Role of ATP-dependent potassium channels in pulmonary vascular tone of fetal lambs with congenital diaphragmatic hernia.

High mortality in newborn babies with congenital diaphragmatic hernia (CDH) is principally due to persistent pulmonary hypertension. ATP-dependent potassium (K(ATP)) channels might modulate pulmonary vascular tone. We have assessed the effects of Pinacidil, a K(ATP) channel opener, and glibenclamide (GLI), a K(ATP) channel blocker, in near full-term lambs with and without CDH. In vivo, pulmonary hemodynamics were assessed by means of pressure and blood flow catheters. In vitro, we used isolated pulmonary vessels and immunohistochemistry to detect the presence of K(ATP) channels in pulmonary tissue. In vivo, pinacidil (2 mg) significantly reduced pulmonary vascular resistance (PVR) in both controls and CDH animals. GLI (30 mg) significantly increased pulmonary arterial pressure (PAP) and PVR in control animals only. In vitro, pinacidil (10 microM) relaxed, precontracted arteries from lambs with and without CDH. GLI (10(-5) microM) did not raise the basal tone of vessels. We conclude that activation of K(ATP) channels could be of interest to reduce pulmonary vascular tone in fetal lambs with CDH, a condition often associated with persistent pulmonary hypertension of the newborn.

Decreased blood pressure response in mice deficient of the alpha1b-adrenergic receptor.

To investigate the functional role of different alpha1-adrenergic receptor (alpha1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the alpha1b-AR (alpha1b-/-). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the alpha1-AR subtypes in various tissues of alpha1b +/+ and -/- mice. Total alpha1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the alpha1b -/- as compared with +/+ mice. Because of the large decrease of alpha1-AR in the heart and the loss of the alpha1b-AR mRNA in the aorta of the alpha1b-/- mice, the in vivo blood pressure and in vitro aorta contractile responses to alpha1-agonists were investigated in alpha1b +/+ and -/- mice. Our findings provide strong evidence that the alpha1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by alpha1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in alpha1b -/- as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in alpha1b-/- mice. The alpha1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different alpha1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice.

The bioavailability of nitric oxide (NO) within the vascular wall is limited by superoxide anions (O2.-). The relevance of extracellular superoxide dismutase (ecSOD) for the detoxification of vascular O2.- is unknown. We determined the involvement of ecSOD in the control of blood pressure and endothelium-dependent responses in angiotensin II-induced hypertension and renovascular hypertension induced by the two-kidney, one-clip model in wild-type mice and mice lacking the ecSOD gene. Blood pressure was identical in sham-operated ecSOD+/+ and ecSOD-/- mice. After 6 days of angiotensin II-treatment and 2 and 4 weeks after renal artery clipping, blood pressure was significantly higher in ecSOD-/- than ecSOD+/+ mice. Recombinant ecSOD selectively decreased blood pressure in hypertensive ecSOD-/- mice, whereas ecSOD had no effect in normotensive and hypertensive ecSOD+/+ mice. Compared with sham-operated ecSOD+/+ mice, sham-operated ecSOD-/- mice exhibited attenuated acetylcholine-induced relaxations. These responses were further depressed in vessels from clipped animals. Vascular O2.-, as measured by lucigenin chemiluminescence, was higher in ecSOD-/- compared with ecSOD+/+ mice and was increased by clipping. The antioxidant tiron normalized relaxations in vessels from sham-operated and clipped ecSOD-/-, as well as from clipped ecSOD+/+ mice. In contrast, in vivo application of ecSOD selectively enhanced endothelium-dependent relaxation in vessels from ecSOD-/- mice. These data reveal that endogenous ecSOD is a major antagonistic principle to vascular O2.-, controlling blood pressure and vascular function in angiotensin II-dependent models of hypertension. ecSOD is expressed in such an abundance that even in situations of high oxidative stress no relative lack of enzyme activity occurs.

Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes.

