L-arginase induces vascular dysfunction in old spontaneously hypertensive rats.

BACKGROUND: Aging is a major non-modifiable risk factor for hypertension. Changes in aging are similar to those seen in hypertension in the vasculature. Also, aging increases the vascular dysfunction that occurs in hypertension. L-arginase action reduces substrate (L-arginine) availability for the formation of nitric oxide (NO). This reduces the level of NO and leads to reduced vasodilation and ultimately, vascular dysfunction. This study examines the hypothesis that age-dependent vascular dysfunction in SHRs is mediated by arginase. METHODS: Young (12-14 weeks) and old (11-12 months) male Wistar and spontaneously hypertensive rats (SHR) were used. Mean arterial pressure (MAP) was measured in the rats. They were then euthanized and mesenteric resistance arteries (MRAs) and thoracic aortae were excised and placed in ice-cold physiological salt solution (PSS). Arterial segments were either snap-frozen in liquid nitrogen and stored for immunoblotting studies or cut into 2mm rings for reactivity studies. Cumulative concentration-response curves to acetylcholine (Ach: 10-9 - 3x10-5M) and sodium nitroprusside (SNP: 10-12 - 3x10-5 M) were performed in the absence or presence (30-minute exposure) of L-arginase, 0.05U/ML (MRA) or 0.5U/ML (aorta). Vessels were pre-contracted with phenylephrine (PE; 3x10-6M). RESULTS: MAP increased during aging in the SHRs p<0.05 but not in the Wistar rats. Arginase impaired the endothelium-dependent relaxation responses of thoracic aortic and MRA arterial rings to Ach in the old Wistars and SHRs (Emax aorta: 29.42±2.19% vs 7.94±1.86%). Arginase also impaired endothelium-independent relaxation response to SNP in the old SHRs only (Emax aorta: 88.62±4.10% vs 31.45±10.61%). We also observed no differences in the serum arginase activity in the four groups of rats. On the contrary, arginase activity in the aortae of young Wistar rats was reduced compared to other groups. CONCLUSIONS: Arginase impairs both endothelium-dependent and -independent vasorelaxation responses, through the NO signaling pathway.

Experimental malaria: the in vitro and in vivo blood pressure paradox.

OBJECTIVE: Malaria causes more deaths worldwide than any other parasitic disease. Many aspects of the biology that governs the pathogenesis of this parasite are still unclear. Therefore insight into the complexity of the pathogenesis of malaria is vital to understand the disease, particularly as it relates to blood pressure. METHODS: In vivo and in vitro experimental models were used for this study. In the in vivo study, mean arterial pressure, pulse rates and heart rates were recorded by cannulation of the carotid artery of rats. In the in vitro study, ring preparations of blood vessels from the rat aorta were studied using standard organ bath techniques. Dose-response curves for phenylepherine (PE) - and acetylcholine (Ach) -induced relaxation were constructed for rings pre-contracted with PE. RESULTS: Our results showed a significant (p < 0.05) reduction in the mean arterial pressure and pulse rates, while the heart rates remained unaltered in rats with malaria parasites, compared with the controls. Incubation of rat aortic rings with parasitised blood resulted in a significant (p < 0.05) increase in maximum contractile response to phenylephrine in the rat aortic rings but there was no effect on the baseline. The dose-response curve showed a significant (p < 0.05) leftward shift following the addition of parasitised blood and the EC(70) (M) values increased from 7 × 10(- 7) to 5 × 10(-6) M. Following exposure to parasitised blood, the magnitude of Ach-induced relaxation responses reduced significantly (p < 0.05) from 73 ± 3.6 to 24.75 ± 7.25% in the rat aortic rings. CONCLUSIONS: The results suggest that malaria parasitaemia caused in vivo reduction in blood pressure, and enhanced the responses to contractile agents and reduced relaxation responses to acetylcholine in vitro. This appears to be a paradox but is explainable by the complex cardiovascular control mechanisms in vivo. This may be independent of direct action on vascular smooth muscle.

Altered vascular reactivity induced by malaria parasites.

