Imaging of the buffering effect of insulin antibodies in the autoimmune hypoglycemic syndrome

The activity and protein expression of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) were investigated during the development of hypertension in spontaneously hypertensive rats (SHR). SHR and Wistar-Kyoto rats (WKY) were studied at three different ages: 4, 14 to 17, and 63 weeks of age. After treatment with saline or lipopolysaccharide (LPS, 10 mg/kg IV) for 3 hours, the aortas were removed for measurement of NOS activity and protein expression assay by [3H]-L-citrulline formation method and Western blot analysis, respectively. Plasma levels of nitrite/nitrate (NO2-/NO3-) and tumor necrosis factor-alpha (TNF-alpha) were also determined. At 14 to 17 weeks and 63 weeks, the basal activity and protein expression of eNOS in the aortas were significantly lower in SHR than in WKY. In addition, the aged WKY exhibited lower eNOS activity than that of adult WKY, but this change was not seen in SHR. By comparison, the basal activity and protein expression of iNOS were only observed in SHR of the 14-to-17-week group and in the 63-week group; SHR still exhibited higher activities, and these differences were further exaggerated by treatment with LPS. The basal and LPS-induced NO2-/NO3- and TNF-alpha levels in the plasma were also higher in the SHR except the 4-week group. After treatment with quinapril, the basal and LPS-induced expressions of iNOS in SHR were significantly attenuated. Our results demonstrated that alterations of activity and protein expression of eNOS and iNOS occurred in SHR. In addition, aging may reduce the activity of eNOS in WKY but not in SHR. The decline of eNOS activity and/or expression may contribute to the development of hypertension, whereas the increase of iNOS expression may be a consequence of the pathological state of vessels associated with hypertension in SHR. However, the augmented expression of iNOS in SHR was attenuated by antihypertensive therapy, suggesting that the abnormal expression of iNOS is associated with hypertension.     

Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.     

Calcium channel blockade enhances nitric oxide synthase expression by cultured endothelial cells

In a recent study, we found marked increases in nitric oxide (NO) production and endothelial and inducible NO synthase (eNOS and iNOS) expressions with calcium channel blockade in rats with chronic renal failure. This study was undertaken to determine whether enhanced NO production with calcium channel blockade is a direct effect of this therapy or a consequence of the associated hemodynamic and humoral changes. We tested the effects of a calcium channel blocker, felodipine (10(-5), 10(-6), and 10(-7) mol/L), on nitrate and nitrite (NOx) generation, Ca2+-dependent and -independent NOS activity, and eNOS and iNOS protein masses in proliferating and quiescent rat aortic endothelial cells in culture. Compared with vehicle alone, felodipine significantly increased NOx generation, Ca2+-dependent NOS activity, and eNOS protein mass in proliferating and quiescent endothelial cells. Felodipine did not modify the stimulatory action of 10% fetal calf serum on DNA synthesis (thymidine incorporation) and cell proliferation. Ca2+-independent NOS activity and iNOS protein expression were negligible and unaffected by calcium channel blockade. NOx production and NOS expression were greater in proliferating cells than in quiescent cells. Thus, calcium channel blockade upregulates endothelial NO production in vitro, confirming our previous in vivo study. This observation indicates that the reductions in cytosolic [Ca2+] and vasodilation with calcium channel blockade are not only due to inhibition of Ca2+ entry but also to an NO-cGMP mediated mechanism.     

