Larry P. Solomonson

The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells

SUMMARY The enzyme endothelial nitric oxide synthase (eNOS) catalyzes the conversion of arginine, oxygen and NADPH to NO and citrulline. Previous results suggest an efficient, compartmentalized system for recycling of citrulline to arginine utilized for NO production. In support of this hypothesis, the recycling enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase (AL), have been shown to colocalize with eNOS in caveolae, a subcompartment of the plasma membrane. Under unstimulated conditions, the degree of recycling is minimal. Upon stimulation of NO production by bradykinin, however, recycling is co-stimulated to the extent that more than 80% of the citrulline produced is recycled to arginine. These results suggest an efficient caveolar recycling complex that supports the receptor-mediated stimulation of endothelial NO production. To investigate the molecular basis for the unique location and function of endothelial AS and AL, endothelial AS mRNA was compared with liver AS mRNA. No differences were found in the coding region of the mRNA species, but significant differences were found in the 5′-untranslated region (5′-UTR). The results of these studies suggest that sequence in the endothelial AS-encoding gene, represented by position -92 nt to -43 nt from the translation start site in the extended AS mRNA 5′-UTRs, plays an important role in differential and tissue-specific expression. Overall, a strong evidential case has been developed supporting the proposal that arginine availability, governed by a caveolar-localized arginine regeneration system, plays a key role in receptor-mediated endothelial NO production.

Properties of a nitrate reductase of Chlorella

Abstract 1. An NADH-nitrate oxidoreductase (EC 1.6.6.1) of Chlorella has the unusual property of existing in cell-free extracts mainly in the form of an inactive precursor which can be activated by a variety of procedures. This enzyme is associated with a cytochrome of the b type. 2. The inhibitors, azide, cyanate, thiocyanate and nitrite, react rapidly with the enzyme, with kinetics which show that they are competitive with nitrate. 3. The inhibitors, cyanide and hydroxylamine, react slowly with the reduced form of the enzyme to give an inactive product which can slowly be reactivated in the presence of nitrate. There is at least a superficial similarity between the reactivation of the inhibited enzyme and the activation of the enzyme precursor in fresh extracts. 4. Mammalian cytochrome c , dichlorophenolindophenol and ferricyanide can substitute for nitrate as oxidants for NADH in the presence of the enzyme. This “diaphorase” reaction does not require activation, but is fully active in fresh extracts. It is not inhibited by cyanide, hydroxylamine, azide, cyanate, thiocyanate, or by the substrate, nitrate. Oxidized cytochrome c , on the other hand, inhibits the reduction of nitrate by NADH in the presence of the enzyme. 5. Pyridoxal phosphate inhibits both nitrate reductase and cytochrome c reductase to about the same extent.

Nitric oxide release from resting human platelets

In this study, we have investigated the release of nitric oxide from resting human platelets. Nitric oxide was detected and quantitated by either measuring the conversion of oxy-hemoglobin to met-hemoglobin or generation of nitrite and nitrate by the cells. Nitric oxide was released from both intact resting platelets and platelets activated by collagen. Nitric oxide release was proportional to platelet concentration, and was equivalent to approximately 4.5 +/- 0.6 pmol (or 2.8 +/- 0.3 pmol in the presence of prostaglandin I2) and 11.2 +/- 1.3 pmol nitric oxide released per minute per 10(8) cells at 37 degrees C for resting platelets and platelets activated by collagen, respectively. The generation of nitric oxide by resting platelets was linear with respect to time over a two hour period, while the release of nitric oxide from platelets following activation was transient and was linear for only the first 10 min, after which it slowed to completion at approximately 30 min. The release of nitric oxide was it slowed to completion at approximately 30 min. The release of nitric oxide was stimulated by L-arginine, but was inhibited by L-nitro-arginine methyl ester (L-NAME). The inhibitory effect of L-NAME could be reversed by addition of L-arginine. The release of nitric oxide from platelets was also partially inhibited by prostaglandin I2, prostaglandin E1, aspirin and EDTA. The amount of nitric oxide released from resting platelets compared with that released from endothelial cells suggests that platelet-derived nitric oxide may play a significant role in the maintenance of vascular tone and blood flow.

Regulation of nitrate reductase activity by NADH and cyanide

Abstract NADH:nitrate oxidoreductase (EC 1.6.6.1) of Chlorella vulgaris is regulated by oxidation-reduction reactions. With a highly purified preparation of nitrate reductase, a second factor such as cyanide is required in addition to a reductant such as NADH to effect a rapid, reversible conversion of the active form to the inactive form. NADH and cyanide are the most effective combination yet tested, either reagent being effective at concentrations equivalent to the concentration of enzyme. Other reductants such as NADPH or dithionite may be substituted for NADH; and ferrocyanide, sulfide or hydroxylamine may be substituted for cyanide but higher concentrations of these reagents are required to effect a rapid inactivation of the enzyme. The inactive form of the enzyme is rapidly converted to the active form by the addition of ferricyanide. Regulation occurs on the nitrate-reducing moiety. Diaphorase activity is the same in the active and inactive forms. The conversion of the active form to the inactive form is prevented by nitrate, nitrite or flavins. No detectable changes in the absorption spectrum between 240 and 650 nm, or in molecular size, are associated with the inactivation-activation reaction. Both forms of the enzyme are stable after removal of effectors by gel filtration. No correlation was found between the state of activation and the state of oxidation of the cytochrome b associated with nitrate reductase.