OBJECTIVE: In this study, we have examined the possibility that there is altered vascular reactivity due to the direct interaction between parasitized erythrocytes and vascular endothelial cells. METHOD: Ring preparations of rat aorta were studied using standard in vitro techniques, the rings were mounted in 20 ml organ baths containing PSS under an initial load of 1 g, maintained at 37 degrees C at pH 7.4 and isometric contractions were recorded electronically. Rings were allowed 90 minutes to equilibrate before the commencement of the various protocols: Dose responses to phenylephrine (PE) and other vasoactive agents (high-K+). Acetylcholine (Ach)--induced relaxation in phenylephrine-contracted rings (pre-contraction was induced by EC70 concentration of phenylephrine). Ach-induced relaxation in PE-precontracted, endothelium-denuded rings. Also, relaxation responses to acetylcholine was investigated through application of a single. (EC70) concentration of acetylcholine in rings exposed to blood with varying concentrations and dilutions of parasitized blood and varying durations of exposure. RESULTS: Incubation with parasitized blood resulted in a significant increase in maximum contractile response to phenylephrine in the rat aortic rings (p < 0.05) but no effect to the base line. Analysis of the whole dose-response curve (using paired t-test) showed a significant left-ward shift following the addition of parasitized blood (p < 0.05), EC70 (M) values increasing from 7 x 10(-7) to 5 x 10(-6)M. Following exposure to parasitized blood, the magnitude of Ach-induced relaxation responses reduced significantly from 73 +/- 3.6 to 24.75 +/- 7.25% in rat aortic rings (p < 0.05). Ach relaxations were significantly enhanced (p < 0.05) at 5-minute exposure; however at longer durations, Ach-relaxations were variable and inconsistent. The lesser the dilution, due to increased volume of parasitized blood, the lesser the relaxation response. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh in both the control and incubated vessels. Exposure to parasitized blood did not significantly alter contractile responses induced by potassium depolarization. CONCLUSIONS: This gives evidence in support of an endothelium-dependent action of malaria parasites as vascular effects of malaria parasites are mediated, at least in part, via endothelium-dependent mechanism(s).

Specificity of vascular reactivity and altered response in experimental malaria.

OBJECTIVE: Adherence of erythrocytes infected with Plasmodium falciparum (P falciparum) to microvascular endothelial cells (sequestration) is considered to play an important role in parasite virulence and pathogenesis. In this study, we have examined the possibility that there is altered vascular reactivity due to the direct interaction between the parasitized erythrocytes and vascular endothelial cells and that it could be tissue specific. METHOD: Ring preparations of blood vessels from the rabbit carotid and rat aorta were studied using standard organ bath techniques. Dose response curves for phenylephrine (PE) and acetylcholine (Ach)-induced relaxation were constructed in rings pre-contracted with PE. RESULTS: Incubation of rat aortic rings with parasitized blood resulted in a significant (p < 0.05) increase in maximum contractile response to phenylephrine in the rat aortic rings but there was no effect on the rabbit carotid artery. The dose-response curve showed a significant (p < 0.05) left-ward shift following the addition of parasitized blood. Parasitised blood had no effect on baseline in both tissues. Following exposure to parasitized blood, the magnitude of Ach-induced relaxation responses reduced significantly (p < 0.05) in rat aortic rings and (p < 0.05) in rabbit carotid rings; relaxations to acetylcholine was more pronounced in the aortic compared to the carotid rings. CONCLUSIONS: Malaria altered vascular reactivity through an endothelium-dependent mechanism. The regulation of vascular tone by various vasoactive agents following exposure to malaria parasites might be altered in a vessel-specific manner. This may contribute to or exacerbate the abnormal haemodynamics observed in the microcirculation of numerous vascular beds in malaria.

Vascular effect of ketamine in isolated rabbit aortic smooth muscle.