HMN-1180, a small molecule inhibitor of neuronal nitric oxide synthase

A newly synthesized isoquinolinesulfonamide, HMN-1180 (1-(5-isoquinolinylsulfonyl)-7-methylhomopiperazine), was shown to have selective inhibitory action against rat neuronal nitric oxide synthase (nNOS) with a Ki value of 5.4 microM. Kinetic analysis indicated that the inhibition was competitive with respect to L-arginine but not to calmodulin (CaM). However HMN-1180 exhibited no significant influence up to a concentration of 1 mM on activity of endothelial NOS (eNOS) and it was less active toward inducible NOS (iNOS) (IC50 > 100 microM). Moreover, nNOS bound to a HMN-1180-coupled Sepharose column, but eNOS and iNOS did not. These results suggest that inhibition of nNOS activity is due to direct binding of the compound to the L-arginine binding site of the synthase. Several HMN-1180 derivatives were synthesized and analyzed for their inhibitory actions against nNOS, eNOS and iNOS to cast light on its structure-activity relationships. The potency of inhibition proved dependent on the position of methyl group in the homopiperazine molecule. HMN-1180 was also found to inhibit glutamate stimulated NO production generated by nNOS in the human neuroblastoma cell line SK-N-MC, thus indicating that it is useful tool for elucidating the physiological role of nNOS in neuronal function.     

Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis

The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.     

Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies

Guanidines, amidines, S-alkylisothioureas, and other compounds containing the amidine function (-C(=NH)NH2) have been described as inhibitors of the generation of nitric oxide (NO) by NO synthase (NOS). Here we report on the inhibition of the activity of NOS isoforms by compounds in which the amidine function is attached to a nitrogen of 1,2-diazo heterocycles to form N-carboxamidines and related compounds. 1H-Pyrazole-1-carboxamidine HCl (PCA) inhibited the activity of purified inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) isoforms to a similar extent (IC50 = 0.2 microM). 3-Methyl-PCA and 4-methyl-PCA showed reduced potencies, but a preference for iNOS [IC50 = 5 and 2.4 microM, respectively; cf. N(G)-methyl-L-arginine (NMA) IC50 = 6 microM]. Inhibition of purified iNOS by PCAs could be reversed completely by excess L-arginine, while their inhibition of NO production by stimulated RAW macrophages could be reversed by transfer to a drug-free medium. This suggests a competitive mode of inhibition. PCA caused potent concentration-dependent inhibition of the acetylcholine-induced, endothelium-dependent relaxations of precontracted rat thoracic aorta (IC50 = 30 microM). 4-Methyl-PCA inhibited the relaxations only at > or = 300 microM. In contrast, 4-methyl-PCA was more effective than both PCA and NMA in restoring the ex vivo contractility of aortic rings taken from lipopolysaccharide-treated rats. PCA and NMA, but not 4-methyl-PCA, caused marked increases in mean arterial pressure when administered i.v. to anesthetized rats. In conclusion, PCA and related compounds caused potent inhibition of NOS. Substitution of the pyrazole ring reduced potency, but improved selectivity towards iNOS as exemplified by 4-methyl-PCA.     

The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle

Beta1- and beta2-adrenoceptors in heart muscle cells mediate the catecholamine-induced increase in the force and frequency of cardiac contraction. Recently, in addition, we demonstrated the functional expression of beta3-adrenoceptors in the human heart. Their stimulation, in marked contrast with that of beta1- and beta2-adrenoceptors, induces a decrease in contractility through presently unknown mechanisms. In the present study, we examined the role of a nitric oxide (NO) synthase pathway in mediating the beta3-adrenoceptor effect on the contractility of human endomyocardial biopsies. The negative inotropic effects of a beta3-adrenoceptor agonist, BRL 37344, and also of norepinephrine in the presence of alpha- and beta1-2-blockade were inhibited both by a nonspecific blocker of NO, methylene blue, and two NO synthase (NOS) inhibitors, L-N-monomethyl-arginine and L-nitroarginine-methyl ester. The effect of the NOS inhibitors was reversed by an excess of L-arginine, the natural substrate of NOS, but not by D-arginine. Moreover, the effects of the beta3-adrenoceptor agonist on contractility were associated with parallel increases in the production of NO and intracellular cGMP, which were also inhibited by NOS inhibitors. Immunohistochemical staining of human ventricular biopsies showed the expression of the endothelial constitutive (eNOS), but not the inducible (iNOS) isoform of NOS in both ventricular myocytes and endothelial cells. These results demonstrate that beta3-adrenoceptor stimulation decreases cardiac contractility through activation of an NOS pathway. Changes in the expression of this pathway may alter the balance between positive and negative inotropic effects of catecholamines on the heart potentially leading to myocardial dysfunction.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Inhibition of nitric oxide synthesis enhances the expression of inducible nitric oxide synthase mRNA and protein in a model of chronic liver inflammation

