Andrey C. da Costa Goncalves

Interaction Between P450 Eicosanoids and Nitric Oxide in the Control of Arterial Tone in Mice

Objective—Epoxyeicosatrienoic acids (EETs) serve as endothelial-derived hyperpolarizing factors (EDHF), but may also affect vascular function by other mechanisms. We identified a novel interaction between EETs and endothelial NO release using soluble epoxide hydrolase (sEH) −/− and +/+ mice. Methods and Results—EDHF responses to acetylcholine in pressurized isolated mesenteric arteries were neither affected by the sEH inhibitor, N-adamantyl-N′-dodecylurea (ADU), nor by sEH gene deletion. However, the EDHF responses were abolished by catalase and by apamin/charybdotoxin (ChTx), but not by iberiotoxin, nor by the cytochrome P450 inhibitor PPOH. All four EETs (order of potency: 8,9-EET >14,15-EET≈5,6-EET >11,12-EET) and all 4 dihydroxy derivatives (14,15-DHET≈8,9-DHET≈11,12-DHET >5,6-DHET) produced dose-dependent vasodilation. Endothelial removal or L-NAME blocked 8,9-EET and 14,15-DHET-dependent dilations. The effects of apamin/ChTx were minimal. 8,9-EET and 14,15-DHET induced NO production in endothelial cells. ADU (100 &mgr;g/mL in drinking water) lowered blood pressure in angiotensin II–infused hypertension, but not in L-NAME–induced hypertension. Blood pressure and EDHF responses were similar in L-NAME–treated sEH +/+ and −/− mice. Conclusions—Our data indicate that the EDHF response in mice is caused by hydrogen peroxide, but not by P450 eicosanoids. Moreover, P450 eicosanoids are vasodilatory, largely through their ability to activate endothelial NO synthase (eNOS) and NO release.

Diabetic Hypertensive Leptin Receptor–Deficient db/db Mice Develop Cardioregulatory Autonomic Dysfunction

Leptin receptor–deficient db/db mice develop human type 2 diabetes mellitus, hypertension, and obesity with disrupted circadian blood pressure (BP) rhythm. Whether leptin is the sole mechanism mediating autonomic imbalance and hypertension is unclear. To explore this notion further, we measured BP by radiotelemetry combined with fast Fourier transformation and assessed autonomic function pharmacologically before and after renin-angiotensin system blockade with enalapril. The resting period BP (117±3 versus 108±1.0 mm Hg) and heart rate (HR; 488±12 versus 436±8 bpm) were higher in db/db mice compared with db/+ mice. BP and HR amplitudes were lower in db/db mice compared with db/+ mice. BP response to trimetaphan (−43±5 versus −27±3 mm Hg) and HR response to metoprolol (−59±12 versus −5±4 bpm) were greater in db/db mice than in db/+ mice. The HR response to atropine was blunted in db/db mice (59±17 versus 144±24 bpm), as were baroreflex sensitivity and HR variability. Enalapril improved autonomic regulation in db/db mice. Stimulation of central &agr;-2 adrenoreceptors enhanced both parasympathetic HR control and baroreflex sensitivity in db/db mice. We suggest that functional, rather than structural, &agr;-2 adrenoceptor changes and the renin-angiotensin system are involved in the increased sympathetic and decreased parasympathetic tones in db/db mice. Our data suggest that db/db mice exhibit features found in humans with type 2 diabetic autonomic neuropathy and could serve as a model for this complication.

Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

Regulator of G protein signalling 2 ameliorates angiotensin II-induced hypertension in mice

Angiotensin II (Ang II) activates signalling pathways predominantly through the G‐protein‐coupled Ang II type 1 receptor (AT1R). The regulator of G protein signalling 2 (RGS2) is a negative G protein regulator. We hypothesized that RGS2 deletion changes blood pressure regulation by increasing the response to Ang II. To address this issue, we infused Ang II (0.5 mg kg−1 day−1) chronically into conscious RGS2‐deleted (RGS2−/−) and wild‐type (RGS2+/+) mice, measured mean arterial blood pressure and heart rate (HR) with telemetry and assessed vasoreactivity and gene expression of AT1A, AT1B and AT2 receptors. Angiotensin II infusion increased blood pressure more in RGS2−/− than in RGS2+/+ mice, while HR was not different between the groups, indicating a resetting of the baroreceptor reflex. Urinary catecholamine excretion was similar in Ang II‐infused RGS2−/− and RGS2+/+ mice, indicating a minor role of sympathetic tone for blood pressure differences. Myogenic tone and vasoreactivity in response to Ang II, endothelin‐1 and phenylephrine were increased in isolated renal interlobar arterioles of RGS2−/− mice compared with RGS2+/+ mice. The AT1A, AT1B and AT2 receptor gene expression was not different between RGS2−/− and RGS2+/+ mice. Our findings suggest that RGS2 deletion promotes Ang II‐dependent hypertension primarily through an increase of myogenic tone and vasoreactivity, probably by sensitization of AT1 receptors.