The precise mechanism by which ketamine induces relaxation of vascular smooth muscle is not clear. The goal of this study was to further characterize the vascular actions of ketamine in rabbit aortic smooth muscles. Ring segments (2mm) of rabbit aortae were suspended in 20ml organ baths containing physiological salt solution (PSS) and isometric contractions were recorded at 37 degrees C and pH 7.4. The medium was bubbled with O(2) 95 % and CO(2) 5 % mixture and rings were given an initial load of 2g. An equilibration period of 90 minutes was allowed. Three protocols were examined: (a) Effect of ketamine on baseline tension (b) relaxation-responses to ketamine following precontractions induced by 10(-7)M phenylephrine or high K(+) (40mM) PSS and (c) influence of presence or absence of endothelium on the relaxation response to ketamine. Ketamine produced relaxation of contractile responses induced by both phenylephrine and high K(+). The respective maximum relaxation responses induced by ketamine following precontractions by phenylephrine and high-K(+) were 76.8 +/- 2.3 and 71.2 +/- 8.0 (p > 0.05). Ach-induced relaxation was observed only in rings with intact endothelium whereas ketamine-induced relaxation was observed in intact as well as endothelium-denuded rings; this suggests that ketamine-induced relaxation of rabbit aortic smooth muscle is independent of vascular endothelium.

Effects of sub-chronic oral cyanide on endothelial function in rabbit aortic rings.

We have investigated how the endothelium affects vascular responses following sub-chronic low dose cyanide administration. Cyanide exists in low levels in cassava foods, which are widely consumed in tropical Africa. Adult rabbits were administered 0.38 mg/kg per day KCN po for 25 days, and responses of the isolated aortic rings to noradrenaline (NA), calcium chloride (Ca2+) and acetylcholine (ACh) were measured in vitro in the presence and absence of the endothelium. In order to establish that the dose was not toxic, animal weight, some haematological indices, plasma alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured. Results show that endothelium denudation significantly (P <0.05) attenuates NA-induced contraction in rings from cyanide-treated rabbits. There was a similar reduction in response in Ca2+-depleted NA-precontracted endothelium-denuded aortic rings from cyanide-treated rabbits. Endothelium-denuded rings from cyanide-treated rabbits showed significantly (P <0.05) enhanced relaxation to ACh. In rings from control animals, the responses to NA and Ca2+ were not significantly altered, whether in the presence or absence of the endothelium. There were no significant changes in the studied toxicological indices. We conclude that endothelial compromise is necessary for low-dose sub-chronic cyanide-induced to alter vascular reactivity to NA and ACh.

Effects of potassium adaptation on blood pressure and pressor responses in normotensive and renal hypertensive Wistar rats.

Potassium adaptation reduces blood pressure (BP) in hypertensive humans and animals but its effects on normotensive BP and the nature of pressor responses to vasoactive drugs are not known. We measured directly, the mean arterial pressure (MAP) of normotensive control, normotensive potassium-adapted (given 0.75% potassium chloride solution for 5 weeks), renal hypertensive (RHP), and renal hypertensive Wistar rats later adapted to potassium. The maximum percentage change, the ED25, and recovery times after bolus injections of noradrenaline (NA), angiotensin II (Ang. II), sodium nitroprusside (SNP), and acetylcholine (ACh) were compared. The MAP of normotensive potassium-adapted rats was significantly lower than that of the normotensive controls (95.6+/-5.0 vs. 110.8+/-2.8 mmHg, p<0.05). The potassium-adapted hypertensive rats (RHP-A) also had significantly lower MAP values than the non-adapted hypertensive ones (116.0+/-4.4 vs. 138.2+/-4.1 mmHg, p<0.01). Potassium adaptation significantly blunted responses to NA and augmented responses to SNP but while the duration of action of Ang. II was significantly shortened, that of SNP was significantly increased. We conclude that potassium adaptation reduces BP in the normotensive and hypertensive rats and may influence both the degree and duration of action of vasoactive drugs given as bolus injections.

Haematological influences of potassium adaptation in normotensive and renally-hypertensive Wistar rats.