In septic shock the inhibition of inducible nitric oxide synthase (iNOS) could be of therapeutic value. However, side effects have to be investigated. Therefore we studied the effects of chronic NOS inhibition on the level of iNOS expression in a model of chronic liver inflammation induced by Corynebacterium parvum (C. parvum) which causes sustained iNOS expression in the liver. NOS inhibitors decreased the rise in plasma levels and urinary excretion of nitrite/nitrate by about 50%; however, iNOS mRNA and protein were increased to 200% and 150%, respectively. Thus chronic inhibition of NOS can result in an increase in iNOS mRNA level and protein under conditions when iNOS is expressed. This could result in an overproduction of NO upon removal of the NOS-inhibitor.     

Enhanced ultrasonography in the diagnosis of renal tumors

Possible impairment of the L-arginine-nitric oxide (NO) pathway in the rostral ventrolateral medulla of adult spontaneously hypertensive rats (SHR) was investigated by microinjecting N(G)-nitro-L-arginine methyl ester (L-NAME), NOC 18 (an NO donor), or L-arginine. Unilateral injection of L-NAME (10 nmol/50 nL) into the rostral ventrolateral medulla significantly increased mean arterial pressure (MAP) in both SHR and Wistar-Kyoto rats (WKY). The increases in MAP did not differ significantly between the two strains (15+/-3 versus 10+/-2 mm Hg, respectively; n=8). In contrast, microinjection of L-arginine elicited significant (P<.05) dose-dependent decreases in MAP in both strains, and these depressor responses were significantly greater in SHR than in WKY (in 10 nmol of L-arginine: -29+/-2 versus -15+/-2 mm Hg, respectively; n=8, P<.01). Similarly, microinjection of NOC 18 (10 nmol/50 nL) reduced MAP in both strains, and the depressor response was also significantly greater in SHR than in WKY (-38+/-7 versus -22+/-3 mm Hg, respectively; n=8, P<.05). These results suggest that the L-arginine-NO pathway in the rostral ventrolateral medulla is impaired in SHR and that this impairment may contribute to the increase in arterial pressure in this animal model of genetic hypertension.     

Corneal penetration of diclofenac from a fixed combination of diclofenac-gentamicin eyedrops

Recent studies suggest a dual role for nitric oxide (NO) in tumour biology. High concentrations of NO can mediate tumouricidal activity, whereas lower concentrations have been shown to promote tumour growth. In this study, NO synthase (NOS) activity was investigated in cells that were prepared from tissue from primary and metastatic sites and from malignant effusions in 41 cases of suspected ovarian cancer. NO biosynthesis, determined by nitrite + nitrate (NOx) accumulation in medium from cultured cells prepared from disaggregated tumours or effusions and indicative of the inducible NO synthase isoform, was detected in 37% of the cases investigated (range 10.2-114 microM). There was a significant relationship between NOx and tumour differentiation (P = 0.014), with NOx being significantly higher for the more differentiated tumours. NOS activity, determined by the conversion of radiolabelled L-arginine to citrulline by tissue or cell extracts, was detected in 29% of cases (range 0.9-6.9 pmol/min per mg of protein), with all samples tested being moderately or poorly differentiated. Seventy percent of this activity was calcium dependent, indicative of constitutive NOS isoforms. Morphological and immunohistochemical assessment of tumour samples indicated a significant relationship between high macrophage content and NOS activity (as NOx biosynthesis) (rs = 0.726, N = 16, P < 0.01). The relationship between NOS expression, immune response, and disease progression is complex and not simply dependent on the differentiation status of ovarian cancer.     

