of excess angiotensin II (ANG II) on [B.sub.1] receptor and [B.sub.2] receptor gene expression in mouse cardiomyocytes and rat vascular smooth muscle cells (VSMC) in vivo (after a 3-day infusion of 30 ng/min ANG II in 11 wild-type and in 13 genetically engineered mice with deleted [B.sub.2] receptor gene) and in vitro (ANG II added in rat VSMC culture in the presence or absence of [AT.sub.1] or [AT.sub.2] receptor antagonist). Expression of [B.sub.1] and [B.sub.2] receptor mRNA was assessed by reverse transcriptase-polymerase chain reaction. ANG II infusion caused upregulation by 30% of the already significantly overexpressed [B.sub.1] receptors in cardiomyocytes of the [B.sub.2] receptor gene knockout mice, but in the wild-type mice it upregulated only the [B.sub.2] receptor mRNA by 47%. The addition of ANG II in VSMC culture produced a time-dependent induction of [B.sub.2] and upregulation of [B.sub.2] receptor gene expression, maximal at 3 h (by fivefold), declining almost to baseline by 24 h. The addition of losartan completely blocked this effect, whereas the [AT.sub.2] blocker PD-123319 made no difference, indicating that this is an [AT.sub.1]-mediated effect of ANG II. The data indicate that excess ANG II in subpressor doses in vivo upregulates expression of the [B.sub.2] receptor, but in its absence, the already overexpressed [B.sub.1] receptor is further upregulated, evidently assuming a counterregulatory response; in vitro, it transiently upregulates both bradykinin receptors. angiotensin infusion; [B.sub.2] gene knockout mice; cell culture

Background: Salt-induced hypertension is mediated via the α2B-adrenergic receptor (AR) subtype. In α2B-AR gene knockout mice, blood pressure (BP) does not rise with salt loading, and in rats with salt-induced hypertension, BP decreases transiently with antisense (AS) treatment targeting the α2B-AR gene. The present experiments were designed to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA via adeno-associated virus (AAV) to prolong α2B-AR inhibition and hence reversal of salt-dependent hypertension. Methods: A recombinant AAV (rAAV) vector preparation encoding the α2B-AS fragment (previously tested in vitro for inhibition of α2B-AR protein production in cells) and containing green fluorescence protein (GFP) for visualization was injected ICV into subtotally nephrectomized, salt-fed rats. Control rats received rAAV-GFP (n = 8 per group). Results: We observed that BP rose from a baseline of 120 ± 10 to 184 ± 12 mm Hg. Injection of rAAV-α2B-AS produced a 35 ± 12 mm Hg fall in BP, lasting without evidence of diminishing for at least 16 days, whereas rAAV-GFP–injected rats showed a continued rise in BP. Rats treated with rAAV-α2B-AS treated had a 45% to 65% decrease in α2B-AR protein levels in key regulatory regions of the brain. Neither group had signs of immunologic response to the virus injection. Conclusions: These results indicate that our construct, when given by ICV means, could reach multiple sites of the central nervous system relevant to BP regulation and could safely inhibit the central α2B-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension. [Copyright &y& Elsevier]

Objective: In previous studies using serial analysis of gene expression for elucidation of the molecular pathways of angiotensin II (Ang II)-induced hypertensive/ischemic cardiomyopathy in mice, we found that a hitherto unknown transcript, designated initially as 2310008C07Rik, an unknown expressed sequence tag (EST), was highly significantly upregulated in myocardial tissue. The current experiments were designed to further characterize this gene and to evaluate its expression in various types of hypertension. Methods: Mice rendered hypertensive by Ang II infused intravenously at 30 ng/min for 6 h or by osmotic minipump at 0.9 μg/h for 7 or 14 days, were compared to saline-infused normotensive controls and to mice with hypertension induced by subtotal nephrectomy and 1% saline as drinking water. At end point, mice were euthanized, their tissues processed for gene expression analysis, and results were confirmed by ribonuclease protection assay. Results: The Ang II-infused mice developed systolic blood pressure (BP) of 134 ± 7, 158 ± 13, and 149 ± 15 mm Hg at 6 h, 7days, and 14 days, respectively, compared to 102 ± 9, 110 ± 8, and 114 ± 7 mm Hg in their respective controls and subtotally nephrectomized salt-fed mice had end point blood pressure of 153 ± 5 v 112 ± 7 mm Hg in controls. Through sequencing and expression analysis we found that the unknown transcript is part of the cardiomyopathy associated 3 (Cmya3) gene, being overexpressed in Ang II-induced but not salt-induced hypertension. Conclusions: The highly expressed 2310008C07Rik EST was found to be part of Cmya3 and its upregulation is due to Ang II-induced myocardial damage and not to BP elevation per se. [Copyright &y& Elsevier]