Jaroslav Kuneš

Abnormalities of membrane function and lipid metabolism in hypertension* A review

Hypertension, which is characterized by multiple alterations in the structure and function of the cell membrane, is often associated with important metabolic abnormalities including those concerning lipid metabolism. Dyslipidemia accompanying essential hypertension consists of elevated plasma triglycerides, low HDL cholesterol, and increased levels of atherogenic LDL cholesterol particles. The altered membrane microviscosity seen in hypertensive subjects reflects the changes of membrane lipid composition resulting from intensive exchange between circulating and membrane lipids, as well as from abnormal cellular lipid synthesis and metabolism. The changes of membrane microviscosity are known to modulate the activity of proteins involved in ion transport, signal transduction, cell Ca2+ handling, intracellular pH regulation, etc. Alterations in plasma or membrane lipids are indeed closely associated with ion transport abnormalities as well as with impaired control of cytosolic Ca2+ and pH in various forms of hypertension in both humans and rats. Such lipid-dependent modifications of membrane properties in cells participating in the cardiovascular regulation might be a part of pathogenetic mechanisms responsible for chronic blood pressure elevation. Thus nutritional and pharmacologic interventions aiming to normalize abnormal lipid metabolism could be useful for amelioration of membrane abnormalities and lowering of high blood pressure. Future studies of functional membrane alterations in hypertension or dyslipidemia will therefore require the detailed determination of membrane lipid composition and the measurement of microviscosity in particular membrane domains.

An analysis of spontaneous hypertension in spontaneously hypertensive rats by means of new recombinant inbred strains.

The mode of blood pressure inheritance and some genetic markers of spontaneous hypertension were evaluated in a new set of recombinant inbred (RI) strains obtained by crossing of normotensive (BN.lx) and hypertensive (SHR) progenitor strains. Blood pressure values of RI strains were continuously distributed between both progenitor strains, although normotensive strains slightly prevailed. Statistical analysis suggested that there are three major genes and multiple minor genes responsible of the determination of spontaneous hypertension. The association between blood pressure and gene(s) within RT1 complex or gene(s) closely linked to it was found by RI strain analysis. This suggestion was confirmed by the detection of significant difference in blood pressure between SHR and SHR.1N congenic strains. Our results indicate that RT1 complex gene(s) may be involved in the development of high blood pressure.

Relative deficiency of nitric oxide-dependent vasodilation in salt-hypertensive Dahl rats: the possible role of superoxide anions.

Objective The contribution of major vasoactive systems (renin–angiotensin system, sympathetic nervous system and nitric oxide) to blood pressure maintenance and the possible involvement of superoxide anions in the reduced efficiency of nitric oxide (NO)-dependent vasodilation to counterbalance sympathetic vasoconstriction were studied in salt-hypertensive Dahl rats. Design and methods We used Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) female rats kept on a low-salt (0.3% NaCl) or high-salt diet (8% NaCl) for 6 weeks since weaning. Mean arterial pressure (MAP) was measured in conscious animals subjected to acute consecutive blockade of the renin–angiotensin system (RAS) [captopril, 10 mg/kg intravenously (i.v.)], the sympathetic nervous system (SNS) (pentolinium, 5 mg/kg i.v.) and NO synthase (Nω-nitro-l-arginine methyl ester (l-NAME), 30 mg/kg i.v.). Before the consecutive blockade of vasoactive systems one-half of the animals in each experimental group was pre-treated with a stable membrane-permeable mimetic of superoxide dismutase (tempol, 25 mg/kg i.v.) which functions as a superoxide scavenger. Results Compared to normotensive SR/Jr animals, salt-hypertensive SS/Jr rats were characterized by an enhanced blood pressure (BP) fall after ganglionic blockade (− 104 ± 8 versus − 62 ± 5 mm Hg, P < 0.001) and by higher residual blood pressure recorded after the blockade of both RAS and SNS (70 ± 3 versus 43 ± 3 mmHg, P < 0.01), but there was only a borderline elevation of their BP response to acute NO synthase inhibition (67 ± 6 versus 49 ± 4 mmHg, P < 0.05). The acute tempol pre-treatment elicited the most pronounced reduction of basal BP (− 13 ± 1 mmHg, P < 0.001) in the salt-hypertensive SS/Jr group in which the BP rise after l-NAME administration was augmented by about 50%. On the contrary, tempol pre-treatment did not affect norepinephrine- or angiotensin II-dependent vasoconstriction. Conclusions The NO system is not able to counterbalance effectively the hyperactivity of the sympathetic nervous system in salt-hypertensive Dahl rats. The predominance of sympathetic vasoconstriction over NO-dependent vasodilation could be explained partially by enhanced NO inactivation due to augmented superoxide anion formation in hypertensive animals.

