Pressor recovery after acute stress is impaired in high fructose-fed Lean Zucker rats.

Insulin resistance is a powerful predictor of cardiovascular disease; however, the mechanistic link remains unclear. This study aims to determine if early cardiovascular changes associated with short-term fructose feeding in the absence of obesity manifest as abnormal blood pressure control. Metabolic dysfunction was induced in Lean Zucker rats by short-term high-fructose feeding. Rats were implanted with telemetry devices for the measurement of mean arterial blood pressure (MAP) and subjected to air jet stress at 5 and 8 weeks after feeding. Additional animals were catheterized under anesthesia for the determination of MAP and blood flow responses in the hind limb and mesenteric vascular beds to intravenous injection of isoproterenol (0.001-0.5 µm), a ß-adrenergic agonist. Metabolic dysfunction in high-fructose rats was not accompanied by changes in 24-h MAP Yet, animals fed a high-fructose diet for 8 weeks exhibited a marked impairment in blood pressure recovery after air-jet stress. Dose-dependent decreases in MAP and peripheral blood flow in response to isoproterenol treatment were significantly attenuated in high-fructose rats. These data suggest that impaired blood pressure recovery to acute mental stress precedes the onset of hypertension in the early stages of insulin resistance. Further, blunted responses to isoproterenol implicate ß2-adrenergic sensitivity as a possible mechanism responsible for altered blood pressure control after short-term high-fructose feeding.

High salt diet increases the pressor response to stress in female, but not male ETB-receptor-deficient rats.

Acute stress in both rodents and humans causes a transient rise in blood pressure associated with an increase in plasma endothelin-1 (ET-1). High salt (HS) intake also increases ET-1 production, and interestingly, blunts the pressor response to acute air jet stress in rats. We previously reported that female rats lacking functional ETB receptors everywhere except sympathetic nerves (ETB def) had a greater degree of hypertension in response to a HS diet compared to their male counterparts when measured by the tail cuff method. However, we now report that salt-induced hypertension is not different between sexes when measured by telemetry. Therefore, additional experiments were designed to test the hypothesis that female ETB def rats are more sensitive to acute stress when on a HS diet. The pressor response, measured by telemetry, to acute air jet stress was similar between male transgenic control (Tg control) and ETB def rats following chronic HS intake. In contrast, female ETB def rats had a significantly greater pressor response (about twofold higher) than female or male Tg control or male ETB def rats maintained on HS, a finding that cannot be explained by increased vascular reactivity to ET-1 in female rats as we observed that male ETB def rats had a greater pressor response to i.v. infusion of ET-1 compared to females. Furthermore, HS feeding exacerbated the pressor response to ET-1 in both male and female ETB def rats. Given our previous studies demonstrating that the ETA receptor functions to reduce the pressor response to acute stress, these findings further support a role for the ET receptor system in the pressor response to acute stress and that female rats have reduced ETA receptor activity when on a HS diet compared to males.

Discovery of (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid and congeners as highly potent inhibitors of human arginases I and II for treatment of myocardial reperfusion injury.

Recent efforts to identify treatments for myocardial ischemia reperfusion injury have resulted in the discovery of a novel series of highly potent α,α-disubstituted amino acid-based arginase inhibitors. The lead candidate, (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, compound 9, inhibits human arginases I and II with IC50s of 223 and 509 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). It is 28% orally bioavailable and significantly reduces the infarct size in a rat model of myocardial ischemia/reperfusion injury. Herein, we report the design, synthesis, and structure-activity relationships (SAR) for this novel series of inhibitors along with pharmacokinetic and in vivo efficacy data for compound 9 and X-ray crystallography data for selected lead compounds cocrystallized with arginases I and II.

Endothelin activation of reactive oxygen species mediates stress-induced pressor response in Dahl salt-sensitive prehypertensive rats.