Regulation of the endogenous NO pathway by prolonged inhaled NO in rats

Nitric oxide (NO) modulates the endogenous NO-cGMP pathway. We determined whether prolonged inhaled NO downregulates the NO-cGMP pathway, which may explain clinically observed rebound pulmonary hypertension. Rats were placed in a normoxic (N; 21% O2) or hypoxic (H; 10% O2) environment with and without inhaled NO (20 parts/million) for 1 or 3 wk. Subsequently, nitric oxide synthase (NOS) and soluble guanylate cyclase (GC) activity and endothelial NOS (eNOS) protein levels were measured. Perfusate cGMP levels and endothelium-dependent and -independent vasodilation were determined in isolated lungs. eNOS protein levels and NOS activity were not altered by inhaled NO in N or H rats. GC activity was decreased by 60 +/- 10 and 55 +/- 11% in N and H rats, respectively, after 1 wk of inhaled NO but was not affected after 3 wk. Inhaled NO had no effect on perfusate cGMP in N lungs. Inhaled NO attenuated the increase in cGMP levels caused by 3 wk of H by 57 +/- 11%, but there was no rebound in cGMP after 24 h of recovery. Endothelium-dependent vasodilation was not altered, and endothelium-independent vasodilation was not altered (N) or slightly increased (H, 10 +/- 3%) by prolonged inhaled NO. In conclusion, inhaled NO did not alter the endogenous NO-cGMP pathway as determined by eNOS protein levels, NOS activity, or endothelium-dependent vasodilation under N and H conditions. GC activity was decreased after 1 wk; however, GC activity was not altered by 3 wk of inhaled NO and endothelium-independent vasodilation was not decreased.     

Role of NO on pressure-natriuresis in Wistar-Kyoto and spontaneously hypertensive rats

We investigated the role of the endothelium-derived relaxing factor nitric oxide (NO) on pressure-natriuresis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) using in vivo perfusion studies. Differences in the neural and hormonal background to the kidney were minimized by renal denervation and by holding plasma vasopressin, aldosterone, corticosterone, and norepinephrine levels constant by intravenous infusion. In WKY, elevation of renal perfusion pressure (RPP) from 115 to 157 mm Hg increased urinary sodium excretion 4.5 to 14.8 microEq/min/g kidney wt, and the slope of its linear regression was 0.21 microEq/min/g kidney wt/mm Hg. Infusion of an inhibitor of NO synthase, L-NMMA (1 mg/min/kg), lowered this slope (P < 0.05) but L-arginine (3 mg/min/kg) did not change it. By contrast, the impaired pressure-natriuresis response of SHR was ameliorated by L-arginine (slope: 0.08 to 0.16; P < 0.05), while L-NMMA did not blunt it further. GFR and renal plasma flow (RPF) were well autoregulated in both strains, but L-NMMA lowered RPF significantly (SHR: from 4.2 to 2.6 ml/min/g kidney wt; WKY: 4.5 to 2.5 ml/min/g kidney wt). Moreover, when infused simultaneously, all these individual effects of L-NMMA and L-arginine were nullified. These results suggest that NO plays an important role in the pressure-natriuresis mechanism.     

Apoptosis and expression of inducible nitric oxide synthase are mutually exclusive in renal mesangial cells

