A low concentration of ouabain (0.18 µg/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 μg/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 ± 5.1 to 150 ± 4.7; DAP: 93.7 ± 7.7 to 116 ± 3.5 mmHg; P < 0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

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.

Acute lead-induced vasoconstriction in the vascular beds of isolated perfused rat tails is endothelium-dependent

Chronic lead exposure induces hypertension in humans and animals, affecting endothelial function. However, studies concerning acute cardiovascular effects are lacking. We investigated the effects of acute administration of a high concentration of lead acetate (100 µΜ) on the pressor response to phenylephrine (PHE) in the tail vascular bed of male Wistar rats. Animals were anesthetized with sodium pentobarbital and heparinized. The tail artery was dissected and cannulated for drug infusion and mean perfusion pressure measurements. Endothelium and vascular smooth muscle relaxation were tested with acetylcholine (5 µg/100 µL) and sodium nitroprusside (0.1 µg/100 µL), respectively, in arteries precontracted with 0.1 µM PHE. Concentration-response curves to PHE (0.001-300 µg/100 µL) were constructed before and after perfusion for 1 h with 100 µΜ lead acetate. In the presence of endothelium (E+), lead acetate increased maximal response (Emax) (control: 364.4 ± 36, Pb2+: 480.0 ± 27 mmHg; P < 0.05) and the sensitivity (pD2; control: 1.98 ± 0.07, 2.38 ± 0.14 log mM) to PHE. In the absence of endothelium (E-) lead had no effect but increased baseline perfusion pressure (E+: 79.5 ± 2.4, E-: 118 ± 2.2 mmHg; P < 0.05). To investigate the underlying mechanisms, this protocol was repeated after treatment with 100 µM L-NAME, 10 µM indomethacin and 1 µM tempol in the presence of lead. Lead actions on Emax and pD2 were abolished in the presence of indomethacin, and partially abolished with L-NAME and tempol. Results suggest that acute lead administration affects the endothelium, releasing cyclooxygenase-derived vasoconstrictors and involving reactive oxygen species.

Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

Lead (Pb2+) poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g) before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM) to the bath. Changes in rate of stimulation (0.1-1.5 Hz), relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM), and the effect of isoproterenol (20 ng/mL) were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz) and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

Eucalyptol, an essential oil, reduces contractile activity in rat cardiac muscle

Eucalyptol is an essential oil that relaxes bronchial and vascular smooth muscle although its direct actions on isolated myocardium have not been reported. We investigated a putative negative inotropic effect of the oil on left ventricular papillary muscles from male Wistar rats weighing 250 to 300 g, as well as its effects on isometric force, rate of force development, time parameters, post-rest potentiation, positive inotropic interventions produced by Ca2+ and isoproterenol, and on tetanic tension. The effects of 0.3 mM eucalyptol on myosin ATPase activity were also investigated. Eucalyptol (0.003 to 0.3 mM) reduced isometric tension, the rate of force development and time parameters. The oil reduced the force developed by steady-state contractions (50% at 0.3 mM) but did not alter sarcoplasmic reticulum function or post-rest contractions and produced a progressive increase in relative potentiation. Increased extracellular Ca2+ concentration (0.62 to 5 mM) and isoproterenol (20 nM) administration counteracted the negative inotropic effects of the oil. The activity of the contractile machinery evaluated by tetanic force development was reduced by 30 to 50% but myosin ATPase activity was not affected by eucalyptol (0.3 mM), supporting the idea of a reduction of sarcolemmal Ca2+ influx. The present results suggest that eucalyptol depresses force development, probably acting as a calcium channel blocker.

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.

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.

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.

Ambulatory blood pressure and Doppler echocardiographic indexes of borderline hypertensive men presenting an exaggerated blood pressure response during dynamic exercise

Borderline hypertension (BH) has been associated with an exaggerated blood pressure (BP) response during laboratory stressors. However, the incidence of target organ damage in this condition and its relation to BP hyperreactivity is an unsettled issue. Thus, we assessed the Doppler echocardiographic profile of a group of BH men (N = 36) according to office BP measurements with exaggerated BP in the cycloergometric test. A group of normotensive men (NT, N = 36) with a normal BP response during the cycloergometric test was used as control. To assess vascular function and reactivity, all subjects were submitted to the cold pressor test. Before Doppler echocardiography, the BP profile of all subjects was evaluated by 24-h ambulatory BP monitoring. All subjects from the NT group presented normal monitored levels of BP. In contrast, 19 subjects from the original BH group presented normal monitored BP levels and 17 presented elevated monitored BP levels. In the NT group all Doppler echocardiographic indexes were normal. All subjects from the original BH group presented normal left ventricular mass and geometrical pattern. However, in the subjects with elevated monitored BP levels, fractional shortening was greater, isovolumetric relaxation time longer, and early to late flow velocity ratio was reduced in relation to subjects from the original BH group with normal monitored BP levels (P<0.05). These subjects also presented an exaggerated BP response during the cold pressor test. These results support the notion of an integrated pattern of cardiac and vascular adaptation during the development of hypertension.

Diverse effects of renal denervation on ventricular hypertrophy and blood pressure in DOCA-salt hypertensive rats

Cardiac hypertrophy that accompanies hypertension seems to be a phenomenon of multifactorial origin whose development does not seem to depend on an increased pressure load alone, but also on local growth factors and cardioadrenergic activity. The aim of the present study was to determine if sympathetic renal denervation and its effects on arterial pressure level can prevent cardiac hypertrophy and if it can also delay the onset and attenuate the severity of deoxycorticosterone acetate (DOCA)-salt hypertension. DOCA-salt treatment was initiated in rats seven days after uninephrectomy and contralateral renal denervation or sham renal denervation. DOCA (15 mg/kg, sc) or vehicle (soybean oil, 0.25 ml per animal) was administered twice a week for two weeks. Rats treated with DOCA or vehicle (control) were provided drinking water containing 1% NaCl and 0.03% KCl. At the end of the treatment period, mean arterial pressure (MAP) and heart rate measurements were made in conscious animals. Under ether anesthesia, the heart was removed and the right and left ventricles (including the septum) were separated and weighed. DOCA-salt treatment produced a significant increase in left ventricular weight/body weight (LVW/BW) ratio (2.44 ± 0.09 mg/g) and right ventricular weight/body weight (RVW/BW) ratio (0.53 ± 0.01 mg/g) compared to control (1.92 ± 0.04 and 0.48 ± 0.01 mg/g, respectively) rats. MAP was significantly higher (39%) in DOCA-salt rats. Renal denervation prevented (P>0.05) the development of hypertension in DOCA-salt rats but did not prevent the increase in LVW/BW (2.27 ± 0.03 mg/g) and RVW/BW (0.52 ± 0.01 mg/g). We have shown that the increase in arterial pressure level is not responsible for cardiac hypertrophy, which may be more related to other events associated with DOCA-salt hypertension, such as an increase in cardiac sympathetic activity.