Experiments were designed to test the hypothesis that endothelin (ET) and/or reactive oxygen species contribute to the pressor response induced by acute air jet stress in normotensive Dahl salt-sensitive rats maintained on a normal salt diet (prehypertensive). Mean arterial pressure was chronically monitored by telemetry before and after 3-day treatment with the free radical scavenger 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (Tempol) or ET receptor antagonists ABT-627 (ET A antagonist) or A-182086 (ET A/B antagonist) supplied in the drinking water. Rats were restrained and subjected to pulsatile air jet stress (3 minutes). Plasma samples at baseline and during acute stress were analyzed for 8-isoprostane (measure of reactive oxygen species production) and ET. Neither Tempol nor ET receptor antagonist treatment had an effect on baseline mean arterial pressure or plasma 8-isoprostane. The pressor response to acute stress was accompanied by significant increases in plasma 8-isoprostane and ET. Tempol significantly reduced both the total pressor response (area under the curve) and the stress-mediated increase in plasma 8-isoprostane; conversely, Tempol had no effect on the stress-induced increase in plasma ET. Combined ET(A/B) antagonism, but not selective ET(A) receptor blockade, similarly suppressed the pressor response to stress and stress-mediated rise in 8-isoprostane. Together these results indicate that reactive oxygen species contribute to the pressor response to acute air jet stress. Furthermore, the increase in reactive oxygen species occurs downstream of ET(B) receptor activation.

Early life stress downregulates endothelin receptor expression and enhances acute stress-mediated blood pressure responses in adult rats.

We hypothesized that early life stress enhances endothelin (ET-1)-dependent acute stress responses in adulthood. We utilized a unique rat model, wild-type (WT) and ET(B) receptor-deficient spotting lethal (sl/sl) rats, as well as pharmacological blockade of ET receptors, in a model of early life stress, maternal separation (MS). MS was performed in male WT and sl/sl rats 3 h/day from day 2 to 14 of life. Acute air jet stress (AJS)-induced responses (elevation in blood pressure, plasma corticosterone, and plasma ET-1) were evaluated in adult MS rats compared with the nonhandled littermate (control) rats. MS significantly augmented the acute AJS-induced blood pressure response (area under the curve) in WT rats compared with control, while the AJS-induced pressor responses were similar in sl/sl MS and control rats. ET receptor blockade significantly blunted the AJS-induced pressor response in WT MS and control rats. Moreover, AJS-induced plasma corticosterone levels in control rats were sensitive to ET receptor blockade, yet, AJS did not alter plasma corticosterone levels in MS rats. MS significantly increased circulating ET-1 levels, and AJS-induced plasma ET-1 levels were similarly increased in control and MS rats. MS induced a significant downregulation in expression of ET(A) and ET(B) receptors in aortic tissue compared with control rats. These results indicate that early life stress reduced expression of ET(A) and ET(B) receptors, leading to alterations in the ET pathway, and an exaggerated acute stress-mediated pressor response in adulthood.

Novel use of ultrasound to examine regional blood flow in the mouse kidney.

Conventional methods used for measuring regional renal blood flow, such as laser-Doppler flowmetry, are highly invasive, and each measurement is restricted to a discrete location. The aim of this study was to determine whether ultrasound imaging in conjunction with enhanced contrast agent (microbubbles; Vevo MicroMarker, VisualSonics) could provide a viable noninvasive alternative. This was achieved by determining changes in renal cortical and medullary rate of perfusion in response to a bolus injection of endothelin-1 (ET-1; 0.6, 1.0, or 2.0 nmol/kg) and comparing these responses to those observed in separate groups of mice with conventional laser-Doppler methods. Intravenous infusion of ET-1 in anesthetized male C57bl/6 mice resulted in a dose-dependent increase in mean arterial pressure and a dose-dependent decrease in total renal blood flow as measured by pulse-wave Doppler. ET-1 infusion resulted in a dose-dependent decrease in regional kidney perfusion as measured by both ultrasound with enhanced contrast agent and laser-Doppler measurements, verifying the use of ultrasound to measure regional kidney perfusion. Noted limitations of ultrasound imaging compared with laser-Doppler flowmetry included a lower degree of sensitivity to changes in tissue perfusion and the inability to assess rapid or transient changes in tissue perfusion. In conclusion, ultrasound represents an effective and noninvasive method for the measurement of relatively short-term, steady-state changes in regional blood flow in the mouse kidney.

Exaggerated cardiovascular stress responses and impaired beta-adrenergic-mediated pressor recovery in obese Zucker rats.