Dietary potassium is known to cause reduction in blood pressure in several models of hypertension in human and animal studies but its haematological effects are not known. Here, experiments are designed to study the haematological effects of potassium adaptation (achieved by administering 0.75% KCl solution in drinking water for five weeks) in Wistar rats. The animals are divided into four groups comprising controls, potassium-adapted, renal hypertensive, and renal hypertensive with later adaptation to potassium. Packed cell volume (PCV) and platelet count (PC), whole blood and plasma viscosities, and platelet aggregation in the presence of sodium nitroprusside, levcromakalim, and glibenclamide, are studied. Results showed comparable PCV and PC in all groups. While relative whole blood viscosity was significantly higher (P<0.05) in the hypertensive group, relative plasma viscosity was similar in all groups. Adaptation significantly reduced (P<0.05) the tendency of platelets to aggregate to collagen. Sodium nitroprusside significantly reduced (P<0.05) the pro-aggregatory effects of collagen only in the control group. Neither of the potassium-channel modulators (levcromakalim, glibenclamide) caused any significant alteration in platelet response to collagen at the concentrations studied. Although these results suggest that potassium adaptation may not affect haemorheology, the reduced ability of platelets to aggregate--by mechanisms not clearly understood--has implications for reduced thromboembolism and the attendant cardiovascular sequelae.

Vasorelaxant effect of thiopentone in isolated human epigastric arteries.

The experiments were designed to elucidate the mechanism of thiopentone-induced inhibition of contractile responses in isolated human epigastric arteries. Segments of human epigastric arteries were obtained from patients who underwent elective or emergency caesarean section, placed in standard physiological salt solution (PSS), cut into rings at 3 mm intervals and suspended in organ baths for recording of isometric contractions at 37 degrees C, and pH 7.4. Three protocols were employed to examine the inhibitory effect of thiopentone: (a) concentration-dependent effect on 10(-7) M noradrenaline (NA)- or high-K+ (40 mM)-induced contractions: (b) effect on NA-induced extra-and intracellular Ca(2+)-dependent contractions and (c) effect on the dose-response curve when Ca2+ is restored to Ca(2+)-depleted rings in Ca(2+)-free 40 mM K(+)-depolarizing medium. Thiopentone (1 x 10(-6) -4 x 10(-3) M) caused concentration-dependent relaxation of both NA- and high-K(+)-induced contractions. The magnitudes of both precontractions were not significantly different but the IC50 values for thiopentone relaxation of high-K+ contractions were significantly lower than for NA contractions. Thiopentone (10(-4) M) significantly attenuated the phasic (intracellular Ca2+ dependent) contractile responses to 10(-5) M NA in Ca(2+)-free PSS as well as the tonic (extracellular Ca2+ dependent) contractions upon restoration of Ca2+. In contrast, nifedipine (1 microM) did not modify the phasic response but significantly attenuated the tonic response. Thiopentone (10(-4) M) also almost completely abolished concentration-dependent Ca(2+)-induced contractions in K(+)-depolarized Ca(2+)-depleted rings. The results suggest that in the smooth muscle of human epigastric arteries, thiopentone-induced relaxation is non-specific and is associated with impairment of Ca2+ supply from both extracellular and intracellular pools.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on contractile responses in isolated rat aorta.

The vascular effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a meperidine analog, were studied in vitro on ring preparations of rat aorta for the purpose of characterizing its mode of action. Isometric contractions were evaluated under standard organ bath conditions. Exposure to MPTP (10(-12)-1.6 x 10(-8) M) did not affect basal tension but did cause dose-dependent relaxation of rings precontracted by 10(-7) M noradrenaline (NA) and was ineffective against 30 mM K+ contractions. Removal of endothelium did not significantly modify the relaxation responses. In 10(-5) M NA-stimulated (but not in 100 mM K(+)-stimulated) rings, MPTP significantly (p < 0.05) attenuated contractile responses to calcium chloride following calcium-free exposure. Furthermore, the phasic contractile responses to noradrenaline in calcium-free medium (presumed to be due to mobilization of membrane bound calcium pool) were significantly (p < 0.05) attenuated by MPTP. The results suggest that MPTP relaxes rat aortic smooth muscle by a mechanism mediated, at least in part, by impairment of calcium influx through receptor-operated channels, as well as inhibition of calcium release from a membrane bound pool.

Depressed endothelium-dependent relaxation responses to acetylcholine and histamine in isolated human epigastric arteries from pre-eclamptic women.