OBJECTIVE: Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. However, the role of PPARbeta/delta activators in cardiac hypertrophy is not yet known. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the selective PPARbeta/delta activator L-165041 (10 micromol/L) inhibited phenylephrine (PE)-induced protein synthesis ([(3)H]leucine uptake), induction of the fetal-type gene atrial natriuretic factor (ANF) and cardiac myocyte size. Induction of cardiac hypertrophy by PE stimulation also led to a reduction in the transcript levels of both muscle-type carnitine palmitoyltransferase (50%, P<0.05) and pyruvatedehydrogenase kinase 4 (30%, P<0.05), and these changes were reversed in the presence of the PPARbeta/delta agonist L-165041. Stimulation of neonatal rat cardiomyocytes with PE and embryonic rat heart-derived H9c2 cells with lipopolysaccharide (LPS) enhanced the expression of the nuclear factor (NF)-kappaB-target gene monocyte chemoattractant protein 1 (MCP-1). The induction of MCP-1 was reduced in the presence of L-165041, suggesting that this compound prevented NF-kappaB activation. Electrophoretic mobility shift assay (EMSA) revealed that L-165041 significantly decreased LPS-stimulated NF-kappaB binding activity in H9c2 myotubes. Finally, coimmunoprecipitation studies showed that L-165041 strongly enhanced the physical interaction between PPARbeta/delta and the p65 subunit of NF-kappaB, suggesting that increased association between these two proteins is the mechanism responsible for antagonizing NF-kappaB activation by PPARbeta/delta activators. CONCLUSION: These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.

Baroreflex and chemoreflex dysfunction in streptozotocin-diabetic rats

Several investigators have demonstrated that streptozotocin (STZ) diabetes induces changes in the autonomic control of the cardiovascular system. Changes in cardiovascular function may be related to peripheral neuropathy. The aim of the present study was to analyze changes in heart rate (HR) and arterial pressure (AP) as well as baroreflex and chemoreflex sensitivity in STZ-induced diabetic male Wistar rats (STZ, 50 mg/kg, iv, 15 days). Intra-arterial blood pressure signals were obtained for control and diabetic rats (N = 9, each group). Data were processed in a data acquisition system (CODAS, 1 kHz). Baroreflex sensitivity was evaluated by measuring heart rate changes induced by arterial pressure variation produced by phenylephrine and sodium nitroprusside injection. Increasing doses of potassium cyanide (KCN) were used to evaluate bradycardic and pressor responses evoked by chemoreflex activation. STZ induced hyperglycemia (447 ± 49 vs 126 ± 3 mg/dl), and a reduction in AP (99 ± 3 vs 118 ± 2 mmHg), resting HR (296 ± 11 vs 355 ± 16 bpm) and plasma insulin levels (16 ± 1 vs 57 ± 11 µU/ml). We also observed that the reflex bradycardia (-1.68 ± 0.1 vs -1.25 ± 0.1 bpm/mmHg, in the diabetic group) and tachycardia (-3.68 ± 0.5 vs -1.75 ± 0.3 bpm/mmHg, in the diabetic group) produced by vasopressor and depressor agents were impaired in the diabetic group. Bradycardia evoked by chemoreflex activation was attenuated in diabetic rats (control: -17 ± 1, -86 ± 19, -185 ± 18, -208 ± 17 vs diabetic: -7 ± 1, -23 ± 5, -95 ± 13, -140 ± 13 bpm), as also was the pressor response (control: 6 ± 1, 30 ± 7, 54 ± 4, 59 ± 5 vs diabetic: 6 ± 1, 8 ± 2, 33 ± 4, 42 ± 5 mmHg). In conclusion, the cardiovascular responses evoked by baroreflex and chemoreflex activation are impaired in diabetic rats. The alterations of cardiovascular responses may be secondary to the autonomic dysfunction of cardiovascular control

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the a-subunit of Na+,K+-ATPase inhibiting the Na+-pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension

Reactivity of the isolated perfused rat tail vascular bed

Isolated segments of the perfused rat tail artery display a high basal tone when compared to other isolated arteries such as the mesenteric and are suitable for the assay of vasopressor agents. However, the perfusion of this artery in the entire tail has not yet been used for functional studies. The main purpose of the present study was to identify some aspects of the vascular reactivity of the rat tail vascular bed and validate this method to measure vascular reactivity. The tail severed from the body was perfused with Krebs solution containing different Ca2+ concentrations at different flow rates. Rats were anesthetized with sodium pentobarbital (65 mg/kg) and heparinized (500 U). The tail artery was dissected near the tail insertion, cannulated and perfused with Krebs solution plus 30 µM EDTA at 36oC and 2.5 ml/min and the procedures were started after equilibration of the perfusion pressure. In the first group a dose-response curve to phenylephrine (PE) (0.5, 1, 2 and 5 µg, bolus injection) was obtained at different flow rates (1.5, 2.5 and 3.5 ml/min). The mean perfusion pressure increased with flow as well as PE vasopressor responses. In a second group the flow was changed (1.5, 2, 2.5, 3 and 3.5 ml/min) at different Ca2+ concentrations (0.62, 1.25, 2.5 and 3.75 mM) in the Krebs solution. Increasing Ca2+ concentrations did not alter the flow-pressure relationship. In the third group a similar protocol was performed but the rat tail vascular bed was perfused with Krebs solution containing PE (0.1 µg/ml). There was an enhancement of the effect of PE with increasing external Ca2+ and flow. PE vasopressor responses increased after endothelial damage with air and CHAPS, suggesting an endothelial modulation of the tone of the rat tail vascular bed. These experiments validate the perfusion of the rat tail vascular bed as a method to investigate vascular reactivity

The left ventricular contractility of the rat heart is modulated by changes in flow and a1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on a1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo a1-adrenergic stimulation increases left ventricular-developed pressure (P<0.05) together with arterial blood pressure (P<0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P<0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P<0.01). The largest ISP increase was obtained with PE + NP treatment (P<0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial a1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.

Comparison of three methods for the determination of baroreflex sensitivity in conscious rats

Baroreflex sensitivity was studied in the same group of conscious rats using vasoactive drugs (phenylephrine and sodium nitroprusside) administered by three different approaches: 1) bolus injection, 2) steady-state (blood pressure (BP) changes produced in steps), 3) ramp infusion (30 s, brief infusion). The heart rate (HR) responses were evaluated by the mean index (mean ratio of all HR changes and mean arterial pressure (MAP) changes), by linear regression and by the logistic method (maximum gain of the sigmoid curve by a logistic function). The experiments were performed on three consecutive days. Basal MAP and resting HR were similar on all days of the study. Bradycardic responses evaluated by the mean index (-1.5 ± 0.2, -2.1 ± 0.2 and -1.6 ± 0.2 bpm/mmHg) and linear regression (-1.8 ± 0.3, -1.4 ± 0.3 and -1.7 ± 0.2 bpm/mmHg) were similar for all three approaches used to change blood pressure. The tachycardic responses to decreases of MAP were similar when evaluated by linear regression (-3.9 ± 0.8, -2.1 ± 0.7 and -3.8 ± 0.4 bpm/mmHg). However, the tachycardic mean index (-3.1 ± 0.4, -6.6 ± 1 and -3.6 ± 0.5 bpm/mmHg) was higher when assessed by the steady-state method. The average gain evaluated by logistic function (-3.5 ± 0.6, -7.6 ± 1.3 and -3.8 ± 0.4 bpm/mmHg) was similar to the reflex tachycardic values, but different from the bradycardic values. Since different ways to change BP may alter the afferent baroreceptor function, the MAP changes obtained during short periods of time (up to 30 s: bolus and ramp infusion) are more appropriate to prevent the acute resetting. Assessment of the baroreflex sensitivity by mean index and linear regression permits a separate analysis of gain for reflex bradycardia and reflex tachycardia. Although two values of baroreflex sensitivity cannot be evaluated by a single symmetric logistic function, this method has the advantage of better comparing the baroreflex sensitivity of animals with different basal blood pressures.

Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal (»75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM), prostaglandin F2a (PGF2a; 1 µM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings) to 57 ± 7% (N = 26 rings) and 34.9 ± 6% (N = 13 rings), respectively (P<0.05), and for PGF2a from a control response of 75 ± 10% (N = 16 rings) to 52 ± 8% (N = 19 rings) and 25.9 ± 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings), 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings), and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.

Time sequence of changes in the responsiveness of glycogen breakdown to adrenergic agonists in perfused liver of rats with insulin-induced hypoglycemia

The time-course changes of the responsiveness of glycogen breakdown to a- and ß-adrenergic agonists during insulin-induced hypoglycemia (IIH) were investigated. Blood glucose levels were decreased prior to the alteration in the hepatic responsiveness to adrenergic agonists. The activation of hepatic glucose production and glycogenolysis by phenylephrine (2 µM) and isoproterenol (20 µM) was decreased in IIH. The changes in the responsiveness of glycogen catabolism were first observed for isoproterenol and later for phenylephrine. Hepatic ß-adrenergic receptors showed a higher degree of adrenergic desensitization than did a-receptors. Liver glycogen synthase activity, glycogen content and the catabolic effect of dibutyryl cyclic AMP (the ß-receptor second messenger) were not affected by IIH.

Influence of a multideficient diet from northeastern Brazil on resting blood pressure and baroreflex sensitivity in conscious, freely moving rats

The "regional basic diet" or RBD is a multideficient diet (providing 8% protein) which is known to produce dietary deficiencies in some populations in northeastern Brazil. The present study investigated the effects of RBD-induced malnutrition on resting blood pressure and baroreflex sensitivity in conscious rats. Malnourished rats were obtained by feeding dams the RBD during mating and pregnancy (RBD-1 group) or during nursing and a 10-day period after weaning (RBD-2 group). At 90 days of age, only RBD-2 rats weighed significantly (P<0.001) less than control rats born to dams fed a standard commercial diet (23% protein) during pregnancy and nursing. Baseline mean arterial pressure and heart rate of both RBD-1 and RBD-2 rats were comparable to those of controls. The slopes for both reflex bradycardia and tachycardia (bpm/mmHg) induced by intravenous phenylephrine and sodium nitroprusside, respectively, were unchanged in either RBD-1 (-2.08 ± 0.11 and -3.10 ± 0.43, respectively) or RBD-2 (-2.32 ± 0.30 and -3.73 ± 0.53, respectively) rats, when compared to controls (-2.09 ± 0.10 and -3.17 ± 0.33, respectively). This study shows that, after a prolonged period of nutritional recovery, the patterns of resting blood pressure and baroreflex sensitivity of both pre- and postnatally malnourished rats were similar to those of controls. The decreased body weight and the tendency to increased reflex tachycardia in RBD-2 rats may suggest that this type of maternal malnutrition during lactation is more critical than during pregnancy.

Effects of small doses of ouabain on the arterial blood pressure of anesthetized hypertensive and normotensive rats

Ouabain increases vascular resistance and may induce hypertension by inhibiting the Na+ pump. The effects of 0.18 and 18 µg/kg, and 1.8 mg/kg ouabain pretreatment on the phenylephrine (PHE; 0.1, 0.25 and 0.5 µg, in bolus)-evoked pressor responses were investigated using anesthetized normotensive (control and uninephrectomized) and hypertensive (1K1C and DOCA-salt treated) rats. Treatment with 18 µg/kg ouabain increased systolic and diastolic blood pressure in all groups studied. However, the magnitude of this increase was larger for the hypertensive 1K1C and DOCA-salt rats than for normotensive animals, while the pressor effect of 0.18 µg/kg ouabain was greater only in DOCA-salt rats. A very large dose (1.8 mg/kg) produced toxic effects on the normotensive control but not on uninephrectomized or 1K1C rats. Rat tail vascular beds were perfused to analyze the effects of 10 nM ouabain on the pressor response to PHE. In all animals, 10 nM ouabain increased the PHE pressor response, but this increase was larger in hypertensive DOCA-salt rats than in normotensive and 1K1C rats. Results suggested that a) increases in diastolic blood pressure induced by 18 µg/kg ouabain were larger in hypertensive than normotensive rats; b) in DOCA-salt rats, smaller ouabain doses had a stronger effect than in other groups; c) hypertensive and uninephrectomized rats were less sensitive to toxic doses of ouabain, and d) after treatment with 10 nM ouabain isolated tail vascular beds from DOCA-salt rats were more sensitive to the pressor effect of PHE than those from normotensive and 1K1C hypertensive rats. These data suggest that very small doses of ouabain, which might produce nanomolar plasma concentrations, enhance pressor reactivity in DOCA-salt hypertensive rats, supporting the idea that endogenous ouabain may contribute to the increase and maintenance of vascular tone in hypertension.