Clinical studies have demonstrated that the pressor response to acute stress is larger in obese versus lean individuals. We therefore tested the hypotheses that the pressor response to behavioral stress is greater in obese (OZRs) versus lean Zucker rats (LZRs) and that reduced beta-adrenergic-mediated vasodilation contributes to the enhanced pressor response. Animals were restrained and subjected to acute pulsatile air jet stress (3 minutes), followed by a poststress period of 20 minutes; beta-adrenergic blockade was achieved with propranolol (5 mg/kg, IV) given 15 minutes before the start of air jet stress. Mean arterial pressure (MAP) was continuously monitored by telemetry. Untreated OZRs responded with a greater integrated pressor response (area under the curve [AUC]) to acute stress (41.2+/-6.1 versus 21.2+/-3.3 mm Hgx3 minutes, OZR versus LZR; P<0.05) and significantly reduced poststress recovery of MAP. Beta-adrenergic blockade had no effect on stress AUC in either LZRs or OZRs but significantly attenuated the poststress recovery of MAP in LZRs only (poststress AUC: -100.1+/-48.1 versus 49.0+/-13.5 mm Hgx20 minutes, untreated versus propranolol; P<0.05). In anesthetized animals, significantly smaller increases in mesenteric vascular conductance contributed to blunted depressor responses to isoproterenol in OZRs versus LZRs, suggesting that beta-adrenergic stimulation causes a greater reduction in total peripheral resistance in lean versus obese animals. We conclude that beta-adrenergic-mediated vasodilation facilitates blood pressure recovery after stress and that this pathway is compromised in an animal model of morbid obesity, resulting in the impaired ability to regulate blood pressure during stress.

In vivo evidence for endothelin-1-mediated attenuation of alpha1-adrenergic stimulation.

Experiments were designed to determine the influence of endothelin A (ET(A)) receptors on the pressor response to acute environmental stress in Dahl salt-resistant (DR) and Dahl-sensitive (DS) rats. Mean arterial pressure (MAP) was chronically monitored by telemetry before and after treatment with the selective ET(A) receptor antagonist ABT-627. Rats were restrained and subjected to pulsatile air jet stress (3 min). In untreated animals, the total pressor response (area under the curve) to acute stress was not different between DR vs. DS rats (8.1 +/- 1.7 vs. 15.6 +/- 2.6 mmHg x 3 min, P = 0.10). Conversely, treatment with ABT-627 potentiated the total pressor response only in DR rats (36.3 +/- 6.2 vs. 22.6 +/- 5.9 mmHg x 3 min, DR vs. DS, P < 0.05). Treatment with ABT-627 allowed greater responses in anesthetized DR rats to exogenous phenylephrine (1-4 microg/kg) during ganglionic blockade (P < 0.05) and produced a significant increase in plasma norepinephrine at baseline and during stress in conscious DR rats compared with untreated animals (P < 0.05). ET(A) receptor blockade had no effect on these responses in DS rats. Our results suggest that endothelin-1 can inhibit alpha-adrenergic-mediated effects in DR, but not DS rats, consistent with the hypothesis that ET(A) receptor activation functions to reduce sympathetic nerve activity and responses in vascular smooth muscle to sympathetic stimulation.

Fructose feeding increases insulin resistance but not blood pressure in Sprague-Dawley rats.

Fructose feeding has been widely reported to cause hypertension in rats, as assessed indirectly by tail cuff plethysmography. Because there are potentially significant drawbacks associated with plethysmography, we determined whether blood pressure changes could be detected by long-term monitoring with telemetry in age-matched male Sprague-Dawley rats fed either a normal or high-fructose diet for 8 weeks. Fasting plasma glucose (171+/-10 versus 120+/-10 mg/dL), plasma insulin (1.8+/-0.5 versus 0.7+/-0.1 microg/L), and plasma triglycerides (39+/-2 versus 30+/-2 mg/dL) were modestly but significantly elevated in fructose-fed animals. Using the hyperinsulinemic euglycemic clamp technique, the rate of glucose infusion necessary to maintain equivalent plasma glucose was significantly reduced in fructose-fed compared with control animals (22.9+/-3.6 versus 41.5+/-2.9 mg/kg per minute; P<0.05). However, mean arterial pressure (24-hour) did not change in the fructose-fed animals over the 8-week period (111+/-1 versus 114+/-2 mm Hg; week 0 versus 8), nor was it different from that in control animals (109+/-2 mm Hg). Conversely, systolic blood pressure measured by tail cuff plethysmography at the end of the 8-week period was significantly greater in fructose-fed versus control animals (162+/-5 versus 139+/-1 mm Hg; P<0.001). Together, these data demonstrate that long-term fructose feeding induces mild insulin resistance but does not elevate blood pressure. We propose that previous reports of fructose-induced hypertension reflect a heightened stress response by fructose-fed rats associated with restraint and tail cuff inflation.

