Alberto Nasjletti

Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors

Rat renal interlobar arteries express heme oxygenase 2 (HO-2) and manufacture carbon monoxide (CO), which is released into the headspace gas. CO release falls to 30% and 54% of control, respectively, after inhibition of HO activity with chromium mesoporphyrin (CrMP) or of HO-2 expression with antisense oligodeoxynucleotides (HO-2 AS-ODN). Patch-clamp studies revealed that CrMP decreases the open probability of a tetraethylammonium-sensitive (TEA-sensitive) 105 pS K channel in interlobar artery smooth muscle cells, and that this effect of CrMP is reversed by CO. Assessment of phenylephrine-induced tension development revealed reduction of the EC(50) in vessels treated with HO-2 AS-ODN, CrMP, or TEA. Exogenous CO greatly minimized the sensitizing effect on agonist-induced contractions of agents that decrease vascular CO production, but not the sensitizing effect of K channel blockade with TEA. Collectively, these data suggest that vascular CO serves as an inhibitory modulator of vascular reactivity to vasoconstrictors via a mechanism that involves a TEA-sensitive K channel.

Angiotensin II-induced hypertension in the rat. Effects on the plasma concentration, renal excretion, and tissue release of prostaglandins.

We examined in rats the effects of intraperitoneal angiotensin II (AII) infusion for 12 d on urinary excretion, plasma concentration, and in vitro release of prostaglandin (PG) E2 and 6-keto-PGF1 alpha, a PGI2 metabolite. AII at 200 ng/min increased systolic blood pressure (SBP) progressively from 125 +/- 3 to 170 +/- 9 mmHg (P less than 0.01) and elevated fluid intake and urine volume. Urinary 6-keto-PGF1 alpha excretion increased from 38 +/- 6 to 55 +/- 5 and 51 +/- 7 ng/d (P less than 0.05) on days 8 and 11, respectively, of AII infusion, but urinary PGE2 excretion did not change. Relative to a control value of 129 +/- 12 pg/ml in vehicle-infused (V) rats, arterial plasma 6-keto-PGF1 alpha concentration increased by 133% (P less than 0.01) with AII infusion. Aortic rings from AII-infused rats released more 6-keto-PGF1 alpha (68 +/- 7 ng/mg) during 15-min incubation in Krebs solution than did rings from V rats (40 +/- 3 ng/mg); release of PGE2, which was less than 1% of that of 6-keto-PGF1 alpha, was also increased. Slices of inner renal medulla from AII-infused rats released more 6-keto-PGF1 alpha (14 +/- 1 ng/mg) during incubation than did slices from V rats (8 +/- 1 ng/mg, P less than 0.05), but PGE2 release was not altered. In contrast, AII infusion did not alter release of 6-keto-PGF1 alpha or PGE2 from inferior vena cava segments or from renal cortex slices. Infusion of AII at 125 ng/min also increased SBP, plasma 6-keto-PGF1 alpha concentration, and in vitro release of 6-keto-PGF1 alpha from rings of aorta and renal inner medulla slices; at 75 ng/min AII had no effect. SBP on AII infusion day 11 correlated positively with both 6-keto-PGF1 alpha plasma concentration (r = 0.54) and net aortic ring release (r = 0.70) when data from all rats were combined. We conclude that augmentation of PGI2 production is a feature of AII-induced hypertension. The enhancement of PGI2 production may be an expression of nonspecific alteration in vascular structure and metabolic functions during AII-induced hypertension, as well as the result of a specific effect of the peptide on the arachidonate-prostaglandin system.

Heme oxygenase attenuates angiotensin II-mediated increase in cyclooxygenase-2 activity in human femoral endothelial cells.

Abstract—Heme oxygenase (HO) regulates cellular heme levels and catalyzes the formation of bilirubin and carbon monoxide. We hypothesize that the status of the endothelial HO system influences the angiotensin (Ang) II-induced increase in the endothelial production of prostaglandin I2 (PGI2) (measured as 6-keto-PGF1&agr;) and prostaglandin E2 (PGE2), eicosanoids that modulate the vascular actions of Ang II. In the present study, we determined the effect of interventions that suppress HO activity or induce HO-1 gene expression on Ang II-mediated increase in 6-keto-PGF1&agr;and PGE2 in cultures of human femoral artery endothelial cells. Incubation of endothelial cells with Ang II (100 ng/mL) for 24 hours increased the levels of both 6-keto-PGF1&agr; and PGE2 in the culture media. This effect of Ang II on prostaglandin production by endothelial cells was attenuated in cells treated with SnCl2 (10 &mgr;mol/L), an inducer of HO-1, but was magnified in cells treated with the HO inhibitor ZnDPP or heme. Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene also attenuated heme and Ang II-induced prostaglandin synthesis. Of note, prostaglandin synthesis by lysates of endothelial cells stimulated with heme or Ang II appear to involve COX-2, because it was blunted by NS-398, which is presumed to inhibit COX-2 specifically. These results indicate that overexpression of the HO system exerts an inhibitory influence on Ang II-induced synthesis of prostaglandins by endothelial cells.

