A. López-Farré

Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats

Two NO synthase (NOS) isoforms have been described in vessels, an endothelial constitutive NOS (eNOS) and an inducible NOS (iNOS). The purpose of the present study was to examine the endothelium-dependent and endothelium-independent hypotensive response in aging rats, analyzing the ability of their vessels to produce NO. The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (n=20, 18 months old) and young rats (n=20, 5 months old). The hypotensive responses to acetylcholine, bradykinin, and sodium nitroprusside were determined. Furthermore, the expression of the NOS isoforms by Western blot and the eNOS and iNOS activities, defined as Ca2+-dependent and Ca2+-independent conversion of [14C]L-arginine into [14C]L-citrulline, respectively, were also determined. In the aging rats, we found an impaired hypotensive response to acetylcholine and bradykinin (2 NO- and endothelium-dependent hypotensive agents) that was accompanied by a preserved hypotensive response to sodium nitroprusside. Aging rats also demonstrated an enhanced sensitivity response to the pressor effect of the L-arginine antagonist L-Nomega-nitro-L-arginine and a reduced vasoconstrictor response to angiotensin II. The inhibition of NO synthesis normalized the pressor effect of angiotensin II in the aging animals. Nitrite plus nitrate plasma levels were increased in aging rats. Furthermore, cGMP content was also higher in the aging vessels. In the aging aortas, the expression of both eNOS and iNOS isoforms was enhanced. However, in aging rats, the activity of the eNOS isoform was markedly reduced, a finding that was accompanied by the presence of iNOS activity. The vessel wall of aging rats showed an enhanced expression of eNOS and iNOS isoforms. However, eNOS activity was reduced in the aging animals. These findings could explain the impaired endothelium-dependent hypotensive response associated with aging.

Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha.

Changes in endothelial nitric oxide synthase (eNOS) expression may be involved in the endothelium-dependent vasorelaxation dysfunction associated with several vascular diseases. In the present work, we demonstrate that eNOS mRNA contains a previously undescribed cis element in the 3 untranslated region (3 UTR). A U+C-rich segment in the 3 UTR is critical in complex formation with bovine aortic endothelial cell cytosolic proteins. Tumor necrosis factor alpha (TNF-alpha), which destabilizes eNOS mRNA, increased the binding activity of the cytosolic proteins in a time-dependent manner. These data suggest that endothelial cytosolic proteins bind to the 3 UTR of eNOS mRNA. These proteins may play a role in TNF-alpha-induced eNOS mRNA destabilization.

A role for endothelin in the maintenance of post-ischaemic renal failure in the rat.

1. Endothelin (ET) has been shown to reduce glomerular filtration rate (GFR) and renal blood flow (RBF) and may therefore be a possible mediator of the reduction of GFR and RBF observed in post‐ischaemic acute renal failure. 2. We infused a specific ET antibody, i.v., for 1 h before and 1 h after a 60 min period of renal ischemia by clamping the renal artery, and observed the changes in renal function (acute clearance and long‐term metabolic cage studies) compared with rats infused with non‐immune rabbit serum. 3. In acute and long‐term studies, better renal function, as judged by GFR and RBF was observed in rats treated with the ET antibody. Furthermore, ischaemic rats showed higher levels of plasma immunoreactive ET (7.02 +/‐ 1.17 pg ET (ml plasma)‐1; mean +/‐ S.E.M.) than normal rats where it was undetected. 4. We previously reported that glomeruli and renal platelet‐activating factor (PAF) production were increased after renal ischaemia. So, we studied the possible relationship between ET and glomeruli or renal PAF production in post‐ischaemic acute renal failure. 5. Glomeruli from ischaemic rats produced greater amounts of PAF than glomeruli from normal or anti‐ET antibody‐treated ischaemic rats. In addition, total renal PAF production was higher in ischaemic‐untreated than in non‐ischaemic or anti‐ET‐treated rats. Glomeruli incubated with 10(‐7) M‐endothelin produced much more PAF than those incubated in control conditions (138.4 +/‐ 10.5 vs. 80.2 +/‐ 9.4 pg PAF (mg protein)‐1; means +/‐ S.E.M.; n = 10). 6. In conclusion, the present study suggests that endothelin plays a role in the persistent renal vasoconstriction that characterizes post‐ischaemic acute renal failure. In addition, the observed increase in glomerular PAF production after renal ischaemia may be due to the action of endothelin.

ROLE OF NITRIC OXIDE IN THE CONTROL OF APOPTOSIS IN THE MICROVASCULATURE

Cell death occurs by either apoptosis or necrosis. Apoptosis is a cellular event in which a sequence of biochemical and morphological changes conclude in the death of the cell. Apoptosis is an important mechanism to control the number of cells and maintain tissue architecture. Nitric oxide (NO) is a multifunctional molecule that is synthesized by a family of enzymes, namely nitric oxide synthases (NOS). NO is implicated in several physiological functions within the microvascular environment, i.e. regulation of vascular tone, antiplatelet and antileukocyte properties and modulation of cell growth. Several investigations have demonstrated effects of NO on gene transcription. In this regard, NO has been also implicated in the apoptotic processes. The goal of the present review is to summarize the current knowledge about the relationship between NO and different genes involved in the apoptotic phenomena with focus in the cells of the microvascular environment, i.e. monocytes/macrophages, endothelium and vascular smooth muscle cells. Different studies have revealed that stimulation and inhibition of different genes are required to stimulate apoptosis. NO modulates the expression of bcl-2 family members, p53, interleukin-1 beta-converting enzyme family proteases and the cytokine receptor Fas. Therefore, NO generated from NO donors or synthesized by NOS induces cell death via apoptosis in a variety of different cell types. On the other hand, in the endothelial cells NO seems to have a relevant role in the maintenance of the confluent endothelial monolayer inhibiting apoptotic-related mechanisms. Furthermore, the redox states of the cells play an important role in the effects of NO as promotor of apoptosis. There have been exciting advances in the understanding of the molecular relationship between apoptosis and NO. Therefore, NO could be an important mediator to consider in the context of future therapeutic applications particularly considering apoptosis as a mechanism to maintain vascular architecture.