Estrogen Receptor-β Signals Left Ventricular Hypertrophy Sex Differences in Normotensive Deoxycorticosterone Acetate-Salt Mice

We found earlier that deoxycorticosterone acetate-salt treatment causes blood pressure–independent left ventricular hypertrophy, but only in male mice. To test the hypothesis that the estrogen receptor-&bgr; (ER&bgr;) protects the females from left ventricular hypertrophy, we treated male and female ER&bgr;-deficient (ER&bgr;−/−) mice and their male and female littermates (wild-type [WT]) with deoxycorticosterone acetate-salt and made them telemetrically normotensive with hydralazine. WT males had increased (+16%) heart weight/tibia length ratios compared with WT females (+7%) at 6 weeks. In ER&bgr;−/− mice, this situation was reversed. Female WT mice had the greatest heart weight/tibia length ratio increases of all of the groups (+23%), even greater than ER&bgr;−/− males (+10%). Echocardiography revealed concentric left ventricular hypertrophy in male WT mice, whereas ER&bgr;−/− females developed dilative left ventricular hypertrophy. The hypertrophic response in female ER&bgr;−/− mice was accompanied by the highest degree of collagen deposition, indicating maladaptive remodeling. ER&bgr;+/+ females showed robust protective p38 and extracellular signal–regulated kinase 1/2 signaling relationships compared with other groups. Calcineurin A&bgr; expression and its positive regulator myocyte-enriched calcineurin-interacting protein 1 were increased in deoxycorticosterone acetate-salt female ER&bgr;−/− mice, yet lower than in WT males. Endothelin increased murine cardiomyocyte hypertrophy in vitro, which could be blocked by estradiol and an ER&bgr; agonist. We conclude that a functional ER&bgr; is essential for inducing adaptive p38 and extracellular signal–regulated kinase signaling, while reducing maladaptive calcineurin signaling in normotensive deoxycorticosterone acetate female mice. Our findings address the possibility of sex-specific cardiovascular therapies.

Deoxycorticosterone Acetate-Salt Mice Exhibit Blood Pressure–Independent Sexual Dimorphism

We tested the hypothesis that female and male mice differ in terms of cardiac hypertrophy after deoxycorticosterone acetate (DOCA)+salt hypertension (uninephrectomy and 1% saline in drinking water) and focused on calcineurin signaling. We excluded confounding effects of blood pressure elevation or sex-related blood pressure differences by treating DOCA-salt mice with hydralazine (250 mg/L in drinking water). We found that directly measured mean arterial blood pressure was lowered to control values with hydralazine and corroborated this finding in separate mouse groups with radiotelemetry. Male mice were more responsive to DOCA-salt–related effects. They developed more left ventricular hypertrophy and more renal hypertrophy after 6 weeks of DOCA-salt+hydralazine compared with female mice. In hearts, transcripts for calcineurin A&bgr; and for myocyte-enriched calcineurin interacting protein 1 were upregulated in male but not in female mice. Enhanced activity of calcineurin A&bgr;, as indicated by diminished phosphorylation of NFATc2 in male mice, accounted for this sex-specific difference. Stretch-related, inflammatory, and profibrotic responses were also accentuated in male mice, as shown by higher transcript levels of atrial natriuretic peptide, monocyte chemoattractant protein-1, and transforming growth factor-&bgr;. Our results support sex-specific regulation of the calcineurin pathway in response to largely blood pressure–independent mineralocorticoid action. We suggest that sex-specific calcineurin activation determines the maladaptive cardiac and renal hypertrophic responses and accompanying organ injury in male mice.

Cardiovascular autonomic regulation in Non-Obese Diabetic (NOD) mice

Non-Obese Diabetic (NOD) mice show profound pathomorphological changes in sympathetic ganglia during the development of type 1 diabetes mellitus. We tested the hypothesis that NOD mice represent an experimental model to investigate cardiovascular changes seen in humans with diabetic autonomic neuropathy. Blood glucose (BG) levels were measured once a week. Diabetes mellitus was diagnosed as BG levels exceeded 250 mg/dl twice. NOD mice that did not become diabetic served as control group. Blood pressure (BP) and heart rate (HR) were monitored by telemetry and baroreflex sensitivity (BRS) was calculated with the sequence method or with cross spectral analysis. The measurements were obtained before onset of diabetes and during the 4th week of diabetes. The onset of diabetes was accompanied by a continuous decline in HR (615+/-14 vs. 498+/-23 bpm), whereas BP values remained stable (108+/-2 vs. 111+/-2 mm Hg). The circadian HR rhythm increased in diabetic NOD mice. BRS was higher in diabetic NOD mice than in controls. Atropine reduced BRS more profoundly in diabetic mice compared to non-diabetic mice. Despite pathomorphological similarities of the diabetic autonomic neuropathy between patients with diabetes and diabetic NOD mice, the changes in blood pressure regulation are different. In conclusion the use of diabetic NOD mice as a functional model for human diabetes may be questioned.