The vasorelaxant effects of acetylcholine (Ach) and histamine have been examined on 10(-7) M-noradrenaline (NA)-precontracted ring preparations of epigastric arteries from normotensive and pregnancy-induced hypertensive women. Contractile responses to 10(-7) M-NA were significantly (p < 0.05) enhanced following removal of the endothelium. Both Ach and histamine (in the presence of H1-receptor blockers) elicited methylene blue-sensitive concentration-dependent relaxations only in endothelium-intact rings. The relaxation responses to both agents were significantly attenuated in arterial rings from pre-eclamptic women. Endothelium-independent relaxation responses induced by sodium nitroprusside were comparable in arterial rings from both patient groups. The results suggest an impairment of endothelial function in pre-eclampsia.

Differential effects of prostanoid synthesis inhibitors on cicletanine-induced relaxation of isolated human epigastric arteries.

We have examined the influence of prostanoid synthesis inhibitors on the relaxation responses induced by cicletanine in ring preparations of isolated human epigastric arteries following precontraction induced by 10(-7) M noradrenaline. Cicletanine caused concentration-dependent relaxations, uninfluenced by the cyclooxygenase inhibitors, meclofenamate (1, 10 microM) and indomethacin (1, 10 microM) but significantly (p < 0.05) attenuated by the specific prostacyclin synthesis inhibitor, tranylcypromine (0.1-10 microM) (n > 6 in each). In contrast, cromakalim-induced relaxations were significantly attenuated by indomethacin, meclofenamate and tranylcypromine. The tranylcypromine-sensitive cicletanine-induced relaxation suggests, at least in part, the involvement of prostacyclin in the vasodilator action of cicletanine.

Evidence for preservation of epithelial function in cryopreserved porcine and human bronchi.

Porcine and human bronchi have been investigated in vitro without or after storage at -196 degrees C in Krebs-Henseleit solution containing 2.0 M dimethyl sulphoxide and 0.1 M sucrose as cryoprotectants. In bronchi from both species maximal post-thaw contractile responses to acetylcholine (ACh) were reduced by about 25-30% compared to unfrozen bronchi. To assess the viability of bronchi and endothelium-denuded rat aortic strips, was employed. The release of an epithelium-derived inhibitory factor (EpDIF) was induced by ACh and assessed in terms of concentration-dependent relaxation of the endothelium-denuded rat aortae. Following removal of bronchial epithelium, ACh failed to elicit any relaxation of rat aorta. With cryopreserved bronchi from both pig and human about 5 and 30 times higher concentrations of ACh, respectively, were required to elicit the same relaxant response as with unfrozen bronchi. The results suggest that after the freezing-thawing process both smooth muscle and epithelial function is largely preserved and provide support for the use of cryopreservation for storage of airway preparations for pharmacological studies.

Responses of isolated human epigastric arteries to histamine.

Responses to histamine have been studied on ring preparations of epigastric artery obtained from normal and from pregnancy-induced hypertensive (PIH) women to characterize the mode of action of histamine in this vascular preparation. In non-contracted arterial rings, histamine elicited concentration-dependent H1 receptor-mediated contractions, competitively antagonised by mepyramine and cicletanine with pA2 values of 9.1 and 7.5, respectively. Arterial rings from pregnancy-induced hypertensive patients displayed greater sensitivity to histamine, but no change in maximal contractions, and were (at the EC30 histamine response) more susceptible to antagonism by mepyramine and cicletanine. Following precontraction by noradrenaline, histamine elicited relaxation responses only in the presence of H1 receptor antagonists. Endothelium removal or exposure to methylene blue significantly attenuated histamine-induced relaxation; the residual relaxations under these conditions appear to be due to a direct effect on H2 receptor on smooth muscle cells. The results show that, in human epigastric artery, histamine elicits H1 receptor-mediated contractions and that, following NA precontraction and in the presence of H1 receptor blockade, relaxations occur. Arterial rings from pregnancy-induced hypertensive patients showed modest but somewhat greater sensitivity to histamine, as well as being more susceptible to cicletanine and mepyramine.

Altered responses of aortic smooth muscle from Sprague-Dawley rats with salt-induced hypertension.