Endogenous endothelin attenuates the pressor response to acute environmental stress via the ETA receptor.

Clinical studies have documented an abrupt rise in plasma endothelin-1 (ET-1) coincident with an increase in mean arterial pressure (MAP) during the response to acute stress. We therefore examined the ET(A) and ET(B) receptor-dependent effects of ET-1 on the pressor response to acute environmental stress in ET-1-dependent hypertension. Stress was induced by administration of air jet pulses (3 min) in ET(B) receptor-deficient (ET(B) sl/sl) rats fed normal salt (NS; 0.8% NaCl), high salt (HS; 8% NaCl), and HS plus the ET(A) receptor antagonist ABT-627 (5 mg.kg(-1).day(-1)) on successive weeks. MAP was chronically monitored by telemetry. Total pressor response (area under the curve) was significantly reduced in ET(B) sl/sl rats maintained on a HS vs. NS diet [-6.8 mmHg (SD 18.7) vs. 29.3 mmHg (SD 8.1) x 3 min, P < 0.05]. Conversely, the total pressor response was augmented in both wild-type [34.2 mmHg (SD 29.2) x 3 min, P < 0.05 vs. NS] and ET(B) sl/sl rats [49.1 mmHg (SD 11.8) x 3 min, P < 0.05 vs. NS] by ABT-627. Blockade of ET(B) receptors in Sprague-Dawley rats caused an increase in basal MAP that was enhanced by HS and lowered by mixed ET(A)/ET(B) receptor antagonism; none of these treatments, however, had any effect on the pressor response. These data demonstrate that increasing endogenous ET-1 suppresses the pressor response to acute stress through ET(A) receptor activation in a genetic model of ET-1-dependent hypertension. These results are consistent with reports that ET-1 can attenuate sympathetically mediated responses.

Inhibition of ERK attenuates force development by lowering myosin light chain phosphorylation.

Phosphorylation of the actin-associated protein caldesmon (CaD) by extracellular signal-regulated kinases (ERK1/2) is purported to participate in force maintenance by vascular smooth muscle. We examined the interrelationship among ERK1/2 activity, phosphorylation of the high molecular weight isoform of CaD (h-CaD) and the 20-kDa myosin light chain (LC(20)), and isometric force in strips of porcine carotid artery stimulated with endothelin-1 (ET-1; 50 nM). After an initial delay, ERK1/2 activity increased in parallel with ET-1-mediated force; h-CaD phosphorylation increased modestly. 2-(2'-Amino-3'-methoxyphenyl)-ox-anaphthalen-4-one (PD-098059; 50 microM), an ERK1/2 kinase inhibitor, significantly reduced basal ERK1/2 activity within 1 h, but only partially attenuated h-CaD phosphorylation at 3 h. The mechanisms underlying the temporal dissociation of ERK1/2 activity from h-CaD phosphorylation are unknown, but include the possibility that a kinase other than ERK1/2 phosphorylates h-CaD or, more likely, that phosphate turnover in h-CaD is very slow. PD-098059 partially inhibited the development of ET-1-stimulated force only in Ca(2+)-replete physiological saline solution, primarily by reducing LC(20) phosphorylation, yet had no effect on myosin light chain kinase in vitro. These inhibitory effects were most evident during the early phase of force production. The inhibitory effect of PD-098059 on force could not be correlated with a corresponding effect on ERK1/2-mediated h-CaD phosphorylation because force in arterial strips stimulated with ET-1 in the absence or presence of PD-098059 tended to approximate each other over time despite significant differences in the level of h-CaD phosphorylation. Force and LC(20) phosphorylation in response to KCl depolarization were unaffected by PD-098059. These results show that ERK1/2 may regulate force in arterial smooth muscle, but suggest that the mechanism for this effect is by inhibiting LC(20) phosphorylation.