Experimental hypertension in young and adult animals.

The susceptibility of immature and adult animals to various environmental factors often differs because the response of the young organism can only involve those regulatory mechanisms that are available at the particular stage of development. Increased sensitivity to certain (e.g., hypertensive) stimuli may be limited to a relatively short age period that is usually characterized by the maturation of some important physiological functions. High salt intake seems to influence the animals especially during the weaning period and prepuberty, in the course of which profound developmental changes of circulation, electrolyte metabolism, and neurohumoral regulation have been demonstrated. Indeed, salt-dependent forms of experimental hypertension are more severe when they are induced in immature animals. Moreover, substantial differences in hemodynamics, distribution of body fluids, and involvement of pressor and natriuretic agents indicate that the mechanisms of salt hypertension need not be the same in immature and adult animals. For this reason, increased attention should be paid to developmental factors in the study of induced forms of experimental hypertension.

Obesity-related hypertension: possible pathophysiological mechanisms

Hypertension is one of the major risk factors of cardiovascular diseases, but despite a century of clinical and basic research, the discrete etiology of this disease is still not fully understood. The same is true for obesity, which is recognized as a major global epidemic health problem nowadays. Obesity is associated with an increasing prevalence of the metabolic syndrome, a cluster of risk factors including hypertension, abdominal obesity, dyslipidemia, and hyperglycemia. Epidemiological studies have shown that excess weight gain predicts future development of hypertension, and the relationship between BMI and blood pressure (BP) appears to be almost linear in different populations. There is no doubt that obesity-related hypertension is a multifactorial and polygenic trait, and multiple potential pathogenetic mechanisms probably contribute to the development of higher BP in obese humans. These include hyperinsulinemia, activation of the renin-angiotensin-aldosterone system, sympathetic nervous system stimulation, abnormal levels of certain adipokines such as leptin, or cytokines acting at the vascular endothelial level. Moreover, some genetic and epigenetic mechanisms are also in play. Although the full manifestation of both hypertension and obesity occurs predominantly in adulthood, their roots can be traced back to early ontogeny. The detailed knowledge of alterations occurring in the organism of experimental animals during particular critical periods (developmental windows) could help to solve this phenomenon in humans and might facilitate the age-specific prevention of human obesity-related hypertension. In addition, better understanding of particular pathophysiological mechanisms might be useful in so-called personalized medicine.

Effect of chronic N-acetylcysteine treatment on the development of spontaneous hypertension

The imbalance between NO (nitric oxide) and ROS (reactive oxygen species) is an important factor in the development of hypertension. The aim of the present study was to determine the preventive and therapeutic effects of NAC (N-acetylcysteine) in SHRs (spontaneously hypertensive rats). Young and adult SHRs and WKY (Wistar-Kyoto) rats were treated with NAC (20 g/l in the drinking water). After 8 weeks of treatment, BP (blood pressure) and NOS (NO synthase) activity, conjugated dienes and GSH (reduced glutathione) in the kidney and left ventricle were determined. Protein expression of eNOS (endothelial NOS), inducible NOS and NF-kappaB (nuclear factor kappaB) were also determined in the left ventricle and kidney. Chronic NAC treatment partially attenuated the rise in BP in young SHRs (179+/-6 compared with 210+/-8 mmHg in untreated animals), but it had no significant effect on BP in adult SHRs. The antioxidant action of NAC, measured as a decrease of the concentration of conjugated dienes or inhibition of NF-kappaB expression, was greater in young than in adult SHRs. Similarly, eNOS protein expression was attenuated more in young than in adult SHRs, although NAC treatment increased NOS activity to a similar extent in both young and adult rats. In conclusion, both decreased ROS production and increased NOS activity appear to participate in the BP changes after NAC treatment in young SHRs. In adult SHRs with established hypertension, however, the secondary alterations (such as pronounced structural remodelling of resistance vessels) might attenuate the therapeutic effect of NAC.

