Angiotensin-converting enzyme inhibition after experimental myocardial infarct: role of the kinin B1 and B2 receptors.

We sought to define the contribution of each of the 2 kinin receptors (bradykinin 1 receptor [B(1)R] and bradykinin 2 receptor [B(2)R]) to the cardioprotection of angiotensin-converting enzyme (ACE) inhibition after acute myocardial infarct. Wild-type mice and gene knockout mice missing either B(1)R or B(2)R were submitted to coronary ligation with or without concurrent ACE inhibition and had evaluation of left ventricular systolic capacity by assessment of fractional shortening (FS). Baseline FS was similar in all of the animals and remained unchanged in sham-operated ones. At 3 weeks after myocardial infarct, in the wild-type group there was a 27% reduction of FS (P<0.5) without ACE inhibition and 8% with ACE inhibition; in the B(1)R(-/-) groups the FS was reduced by 24% and was no different (at 28%) with ACE inhibition; in the B(2)R(-/-) groups, however, the FS was decreased by 39% and with ACE inhibition was decreased further by 52%. Analysis of bradykinin receptor gene expression in hearts showed that when one receptor was missing, the other became significantly upregulated; but the B(1)R remained highly overexpressed in the B(2)R(-/-) mice throughout, whereas the overexpressed B(2)R became significantly suppressed in the B(1)R(-/-) mice in a manner quantitatively and directionally similar to that of wild-type mice. We conclude that both bradykinin receptors contribute to the cardioprotective bradykinin-mediated effect of ACE inhibition, not only the B(2)R as believed previously; but, whereas with potentiated bradykinin in the absence of B(1)R, the upregulation of B(2)R is simply insufficient to provide full cardioprotection, in the absence of B(2)R, the upregulated B(1)R actually seems to inflict further tissue damage.

Role of bradykinin B1 and B2 receptors in normal blood pressure regulation.

With inhibition or absence of the bradykinin B2 receptor (B2R), B1R is upregulated and assumes some of the hemodynamic properties of B2R, indicating that both participate in the maintenance of normal vasoregulation or to development of hypertension. Herein we further evaluate the role of bradykinin in normal blood pressure (BP) regulation and its relationship with other vasoactive factors by selectively blocking its receptors. Six groups of Wistar rats were treated for 3 wk: one control group with vehicle alone, one with concurrent administration of B1R antagonist R-954 (70 microg x kg(-1) x day(-1)) and B2R antagonist HOE-140 (500 microg x kg(-1) x day(-1)), one with R-954 alone, one with HOE 140 alone, one with concurrent administration of both R-954 and HOE-140 plus the angiotensin antagonist losartan (5 mg x kg(-1) x day(-1)), and one with only losartan. BP was measured continuously by radiotelemetry. Only combined administration of B1R and B2R antagonists produced a significant BP increase from a baseline of 107-119 mmHg at end point, which could be partly prevented by losartan and was not associated with change in catecholamines, suggesting no involvement of the sympathoadrenal system. The impact of blockade of bradykinin on other vasoregulating systems was assessed by evaluating gene expression of different vasoactive factors. There was upregulation of the eNOS, AT1 receptor, PGE2 receptor, and tissue kallikrein genes in cardiac and renal tissues, more pronounced when both bradykinin receptors were blocked; significant downregulation of AT2 receptor gene in renal tissues only; and no consistent changes in B1R and B2R genes in either tissue. The results indicate that both B1R and B2R contribute to the maintenance of normal BP, but one can compensate for inhibition of the other, and the chronic inhibition of both leads to significant upregulation in the genes of related vasoactive systems.

A novel gene (Cmya3) induced in the heart by angiotensin II-dependent but not salt-dependent hypertension in mice.

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 mug/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, 7 days, 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.

Angiotensin-converting enzyme regulates bradykinin receptor gene expression.

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 B1 and B2. This transcriptional regulatory property of ACE was shown to be unrelated to its known enzymatic properties. Indeed, ACE at 3.75 microg/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 B1 and B2 receptors of bradykinin by 22- and 11-fold, respectively. This phenomenon was not altered by the addition of specific angiotensin II antagonists for the AT1 or AT2 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. Electrophoretic mobility shift assay revealed increase in NF-kappaB 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.

Age-related changes of bradykinin B1 and B2 receptors in rat heart.

Aging is a major risk factor for the development of vascular diseases, such as hypertension and atherosclerosis, that leads to end organ damage and especially heart failure. Bradykinin has been demonstrated to have a cardioprotective role by affecting metabolic processes and tissue perfusion under conditions of myocardial ischemia. Its actions are exerted via the bradykinin B1- and B2-type receptors (B1Rs and B2Rs), but the functional status of these receptors during the aging process is poorly understood. This study aims to investigate whether changes in B1R and B2R gene and protein expression in rat heart are associated with the age-related alterations of cardiac structure and function. Using real-time PCR, we found that B1R mRNA expression increased 2.9-fold in hearts of older rats (24 mo of age) compared with younger rats (3 mo of age), whereas B2R gene expression remained unchanged. Western blot analysis showed that expression of B2R at the protein level is approximately twofold higher in young rats compared with old rats, whereas the B1R protein is approximately twofold higher in old rats compared with young rats. The present results provide clear functional and molecular evidence that indicate age-related changes of bradykinin B1Rs and B2Rs in heart. Because the cardioprotective actions of bradykinin are physiologically mediated via the B2Rs, whereas the B1Rs become induced by tissue damage, these results suggest that age-related decreases in B2R protein levels may leave the heart vulnerable to ischemic damage, and increases in B1R expression and activity may represent a compensatory reaction in aging hearts.

