Nitric oxide-mediated arteriolar dilation after endothelial deformation

Previously, we frequently observed dilation of arterioles after agonist-induced constrictions. We hypothesized that deformation of the endothelium during decreases in diameter of isolated arterioles elicits the release of nitric oxide (NO). In isolated arterioles of rat mesentery, phenylephrine (PE, 10(-7) M)-, U-46619 (10(-7) M)-, and KCl (50 mM)-induced constrictions were followed by potent dilations. Inhibition of NO synthase with N(omega)-nitro-L-arginine (L-NNA, 2 x 10(-4) M) or removal of the endothelium significantly enhanced constriction and reduced the postconstriction dilation. In the presence of 80 mmHg of intraluminal pressure, an increase in extraluminal pressure (P(e)) to 75 mmHg for 20 s and 1 and 2 min decreased vessel diameter. After release of P(e), arterioles dilated as a function of the duration of diameter reduction by P(e). Removal of the endothelium or administration of L-NNA significantly diminished the post-P(e) dilations. In cultured mesenteric arteriolar endothelial cells (EC), PE, U-46619, or KCl did not increase, whereas ACh did increase, the production of NO, as measured by a fluorometric assay for nitrite. Furthermore, when EC, cultured on a stretched silicone membrane, were subjected to deformation by shortening the membrane to 50% of its original length, NO release increased significantly. Based on all of the above, we propose that deformation of EC per se elicits release of NO, a mechanism that modulates arteriolar constriction.     

Altered cardiac metabolic phenotype after prolonged inhibition of NO synthesis in chronically instrumented dogs

Acute inhibition of nitric oxide (NO) synthase causes a reversible alteration in myocardial substrate metabolism. We tested the hypothesis that prolonged NO synthase inhibition alters cardiac metabolic phenotype. Seven chronically instrumented dogs were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1) po) for 10 days to inhibit NO synthesis, and seven were used as controls. Cardiac free fatty acid, glucose, and lactate oxidation were measured by infusion of [(3)H]oleate, [(14)C]glucose, and [(13)C]lactate, respectively. After 10 days of L-NAME administration, despite no differences in left ventricular afterload, cardiac O(2) consumption was significantly increased by 30%, consistent with a marked enhancement in baseline oxidation of glucose (6.9 +/- 2.0 vs. 1.7 +/- 0.5 micromol.min(-1).100 g(-1), P < 0.05 vs. control) and lactate (21.6 +/- 5.6 vs. 11.8 +/- 2.6 micromol.min(-1).100 g(-1), P < 0.05 vs. control). When left ventricular afterload was increased by ANG II infusion to stimulate myocardial metabolism, glucose oxidation was augmented further in the L-NAME than in the control group, whereas free fatty acid oxidation decreased. Exogenous NO (diethylamine nonoate, 0.01 micromol.kg(-1).min(-1) iv) could not reverse this metabolic alteration. Consistent with the accelerated rate of carbohydrate oxidation, total myocardial pyruvate dehydrogenase activity and protein expression were higher (38 and 34%, respectively) in the L-NAME than in the control group. Also, protein expression of the constitutively active glucose transporter GLUT-1 was significantly elevated (46%) vs. control. We conclude that prolonged NO deficiency causes a profound alteration in cardiac metabolic phenotype, characterized by selective potentiation of carbohydrate oxidation, that cannot be reversed by a short-term infusion of exogenous NO. This phenomenon may constitute an adaptive mechanism to counterbalance cardiac mechanical inefficiency.     

Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hyg^r) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hyg^r–β-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hyg^r stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.     

The pentaammineruthenium(III)histidine complex in ribonuclease A: application to the assignment of histidine proton resonances

The assignment of two histidine proton resonances in the proton NMR spectrum of ribonuclease A has been made by forming a paramagnetic complex between pentaammineruthenium(III) and the N-3 nitrogen of a single histidine residue. Reaction of chloropentaammineruthenium(III)dichloride with ribonuclease A in 0.1 m Tris-HCl, pH 7.0, 25°C yields a variety of products in which various histidine residues have been labeled. Cation-exchange chromatography affords the isolation of a specific derivative, labeled at a single histidine residue, that retains 66% of the activity toward the hydrolysis of 2′,3′-cyclic CMP. The site of labeling was determined by peptide mapping to be histidine 105. The binding of ruthenium results in the disappearance of both a histidine C-2 and a C-4 proton resonance from the downfield region of the proton NMR spectrum, as expected from model compound studies. The assignment of these two resonances to histidine 105 is in agreement with a previous assignment (J. L. Markley, 1975, Biochemistry, 14, 3546–3554), thereby demonstrating the potential utility of this ruthenium reagent in the assignment of histidine resonances in the proton NMR spectra of other proteins.     

Commitment to the First Cortical Reorganization During Conjugation in Stylonychia mytilus: An Argument for Homology with Cortical Development During Binary Fission

The asexual nature of the first cortical reorganization of conjugation in Stylonychia was analyzed by comparing the effect of amputation performed at different stages of early conjugation to that performed on vegetative cells at different stages of the cell cycle. Amputation of vegetative cells delineated a point of commitment to binary fission at 0.51–0.57 of the cell cycle. Cells amputated before this point were induced to undergo the regenerative mode of asexual development, but those amputated after this point continued with binary fission. In parallel, during conjugation a similar commitment was made around the time of formation of tight mating-pairs: early conjugants amputated around this time might undergo regeneration, and those operated on after this stage continued with the first cortical reorganization as in typical conjugants. The two mates of a pair might differ in their response to amputation, suggesting that the timing of commitment to the first cortical reorganization is not related to the events of conjugation, but rather is individually determined in the vegetative cycle of the cells before they pair up in mating. These observations provide support for the notion that the first cortical reorganization of conjugants is homologous to the asexual mode of cortical development in dividers, according to the theory of developmental heterochrony in the sexual reproduction of hypotrichs. The timing of commitment to the first cortical reorganization was found to temporally correlate with the entrance of the micronuclei into meiosis. Since the first cortical reorganization can proceed without the micronucleus, this raises the possibility that initiation of micronuclear meiosis is closely coupled with, and may be determined by, the commitment to the first cortical reorganization.     

NO metabolites accumulate in erythrocytes in proportion to carbon dioxide and bicarbonate concentration

It is not known whether the ratio between the concentrations of NO metabolites (NOx) in plasma (pNOx) and in erythrocytes (eNOx) is constant or correlates with chemical parameters of the blood. We measured pH, PO(2), and PCO(2) and calculated bicarbonate concentration in 19 blood samples from the aorta, coronary sinus, and leg veins of 7 dogs. Erythrocytes were then separated by centrifugation and lysed with distilled water, and the lysate was ultrafiltered with a molecular cutoff of 50 kDa to remove the hemoglobin. NOx were measured in plasma and in the ultrafiltrate. NOx concentration was higher in erythrocytes, with eNOx/pNOx ranging from 4.38 to 14.60. Linear and significant correlations were found between the natural logarithm of eNOx/pNOx and PCO(2) (r = 0.70, P < 0.001) or bicarbonate concentration (r = 0.72, P < 0.001). These results demonstrate, for the first time, that plasma NOx cannot be considered as a constant fraction of the total NOx in blood but varies dramatically in proportion to the CO(2)/bicarbonate concentration. To prevent an underestimation of venous-arterial difference of NOx across organs, NOx should be measured in whole blood.