Regression of L-NAME-Induced Hypertension : The Role of Nitric Oxide and Endothelium-Derived Constricting Factor

NG-Nitro-L-arginine-methyl ester (L-NAME)–induced hypertension is a well established model of experimental hypertension. Although regression experiments are effective at approximating a clinical setting the reversal of already established L-NAME hypertension has not been intensively researched. We investigated whether spontaneous regression of L-NAME hypertension after discontinuing the drug administration was associated with recovery of endothelial dysfunction. Special attention was devoted to NO signaling and endothelium-derived constricting factor (EDCF) formation in various parts of the vascular tree. Male adult Wistar rats were divided into 4 groups: an L-NAME (5 weeks), a spontaneous recovery (5 weeks L-NAME + 3 weeks of recovery) and two age-matched control groups (a 5- and 8-week control group). The NO-mediated and EDCF-mediated components of acetylcholine-induced responses were evaluated in preconstricted small mesenteric and femoral arteries. The activity, mRNA and protein expression of NO synthase together with the mRNA expression of cyclooxygenase were determined in the aorta. L-NAME administration caused hypertension, impaired NO signaling (as indicated by the reduced NO component of acetylcholine-induced relaxation and decreased NO synthase activity) in all arteries investigated and reduced the inner diameter of the femoral artery. Moreover, we observed enhanced cyclooxygenase-dependent EDCF formation in the femoral arteries and enhanced cyclooxygenase-2 expression in the aortas of L-NAME–treated rats. During spontaneous recovery a functional restoration of NO signaling took place in all parts of the vascular tree. However, the increases in systolic blood pressure, EDCF formation, and cyclooxygenase expression and the reduction in femoral artery diameter were not completely restored. We conclude that impaired NO signaling was improved after the cessation of L-NAME administration. However, persisting arterial structural alterations and enhanced EDCF formation may decelerate blood pressure reduction even after the restoration of NO synthase activity.

Workshop: excess growth and apoptosis: is hypertension a case of accelerated aging of cardiovascular cells?

Several groups including ours have demonstrated cardiac hyperplasia in neonates from genetically hypertensive rat strains. We have shown that similar problems exist in the kidney as well. More recently, we found that excessive heart and kidney weight is neonatally related to inhibition of apoptosis. Using recombinant inbred strains derived from a reciprocal cross between Brown Norway and spontaneously hypertensive rat progenitor strains, we mapped the inhibition of neonatal apoptosis to 2 distinct loci on chromosomes 1 (Myl 2) and 18 (Abrb 2). Positional candidate genes at these loci are being explored. These studies have also demonstrated that the loci determining kidney and heart weights in neonates are distinct from those determining increased organ weight in adults. The impact of blood pressure per se is also divergent because adult kidney weight is negatively correlated whereas heart weight is positively correlated with it. Analyses by extremes of low and high percentiles from fetal life to adulthood identified a single locus determining heart weight at Acaa on chromosome 8 in newborn (P =0.0003) and adult (P =0.016) rats. The Acaa region contains a DNA mismatch repair gene (hMLH1). The kinetics of neonatal growth through adulthood by prelabeling DNA with [3H]thymidine in pregnant mares showed that although the growth process is complex and nonlinear in the kidney of hypertensive rats, there is an increased turnover of cells, that is, reduced half-life of DNA. This observation is supported by the presence of shorter telomere fragments in kidneys of spontaneously hypertensive rats. These studies suggest that cardiovascular cells from hypertensive animals are subject to accelerated turnover, potentially leading to their accelerated aging.