Mitochondrial genome mutations in hypertensive individuals.

Human essential hypertension (HTN), a polygenic, multifactorial, and highly heterogeneous disorder of unknown etiology, has been shown to have excess maternal transmission in several studies, suggesting a possible mitochondrial involvement. In an effort to assess the contribution of the mitochondrial genome to HTN we initiated a systematic, extended screening of hypertensive individuals to identify potentially pathogenic mtDNA mutations. We applied our newly developed novel class of tests for the detection of mitochondrial mutation involvement in complex diseases to the hypertension data set from 350 pedigrees of white ethnicity and 98 of African American ethnicity ascertained at HTN clinics associated with Boston Medical Center, and we identified families with a likely mitochondrial involvement. We analyzed the sequence of the entire mitochondrial genome in probands from 20 such pedigrees, consisting of 10 African American and 10 white families. Comparison with the reference "Cambridge" sequence revealed a total of 297 base changes, including 24 in the ribosomal RNA (rRNA) genes, 15 in the transfer RNA (tRNA) genes, and 46 amino acid substitutions, with the remainder involving the noncoding regions or synonymous changes. Among the coding region mutations, 30 are novel, with 13 hypertensive probands carrying at least one novel variant, usually in combination with the previously described common polymorphisms, several of which are associated with cardiovascular and renal pathologies. These data will serve as a starting point for large-scale case-control association studies.

Novel targets of ANG II regulation in mouse heart identified by serial analysis of gene expression.

Although the central role of ANG II in cardiovascular homeostasis is well appreciated, the molecular circuitry of its many actions is not completely understood. With the use of serial analysis of gene expression to assess global transcriptional changes in the heart of mice after continuous 7-day ANG II administration, we identified patterns of gene expression indicative of cardiac remodeling, including coordinate regulation of genes previously described in a context of processes associated with hypertrophy and fibrosis. In addition, we discovered several novel ANG II targets, including characterized genes of known function, recently annotated genes of unknown function, and the putative genes not yet present in current databases. The serial analysis of gene expression approach to assess the role of ANG II presented in this report provides new venues for inquiries into ANG II-mediated cardiac function.

Mechanisms mediating the vasoactive effects of the B1 receptors of bradykinin.

Bradykinin normally exerts its vasodilatory effect via the B2 receptor (B2R), but in this receptor's absence, the B1 receptor becomes expressed and activated. To explore the mechanism of B1R-mediated vasodilation, 8 groups of B2R gene-knockout mice received a 2-week infusion of a B1R antagonist (300 microg x kg(-1) x d(-1)) or vehicle (groups 1 and 2), B1R antagonist or vehicle plus NO inhibition with Nomega-nitro-L-arginine methyl ester (groups 3 and 4), B1R antagonist or vehicle plus cyclooxygenase inhibition with indomethacin (groups 5 and 6), or B1R antagonist or vehicle plus blockade of vasoconstricting prostaglandin (PG) H2 and thromboxane A2 (TxA2) with SQ29548 (groups 7 and 8). The B1R antagonist produced significant (P<0.05) blood pressure increases of 17.7+/-3.1 mm Hg in group 1 and 10.4+/-3 mm Hg in group 3, whereas their vehicle-treated respective control groups 2 and 4 had no significant blood pressure changes. Indomethacin abolished the capacity of the B1R antagonist to raise blood pressure, as did blockade of the receptors of PGH2 and TxA2. Injection with the B1R agonist produced a hypotensive response (12+/-1.3 mm Hg), which was further accentuated by TxA2 blockade (21.7+/-4.1 mm Hg). Analysis of B1R gene expression by reverse transcription-polymerase chain reaction (PCR) in cardiac and renal tissues revealed marked expression at baseline, with further upregulation by 1.5- to 2-fold after various manipulations. Expression of the TxA2 receptor gene in renal tissue by quantitative real-time PCR was significantly lower in mice treated with the B1R antagonist, consistent with increased levels of agonist for this receptor. The data confirm that the B1R becomes markedly expressed in the absence of B2R and suggest that it contributes to vasodilation by inhibiting a vasoconstricting product of the arachidonic acid cascade acting via the PGH2/TxA2 receptor.

Serial analysis of gene expression in mouse kidney following angiotensin II administration.

As a new line of inquiry into the molecular mechanisms underlying pathophysiological processes associated with angiotensin (ANG II)-dependent hypertension, we applied the method of serial analysis of gene expression (SAGE) to examine genome-wide transcription changes in the kidneys of mice that developed hypertension in response to chronic ANG II administration. Mice were infused subcutaneously via osmotic minipumps with ANG II for 7 days, and systolic blood pressure was measured by tail-cuff plethysmography. Subsequently, mice were euthanized, and the total RNA isolated from the kidneys was used to construct SAGE libraries. Comparison of 11,447 SAGE tags from the hypertensive kidneys, representing 5,740 unique transcripts, and 11,273 tags from the control kidneys, corresponding to 5,619 different transcripts, identified genes that are significantly (P < 0.05) down- or upregulated in the hypertensive kidney. Our assessment of the genome-wide influence of ANG II resulted in the detection of several novel genes and in a recognition of potential new roles for the previously characterized genes, thus providing new probes with which to further explore the ANG II effects in normal and disease states.