Reflex control of arterial pressure and heart rate in short-term streptozotocin diabetic rats

Impaired baroreflex sensitivity in diabetes is well described and has been attributed to autonomic diabetic neuropathy. In the present study conducted on acute (10-20 days) streptozotocin (STZ)-induced diabetic rats we examined: 1) cardiac baroreflex sensitivity, assessed by the slope of the linear regression between phenylephrine- or sodium nitroprusside-induced changes in arterial pressure and reflex changes in heart rate (HR) in conscious rats; 2) aortic baroreceptor function by means of the relationship between systolic arterial pressure and aortic depressor nerve (ADN) activity, in anesthetized rats, and 3) bradycardia produced by electrical stimulation of the vagus nerve or by the iv injection of methacholine in anesthetized animals. Reflex bradycardia (-1.4 ± 0.1 vs -1.7 ± 0.1 bpm/mmHg) and tachycardia (-2.1 ± 0.3 vs -3.0 ± 0.2 bpm/mmHg) were reduced in the diabetic group. The gain of the ADN activity relationship was similar in control (1.7 ± 0.1% max/mmHg) and diabetic (1.5 ± 0.1% max/mmHg) animals. The HR response to vagal nerve stimulation with 16, 32 and 64 Hz was 13, 16 and 14% higher, respectively, than the response of STZ-treated rats. The HR response to increasing doses of methacholine was also higher in the diabetic group compared to control animals. Our results confirm the baroreflex dysfunction detected in previous studies on short-term diabetic rats. Moreover, the normal baroreceptor function and the altered HR responses to vagal stimulation or methacholine injection suggest that the efferent limb of the baroreflex is mainly responsible for baroreflex dysfunction in this model of diabetes.

Effects of losartan combined with exercise training in spontaneously hypertensive rats

We investigate whether combined treatment with losartan, an angiotensin II receptor blocker, and exercise training (ET) in spontaneously hypertensive rats (SHR) would have an additive effect in reducing hypertension and improving baroreflex sensitivity when compared with losartan alone. Male SHR (8 weeks old) were assigned to 3 groups: sedentary placebo (SP, N = 16), sedentary under losartan treatment (SL, N = 11; 10 mg kg-1 day-1, by gavage), and ET under losartan treatment (TL, N = 10). ET was performed on a treadmill 5 days/week for 60 min at 50% of peak VO2, for 18 weeks. Blood pressure (BP) was measured with a catheter inserted into the carotid artery, and cardiac output with a microprobe placed around the ascending aorta. The baroreflex control of heart rate was assessed by administering increasing doses of phenylephrine and sodium nitroprusside (iv). Losartan significantly reduced mean BP (178 ± 16 vs 132 ± 12 mmHg) and left ventricular hypertrophy (2.9 ± 0.4 vs 2.5 ± 0.2 mg/g), and significantly increased baroreflex bradycardia and tachycardia sensitivity (1.0 ± 0.3 vs 1.7 ± 0.5 and 2.0 ± 0.7 vs 3.2 ± 1.7 bpm/mmHg, respectively) in SL compared with SP. However, losartan combined with ET had no additional effect on BP, baroreflex sensitivity or left ventricular hypertrophy when compared with losartan alone. In conclusion, losartan attenuates hypertension and improves baroreflex sensitivity in SHR. However, ET has no synergistic effect on BP in established hypertension when combined with losartan, at least at the dosage used in this investigation.