Nitric oxide (NO) is a multipurpose messenger molecule, important for blood vessel relaxation, neuronal communication, and antimicrobial activities. The generation of NO from L-arginine is catalyzed by NO synthase (NOS). An inducible form of NOS, iNOS, was first characterized in macrophages and then in many other tissues and cells, including renal mesangial cells. Mesangial cells play a crucial role in the regulation of the glomerular filtration rate as well as in the pathophysiology of certain forms of glomerulonephritis in which mesangial cells and macrophages produce NO in high amounts. Because reports have associated NO production with apoptotic cell death in macrophages and we recently demonstrated NO-mediated apoptosis in mesangial cells, we searched for the relationship between in situ iNOS induction and apoptosis by iNOS immunocytochemistry and terminal desoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. RAW 264.7 macrophages exhibited homogeneous iNOS expression and apoptotic nuclei in the iNOS-containing cells upon stimulation with interferon-gamma and lipopolysaccharide. In contrast, stimulated rat mesangial cells stained heterogeneously for iNOS, depending on cell passage and iNOS-stimulating pathway. Mesangial cells expressing iNOS did not display signs of apoptosis and, vice versa, cells showing characteristic features of apoptosis did not stain for iNOS. Thus, our study suggests that mesangial cells react to stimulation by interleukin-1 and/or cAMP-elevating compounds with mutually exclusive responses, either by expression of iNOS or by undergoing programmed cell death.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Molecular basis of the pleiotropic phenotype of mice carrying the hypervariable yellow (Ahvy) mutation at the agouti locus

Nitric oxide (NO)-related activity has been shown to be protective against Plasmodium falciparum in vitro. It has been hypothesized, however, that excess NO production contributes to the pathogenesis of cerebral malaria. The purpose of this study was to compare markers of NO production [urinary and plasma nitrate + nitrite (NOx)], leukocyte-inducible nitric oxide synthase type 2 (NOS2), and plasma TNF-alpha and IL-10 levels with disease severity in 191 Tanzanian children with and without malaria. Urine NOx excretion and plasma NOx levels (corrected for renal impairment) were inversely related to disease severity, with levels highest in subclinical infection and lowest in fatal cerebral malaria. Results could not be explained by differences in dietary nitrate ingestion among the groups. Plasma levels of IL-10, a cytokine known to suppress NO synthesis, increased with disease severity. Leukocyte NOS2 antigen was detectable in all control children tested and in all those with subclinical infection, but was undetectable in all but one subject with cerebral malaria. This suppression of NO synthesis in cerebral malaria may contribute to pathogenesis. In contrast, high fasting NOx levels and leukocyte NOS2 in healthy controls and asymptomatic infection suggest that increased NO synthesis might protect against clinical disease. NO appears to have a protective rather than pathological role in African children with malaria.     

Aorta and skeletal muscle NO synthase expression in experimental heart failure

Nitric oxide (NO), the free radical that accounts for the biological activity of endothelium-derived relaxing factor, is synthesized from L-arginine by NO synthase (NOS). There is evidence that NO availability is reduced in the peripheral vasculature of patients with congestive heart failure (CHF). The aim of this study was to investigate the expression of NOS in the descending aorta and in the skeletal muscles of rats subjected to heart failure. The alkaloid, monocrotaline, was used to induce pulmonary hypertension and cardiac failure in rats. The expression of both the constitutive (ecNOS) and the inducible (iNOS) isoforms of the enzyme was assessed by Western blot analysis. In CHF animals, the ecNOS location in the aorta is altered: the endothelial protein expression is substantially reduced (from 0.083 +/- 0.012 to 0.003 +/- 0.004 OD/microgram total proteins, P < 0.001) whereas the expression of ecNOS in the smooth muscle is increased (from 0.024 +/- 0.004 to 0.059 +/- 0.009 OD/ microgram total proteins, P < 0.01). The total aortic ecNOS is diminished in CHF respect to control animals (0.062 +/- 0.009 v 0.107 +/- 0.013 OD/microgram total proteins, P < 0.01). On the contrary, no difference in ecNOS protein expression was observed in the extensor digitorum longus and soleus muscles. Furthermore, iNOS was not detected in any of the tissues considered. In conclusion, experimental CHF causes a re-setting of the ecNOS protein expression in the descending aorta but not in skeletal muscles. The reduced abundance of ecNOS in the aortic endothelium is consistent with the impairment of the vasodilating function reported in patients with CHF.