Leptin regulates ACE activity in mice

Leptin is a hormone related to metabolism. It also influences blood pressure, but the mechanisms triggered in this process are not yet elucidated. Angiotensin-I converting enzyme (ACE) regulates cardiovascular functions and recently has been associated with metabolism control and obesity. Here, we used ob/ob mice, a model lacking leptin, to answer the question whether ACE and leptin could interact to influence blood pressure, thereby linking the renin-angiotensin system and obesity. These mice are obese and diabetic but have normal 24 h mean arterial pressure. Our results show that plasma and lung ACE activities as well as ACE mRNA expression were significantly decreased in ob/ob mice. In agreement with these findings, the hypotensive effect produced by enalapril administration was attenuated in the obese mice. Plasma renin, angiotensinogen, angiotensin I, bradykinin, and angiotensin 1–7 were increased, whereas plasma angiotensin II concentration was unchanged in obese mice. Chronic infusion of leptin increased renin activity and angiotensin II concentration in both groups and increased ACE activity in ob/ob mice. Acute leptin infusion restored ACE activity in leptin-deficient mice. Moreover, the effect of an ACE inhibitor on blood pressure was not changed in ob/+ mice during leptin treatment but increased four times in obese mice. In summary, our findings show that the renin–angiotensin system is altered in ob/ob mice, with markedly reduced ACE activity, which suggests a possible connection between the renin–angiotensin system and leptin. These results point to an important interplay between the angiotensinergic and the leptinergic systems, which may play a role in the pathogenesis of obesity, hypertension, and metabolic syndrome.

Autonomic dysregulation in ob/ob mice is improved by inhibition of angiotensin-converting enzyme

The leptin-deficient ob/ob mice are insulin resistant and obese. However, the control of blood pressure in this model is not well defined. The goal of this study was to evaluate the role of leptin and of the renin-angiotensin system in the cardiovascular abnormalities observed in obesity using a model lacking leptin. To this purpose, we measured blood pressure in ob/ob and control animals by radiotelemetry combined with fast Fourier transformation before and after both leptin and enalapril treatment. Autonomic function was assessed pharmacologically. Blood pressure during daytime was slightly higher in the ob/ob compared to control mice, while no difference in heart rate was observed. Blood pressure response to trimetaphane and heart rate response to metoprolol were greater in ob/ob mice than in control littermates indicating an activated sympathetic nervous system. Heart rate response to atropine was attenuated. Baroreflex sensitivity and heart rate variability were blunted in ob/ob mice, while low frequency of systolic blood pressure variability was found increased. Chronic leptin replacement reduced blood pressure and reversed the impaired autonomic function observed in ob/ob mice. Inhibition of angiotensin-converting enzyme by enalapril treatment had similar effects, prior to the loss of weight. These findings suggest that the renin-angiotensin system is involved in the autonomic dysfunction caused by the lack of leptin in ob/ob mice and support a role of this interplay in the pathogenesis of obesity, hypertension, and metabolic syndrome.

Fine tuning of blood pressure by the regulator of G protein signaling (RGS) 2

G protein-coupled receptors (GPCRs) are expressed ubiquitously and involved in a variety of physiologic and pathologic processes. One of the key steps in the GPCR signaling cascade is the phosphorylation of the Galpha-subunit that triggers its dissociation from the Gbetagamma-subunit and from the receptor, allowing both G protein subunits to activate different downstream second messengers. Thereafter, Galpha hydrolyzes the attached guanosine triphosphate (GTP) to guanosine diphosphate (GDP) by its inherent enzymatic activity and terminates signaling. Small/connecting proteins that act as GTPase activating proteins (GAP) accelerate this process. Regulator of G protein signaling (RGS) proteins play a key role in the regulation of GPCRs, by acting as GAP and increasing the rate of GPCRs deactivation. RGS2 affects GPCR-dependent and GPCR-independent pathways. RGS2 -/- displayed an increase of blood pressure (BP) mainly by an increase of total peripheral resistance. After N(omega)-nitro-L-arginine methyl ester (L-NAME) RGS2 -/- mice responded with a smaller BP increase during the day than RGS2 +/+, suggesting an impaired NO signaling. Infusion of angiotensin II increased BP stronger in RGS2 -/- compared with RGS2 +/+. In summary, GPCR-dependent and GPCR-independent pathways are involved in BP changes of RGS2 -/- mice. Interactions between GPCRs and RGS2 represent a regulatory mechanism for fine-tuning of BP which may be important for hypertension and may be considered as a potentially novel drug target.