Purification and characterization of (Na+ + K+-ATPase. II. Preparation by zonal centrifugation of highly active (Na+ + K+-ATPase from the outer medulla of rabbit kidneys

Abstract 1. 1. A method is described for the preparation by zonal centrifugation of highly active (Na + + K + -ATPase (ATP phosphohydrolase, EC 3.6.1.3) from the outer medulla of rabbit kidney. 2. 2. Analysis of the sedimentation properties of (Na + + K + )-ATPase shows that the enzyme activity is associated with particles of different size with a well-defined equilibrium density in sucrose gradients. 3. 3. A large part of the (Na + + K + )-ATPase in the tissue from the outer medulla can be collected by isopycnic-zonal centrifugation at a position in the sucrose gradient where the degree of contamination by other subcellular structures is relatively low. The specific activity of (Na + + K + )-ATPase in the preparation obtained by isopycnic-zonal centrifugation is 900–1200 μmoles Pi per mg protein per h, and the yield is 1.5–2.5 mg protein per g tissue. The preparation is stable at 0°. 4. 4. Further purification to a specific activity (Na + + K + )-ATPase of 1500 μmoles Pi per mg protein per h is achieved by rate-zonal centrifugation. 5. 5. The purity of the preparation, calculated as the fraction of the total protein which consists of enzyme, is estimated to be 49%. 6. 6. The data suggest that the high purity of (Na + + K + )-ATPase in the preparation from the outer medulla is due to the isolation of plasma membranes with a high density of enzymes sites per unit membrane area.

Activation of nitrate reductase by oxidation

Abstract The activation of the inactive form of the nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) present in cell-free extracts of Chlorella vulgaris Beijerinck requires an oxidizing agent. Ferricyanide causes conversion of the proenzyme to active enzyme within a few minutes, even at 0°C. Molecular oxygen causes a slow activation which requires many hours at room temperature, and never reaches the high activity level attained with ferricyanide. In unfractionated extracts, CO inhibits the activation by molecular O2. The sensitivity of this activation to CO may account for the in vivo sensitivity of nitrate reduction to CO in these algae.

Reversible inactivation of the nitrate reductase of Chlorella vulgaris Beijerinck.

Abstract The ferricyanide-activated NADH:nitrate oxidoreductase (EC 1.6.6.1) of Chlorella vulgaris Beijerinck, in extracts freed of low molecular weight components, undergoes a reversible inactivation on addition of an ultrafiltrate containing cellular components in the molecular weight range 1000–10 000. This inactivation reaction, which goes virtually to completion, accounts for the fact that the enzyme in the original cell extracts is present almost entirely in the inactive form. NADH or NADPH mimic the effect of the unknown components of the ultrafiltrate. In all cases, the inactivation occurs more readily at pH 7.6 than at pH 6.7, and is prevented by nitrate. The inactivated enzyme can be reactivated with ferricyanide. A partial, reversible inactivation also occurs in the absence of any additions, when extracts freed of low molecular weight components, are brought to a pH of 8.8. The diaphorase component of the nitrate reductase is not inactivated to a substantial degree by any of these procedures. Partially purified, activated nitrate reductase is only slowly and partially inactivated by added NADH or NADPH. The ultrafiltrate alone has no effect on the partially purified, activated enzyme, but enhances the inactivating effect of the reduced pyridine nucleotides. The inactivated enzyme can be reactivated with ferricyanide.

Regulation of Endothelial Argininosuccinate Synthase Expression and NO Production by an Upstream Open Reading Frame

Argininosuccinate synthase (AS) catalyzes the rate-limiting step in the recycling of citrulline to arginine, which in endothelial cells, is tightly coupled to the production of nitric oxide (NO). In previous work, we established that endothelial AS mRNA can be initiated from multiple start sites, generating co-expressed mRNA variants with different 5′-untranslated regions (5′-UTRs). One of the 5′-UTRs, the shortest form, represents greater than 90% of the total AS mRNA. Two other extended 5′-UTR forms of AS mRNA, resulting from upstream initiations, contain an out-of-frame, upstream open reading frame (uORF). In this study, the function of the extended 5′-UTRs of AS mRNA was investigated. Single base insertions to place the uORF in-frame, and mutations to extend the uORF, demonstrated functionality, both in vitro with AS constructs and in vivo with luciferase constructs. Overexpression of the uORF suppressed endothelial AS protein expression, whereas specific silencing of the uORF AS mRNAs resulted in the coordinate up-regulation of AS protein and NO production. Expression of the full-length of the uORF was necessary to mediate a trans-suppressive effect on endothelial AS expression, demonstrating that the translation product itself affects regulation. In conclusion, the uORF found in the extended, overlapping 5′-UTR AS mRNA species suppresses endothelial AS expression, providing a novel mechanism for regulating endothelial NO production by limiting the availability of arginine.

Spin-labeled erythrocyte membranes: direct identification of nitroxide-conjugated proteins.

The covalent incorporation of a spin-labeled analog of N-ethyl maleimide into erythrocyte membrane proteins has been monitored by electron paramagnetic resonance spectroscopy and the individually labeled proteins detected by immunoblotting techniques, using an anti-nitroxide antibody, following electrophoretic separation of the membrane components. Spin-label was primarily found in the high molecular weight bands (I and II) with no incorporation in proteins with molecular weights less than 35,000. Increasing the reaction time between the spin-label and ghosts altered both the observed labeling pattern and the epr spectra with an increase in the proportion of strongly-immobilized species.

Stimulation of Receptor-Mediated Nitric Oxide Production by Vanadate

Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore– (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to ≈125 kDa and 2 minor bands corresponding to ≈105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.