Regular ArticlesThe Krebs Cycle and Mitochondrial Mass Are Early Victims of Endothelial Dysfunction: Proteomic Approach

Endothelial cell dysfunction is associated with bioavailable nitric oxide deficiency and an excessive generation of reactive oxygen species. We modeled this condition by chronically inhibiting nitric oxide generation with subpressor doses of N(G)-monomethyl-L-arginine (L-NMMA) in C57B6 and Tie-2/green fluorescent protein mouse strains. L-NMMA-treated mice exhibited a slight reduction in vasorelaxation ability, as well as detectable abnormalities in soluble adhesion molecules (soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1, and matrix metalloproteinase 9), which represent surrogate indicators of endothelial dysfunction. Proteomic analysis of the isolated microvasculature using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy revealed abnormal expression of a cluster of mitochondrial enzymes, which was confirmed using immunodetection. Aconitase-2 and enoyl-CoA-hydratase-1 expression levels were decreased in L-NMMA-treated animals; this phenotype was absent in nitric oxide synthase-1 and -3 knockout mice. Depletion of aconitase-2 and enoyl-CoA-hydratase-1 resulted in the inhibition of the Krebs cycle and enhanced pyruvate shunting toward the glycolytic pathway. To assess mitochondrial mass in vivo, co-localization of green fluorescent protein and MitoTracker fluorescence was detected by intravital microscopy. Quantitative analysis of fluorescence intensity showed that L-NMMA-treated animals exhibited lower fluorescence of MitoTracker in microvascular endothelia as a result of reduced mitochondrial mass. These findings provide conclusive and unbiased evidence that mitochondriopathy represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward metabolic hypoxia through the selective depletion of both aconitase-2 and enoyl-CoA-hydratase-1. These findings may contribute to an early preclinical diagnosis of endothelial dysfunction.

Carbon Monoxide Stimulates the Ca2+–Activated Big Conductance K Channels in Cultured Human Endothelial Cells

We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca2+-activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, NG-nitro-l-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with NG-nitro-l-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.

Renal denervation at weaning retards development of hypertension in New Zealand genetically hypertensive rats.

The development of hypertension was studied in 3 to 13-week old New Zealand genetically hypertensive (NZGH) rats subjected to bilateral renal denervation (D) or sham operation (S) at 4 weeks of age. Denervation retarded the development of hypertension and delayed the establishment of stable hypertension; blood pressure of D-NZGH rats was 15–40 mm Hg lower than that of S-NZGH rats from 3 to 7 weeks after surgery, but was similar in the two groups thereafter. D-rat renal catecholaimine content was reduced to 17% of control at 1 week after surgery; by the fourth week post surgery, renal catecholamine content had risen to 40% of control, and blood pressure of the D-group had begun to rise, suggesting that spontaneous renal reinnervation prevented the antihypertensive effect of renal denervation from being a permanent one. During the period when blood pressure of the S-rats was greater than that of the D-rats, urinary sodium excretion of the two groups was not significantly different, suggesting that over this interval the relationship between blood pressure and urinary sodium excretion shifted to the right along the pressure axis in the S-rats but not in the renal-denervated rats. Throughout the 60-day period of observation, urinary excretion of prostaglandin E2 and kallikrein did not differ between the renal-denervated and sham-operated rats. (Hypertension 4: 361–368, 1982)

Interrelationship between central bradykinin and vasopressin in conscious rats

Intracerebroventricular administration of bradykinin (1, 5 and 20 micrograms) into conscious rats resulted in significant dose-dependent increases in the plasma vasopressin concentration, mean arterial blood pressure and heart rate. Peripheral blockade of the pressor action of vasopressin with a vasopressin pressor antagonist (10 micrograms/kg, i.v.) did not cause an attenuation but rather a potentiation and prolongation of the pressor effects of central bradykinin (20 micrograms). Central administration of the vasopressin antagonist (150 ng) caused no peripheral blockade of the pressor effects of exogenous i.v. vasopressin but almost abolished the bradykinin-induced tachycardia, with little effect on the pressor effects of central bradykinin (20 micrograms). The results indicate that centrally administered bradykinin stimulates vasopressin release into the plasma and that central vasopressin may modulate the cardiovascular actions of central bradykinin.