1. The contractile responses of aortic ring preparations from Sprague-Dawley rats made hypertensive by 6-week dietary salt loading were studied. The test and control diet contained 8.0 and 0.3% NaCl, respectively. Aortic rings from salt-loaded rats showed enhanced sensitivity to noradrenaline (NA) but not to serotonin. Contractile responses to CaCl2 in Ca-free NA-containing medium was significantly enhanced in salt-loaded rats, but was unchanged in K(+)-depolarised medium. K(+)-induced relaxation (a functional indicator of Na-K adenosine triphosphatase activity) was sensitive to 10 mumol/L ouabain and was significantly attenuated in aortic rings from salt-loaded rats. The results suggest that hypertension induced by salt-loading is associated with enhanced sensitivity to NA, increased Ca2+ entry through receptor-operated channels, and impairment of Na-K ATPase enzyme activity.

In vitro vascular effects of cicletanine in pregnancy-induced hypertension.

1. The vascular effects of cicletanine have been studied in vitro on ring preparations of inferior epigastric arteries from normotensive human females and human females with pregnancy-induced hypertension (preeclampsia). 2. Cicletanine (10(-7)-10(-3) M) elicited concentration-dependent relaxation of vessels precontracted with 10(-7) M noradrenaline (NA) or 60 mM K+ but was more potent in the former. Relaxation was significantly greater in rings from preeclamptic patients and was uninfluenced by endothelium removal. 3. The intracellular Ca-dependent contractile responses to 10(-5) M NA in Ca-free medium as well as the subsequent extracellular Ca-dependent contractions (on restoration of external Ca) were significantly attenuated dose-dependently by cicletanine (10(-5) M, 3 x 10(-4) M) in arterial rings from both normotensive and preeclamptic patients. Cicletanine also relaxed rings precontracted by 25 mM K+ but was ineffective against 80 mM K(+)-induced contractions. 4. The inhibition of intracellular Ca-dependent contractions was significantly greater in rings from preeclamptic than from normotensive patients whereas extracellular Ca-dependent contractions were comparably inhibited in both groups. Nifedipine, on the other hand, had little effect on the intracellular Ca-dependent contractions but significantly depressed extracellular Ca-dependent contractions. 5. Cicletanine-induced relaxation was uninfluenced by pretreatment with propranolol, ouabain, tetraethylammonium, procaine, indomethacin, cimetidine or tetrodotoxin but was antagonized by glibenclamide. 6. The results show that cicletanine inhibits contractile responses of human isolated inferior epigastric arteries by a mechanism unrelated to endothelial factors but associated with inhibition of calcium metabolism. An action of cicletanine on glibenclamide-sensitive K+ channels is also suggested. Cicletanine-induced inhibition was significantly greater in arteries from preclamptic patients.

Mechanism of dichlorovos-induced inhibition of calcium-dependent contractions in the rat tail artery.

The inhibitory effect of dichlorovos (an organophosphate pesticide) on Ca(2+)-dependent contractions has been studied in isolated helical strips of rat tail arteries. Isometric contractions were evaluated under standard organ bath conditions. Dichlorovos caused concentration-dependent relaxation of 10(-7) M NE-induced contractions as well as inhibition of both phasic and tonic components of NE contractions. Extracellular calcium-dependent contractions in K(+)-depolarized medium as well as NE-induced contractions due to mobilization of membrane-bound calcium pool and intracellular calcium-dependent caffeine contractions were significantly attenuated by dichlorovos. The results strongly support our previous observation that dichlorovos interferes with calcium metabolism in the rat tail artery.

Dietary salt-loading attenuates endothelium-dependent relaxation in response to histamine but not to acetylcholine in rat aortic rings.

Endothelium-dependent relaxation in response to histamine and ACh has been studied on precontracted aortic rings from control and salt-loaded Sprague-Dawley rats. Both ACh and histamine caused relaxation of the noradrenaline-induced precontraction only in the presence of the endothelium. The relaxation response to ACh in rings from control and salt-loaded rats did not differ significantly whereas histamine-induced relaxation was significantly attenuated in aortae from salt-loaded rats. The results suggest that salt-induced hypertension is associated with impairment of endothelium-dependent relaxation to histamine but not to ACh.