Effect of endothelin on renal function in rats

The effect of synthetic porcine endothelin on glomerular filtration rate, renal plasma flow and electrolyte excretion was studied in rats. Endothelin, 1 nmol/kg body weight given as a bolus, induced a transient decrease in glomerular filtration rate (72%) and in renal plasma flow (76%) as well as in sodium excretion, accompanied by a sustained increase in renal vascular resistance. This dose had no sustained effect on mean arterial pressure. It is concluded that endothelin induces a marked decrease in glomerular filtration rate and renal perfusion. This peptide could play a role in the alterations in renal function observed after renal injury.

Aspirin inhibits inducible nitric oxide synthase expression and tumour necrosis factor‐α release by cultured smooth muscle cells

Inflammatory related cardiovascular disease, i.e. cardiac allograft rejection, myocarditis, septic shock, are accompanied by cytokine production, which stimulates the expression of inducible nitric oxide (iNOS).

Glomeruli from ischemic rat kidneys produce increased amounts of platelet activating factor

The production of Platelet Activating Factor (PAF-acether) by glomeruli isolated from rats subjected to a 60 min renal ischemia has been studied. PAF-acether has been purified by Sep-Pak columns and measured by its ability to release serotonin from previously loaded rabbit platelets. Glomeruli from ischemic kidneys had higher amounts of PAF-acether activity than glomeruli from sham operated rats. These data suggest a role for PAF-acether in the renal functional alterations induced by renal ischemia.

Prior aspirin use in unstable angina patients with modified plasma inflammatory markers and endothelial nitric oxide synthase in neutrophils

Background u2002Prior use of aspirin in patients with acute coronary syndrome has been associated with a lower incidence of acute myocardial infarction. The aim of this study was to explore if prior aspirin therapy in unstable angina (UA) patients could modify systemic inflammatory markers such as interleukin‐6 (IL‐6), tumour necrosis factor‐α (TNF‐α) and intercellular adhesion molecule‐1 (ICAM‐1) and the expression of endothelial nitric oxide synthase (eNOS) in neutrophils.

Effect of endothelin on systemic and regional hemodynamics in rats

In this study we analyzed the changes in systemic haemodynamics induced by endothelin, 1 nmol/kg, given as an i.v. bolus to anaesthetized rats. Endothelin induced a dramatic decrease in cardiac output whereas the total peripheral resistance increased more than two times. In addition, renal blood flow decreased while renal vascular resistance increased to a similar extent. A marked decrease in blood flow to the splanchnic organs was also observed. In conclusion, endothelin seems to modulate peripheral vasomotor tone, at least under certain conditions.

Factors associated with hyperdynamic or hypodynamic circulation and role of platelet-activating factor in hemodynamic alterations in bacterial peritonitis in conscious rats

Male Wistar rats injected intraperitoneally (i.p.) with 10(9) U Escherichia coli ATCC 25922 developed acute bacterial peritonitis. Hemodynamic studies, with microspheres labeled with 103Ru 57Co, and 113Sn, were performed before, 30 min after bacterial injection, and 30 min after administration of either the platelet-activating factor (PAF) antagonist BN-52021 (5 mg/kg body weight) or isotonic saline. A blood sample of 0.3 ml was obtained for bacterial culture and endotoxemia measurements. Plasma PAF levels were measured in a different group of 10 control rats and 20 animals with experimental peritonitis. One group of rats injected with E. coli (n = 13) displayed hyperdynamic circulation, with an increase in cardiac output (CO) from 15.1 +/- 1.2 to 19.4 +/- 1.1 ml/min/100 g body weight and a decrease in total peripheral resistance (TPR) from 19.5 +/- 2.4 to 14.9 +/- 1.1 dynes.s.cm-5 10(-4). Furthermore, these rats showed high endotoxin blood concentrations and low hemoculture levels. The remaining 7 peritonitic rats showed a significant decrease in CO from 16.3 +/- 1.6 to 12.7 +/- 1.2 ml/min/100 g body weight and an increase in TPR from 17.3 +/- 1.8 to 22.6 +/- 2.8 dynes.s.cm-5 10(-4). In addition, these rats showed low endotoxin blood concentrations and high hemoculture levels. Endotoxin blood concentrations were positively correlated with the change in CO (r = 0.87, p < 0.05), and cell hemocultures were positively correlated with CO (r = 0.89, p < 0.05). Rats with high endotoxin blood levels showed higher PAF plasma levels than control rats or peritonitic rats with low endotoxin blood levels. When peritonitic rats were injected with the specific PAF-receptor blocker BN-52021 (5 mg/kg body weight) as a bolus, CO and TPR returned to baseline values in both groups of animals. These data suggest that the hemodynamic changes induced by bacterial peritonitis depend on endotoxemia and bacteremia in opposite ways. In addition, PAF appears to be involved in both the hyperdynamic and hypodynamic hemodynamic changes shown by peritonitic rats.