Role of vasopressin in regulation of renal kinin excretion in Long-Evans and diabetes insipidus rats.

To study the relationship between vasopressin and the renal kallikrein-kinin system we measured the rate of excretion of kinins into the urine of anesthetized rats during conditions of increased and decreased vasopressin level. The excretion of immunoreactive kinins in Brattleboro rats with hereditary diabetes insipidus (DI) (24 +/- 3 pg min-1 kg-1) was lower than in the control Long Evans (LE) rats (182 +/- 22 pg min-1 kg-1; P less than 0.05). The DI rats also exhibited negligible urinary excretion of immunoreactive vasopressin, reduced urine osmolality, and increased urine flow and kininogenase excretion. In LE rats, volume expansion by infusion of 0.45% NaCl-2.5% dextrose to lower vasopressin secretion reduced (P less than 0.05) kinin excretion, vasopressin excretion, and urine osmolality to 41, 26, and 15% of their respective control values, while increasing (P less than 0.05) urine flow and kininogenase excretion. On the other hand, the infusion of 5% NaCl, which promotes vasopressin secretion, increased (P less than 0.05) the urinary excretion of kinins and vasopressin to 165 and 396% of control, while increasing (P less than 0.05) urine flow and kininogenase excretion. Infusion of vasopressin (1.2 mU/h, intravenous) enhanced (P less than 0.05) kinin excretion by two to threefold in DI rats and in LE rats during volume expansion with 0.45% NaCl-2.5% dextrose, while decreasing urine flow and increasing urine osmolality. This study demonstrates that the urinary excretion of immunoreactive kinins varies in relation to the urinary level of vasopressin, irrespective of urine volume and osmolality and of the urinary excretions of sodium and kininogenase. The study suggests a role for vasopressin in promoting the activity of the renal kallikrein-kinin system in the rat.

Enhancement of renal medulla prostaglandin synthetase activity by dexamethasone treatment in the rat

We studied in rats the effect of dexamethasone (2.5 mg/kg per week) on the conversion of radiolabeled arachidonic acid to prostaglandins by renal medulla slices, microsomes, and homogenates. The steroid did not affect the rate of conversion of arachidonic acid to prostaglandins by renal medulla slices, but significantly increased the rate of conversion by both the microsomes and the 10,000 X g supernatant of renal medulla homogenates. We conclude (a) that dexamethasone treatment increases the activity of renal medulla prostaglandin synthetase measured in broken cells preparations, and (b) that such a change in enzyme activity is not manifested by augmentation of prostaglandin synthesis in renal medulla slices incubated with exogenous arachidonic acid.

Activation of a latent kinin-generating proteinase in the porcine anterior pituitary

This study was conducted to determine whether a kinin-generating proteinase (kininogenase) previously described in the porcine anterior pituitary exists in a latent form. Porcine anterior pituitaries were homogenized in 0.25 M sucrose (pH 7.5) and sequentially centrifuged at 1000 X g for 5 min, 1500 X g for 20 min, 10 000 X g for 20 min, and 105 000 X g for 60 min. The various fractions were assayed for their ability to generate kinins from kininogen and cleave H-D-Pro-Phe-Arg-p-nitroanilide (S-2302) before or after various activation procedures. Untreated pituitary fractions had a small amount of proteolytic activity. However, large increases in kininogenase and S-2302 hydrolytic activity were observed in the 105 000 X g pellet after dialysis, or incubation with trypsin. Repeated freezing and thawing, detergents, phospholipase A2, melittin, plasmin, thrombin, urokinase and Factor Xa failed to activate kininogenase activity in the 105 000 X g pellet. However, plasmin produced massive increases in S-2302 hydrolytic activity. The kininogenase and S-2302 hydrolytic activity was sensitive to inhibition by soybean trypsin inhibitor and aprotinin, and had a broad pH optimum between 7 and 9. The data indicate that the porcine anterior pituitary kininogenase largely exists in a latent form. Also, the porcine anterior pituitary appears to contain an additional latent proteinase which can hydrolyze S-2302.