Antihypertensive Mechanisms of Chronic Captopril or N -Acetylcysteine Treatment in L-NAME Hypertensive Rats

Hypertension due to chronic inhibition of NO synthase (NOS) by Nω-nitro-L-arginine methyl ester (L-NAME) administration is characterized by both impaired NO-dependent vasodilation and enhanced sympathetic vasoconstriction. The aim of our study was to evaluate changes in the participation of major vasoactive systems in L-NAME–treated rats which were subjected to simultaneous antihypertensive (captopril) or antioxidant (N-acetylcysteine, NAC) treatment. Three-month-old Wistar males treated with L-NAME (60 mg/kg/day) for 5 weeks were compared to rats in which L-NAME treatment was combined with simultaneous chronic administration of captopril or NAC. Basal blood pressure (BP) and its acute responses to consecutive i.v. injections of captopril (10 mg/kg), pentolinium (5 mg/kg), L-NAME (30 mg/kg), tetraethylammonium (TEA, 16 mg/kg) and nitroprusside (NP, 20 μg/kg) were determined in conscious rats at the end of the study. The development of L-NAME hypertension was prevented by captopril treatment, whereas NAC treatment caused only a moderate BP reduction. Captopril treatment normalized the sympathetic BP component and significantly reduced residual BP (measured at full NP-induced vasodilation). In contrast, chronic NAC treatment did not modify the sympathetic BP component or residual BP, but significantly enhanced NO-dependent vasodilation. Neither captopril nor NAC treatment influenced the compensatory increase of TEA-sensitive vasodilation mediated by endothelium-derived hyperpolarizing factor in L-NAME–treated rats. Chronic captopril treatment prevented L-NAME hypertension by lowering of sympathetic tone, whereas chronic NAC treatment attenuated L-NAME hypertension by reduction in the vasodilator deficit due to enhanced NO-dependent vasodilation.

Vasoactive systems in L-NAME hypertension: the role of inducible nitric oxide synthase.

Objectives The contribution of the renin–angiotensin system (RAS) and the sympathetic nervous system (SNS) to blood pressure (BP) maintenance was evaluated in rats with Nω-nitro-l-arginine methyl ester (L-NAME) hypertension. Furthermore, we studied the extent of nitric oxide (NO) synthesis inhibition and the participation of remaining NO in the counterbalance of pressor systems, with a special reference to inducible nitric oxide synthase (iNOS). Methods Wistar rats subjected to chronic L-NAME treatment (40 mg/kg per day for 4 weeks) were used. A consecutive blockade of RAS (captopril) and SNS (pentolinium) was followed by acute L-NAME injection. Dimethylguanidine or aminoguanidine were used to affect NO synthesis by iNOS. Results L-NAME hypertensive rats had borderline augmentation of depressor response to captopril injection, but their BP fall after pentolinium was considerably enhanced compared with controls. Residual BP (recorded after simultaneous blockade of the RAS and the SNS) was elevated by 20–40% in hypertensive rats. Pronounced inhibition of NO synthase activity (50% reduction in the aorta and myocardium) was detected in L-NAME hypertensive rats in which the BP rise elicited by acute L-NAME injection was considerably attenuated (by 60–80%). In contrast, acute administration of dimethylguanidine [mixed endothelial NO synthase (eNOS)/iNOS inhibitor] to hypertensive rats induced a major BP rise similar to that caused by L-NAME injection in controls. Aminoguanidine (a selective iNOS inhibitor) caused a substantial BP rise in L-NAME hypertensive rats only. Conclusion The contribution of SNS to BP maintenance in L-NAME hypertension is more important than that of RAS. In L-NAME hypertensive rats the iNOS becomes a major source of hemodynamically important NO production, which is still insufficient to compensate prevailing vasoconstriction.