Duane C. Eichler

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.

Correlates of adiponectin and the leptin/adiponectin ratio in obese and non-obese children.

Adiponectin is an adipocyte secreted protein that has been reported to increase fatty acid oxidation and improve insulin sensitivity. Our aim was to study the relationship between adiponectin and leptin, body fat, insulin and lipoproteins in obese compared to non-obese children matched for age and gender. Adiponectin serum concentrations were significantly lower in the obese compared to the non-obese children (9.1+/-3.7 vs 17.1+/-12.3 microg/ml, p <0.05), in contrast to serum leptin concentrations which were greater in the obese compared to the non-obese subjects (31.8+/-11.1 vs 8.2+/-5.7 ng/ml, p <0.001). When considered as a single group to assess adiponectin concentrations over a spectrum of body size, adiponectin values correlated inversely with body weight (r = -0.33, p <0.05) and BMI (r = -0.35, p <0.05). Adiponectin values correlated directly with HDL-C (r = 0.47, p <0.005), but not with total cholesterol, IGF-I, or leptin binding activity. Since leptin and adiponectin change inversely in relation to BMI, the leptin/adiponectin (L/A) ratio was determined as a potential index relating adiposity to the development of complications of obesity. The L/A ratio was eight-fold greater in the obese compared to the non-obese children, and correlated more strongly with BMI (r = 0.779, p <0.0001) and with HDL-C (r = -0.53, p <0.001), than did adiponectin alone. The L/A ratio also correlated significantly with triceps skinfold thickness (TSF) (r = 0.77, p <0.001) and percent body fat (r = 0.79, p <0.0001) in non-obese children. These data suggest that adiponectin concentrations are already differentially regulated in childhood obesity. The index of increased leptin concentration corrected by reduced adiponectin values (L/A ratio) merits investigation as a marker for morbidities associated with childhood obesity.

Insulin Regulates Protein Kinase CβII Expression through Enhanced Exon Inclusion in L6 Skeletal Muscle Cells A NOVEL MECHANISM OF INSULIN- AND INSULIN-LIKE GROWTH FACTOR-I-INDUCED 5′ SPLICE SITE SELECTION

The protein kinase Cβ (PKCβ) gene encodes two isoforms, PKCβI and PKCβII, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCβ pre-mRNA was examined in L6 myotubes. Mature PKCβII mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCβI mRNA and protein levels remained unchanged. Mature PKCβII mRNA resulted from inclusion of the PKCβII-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCβII expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCβII mRNA, and suggest that PKCβII expression is regulated primarily at the level of alternative splicing. Insulin effects on exon inclusion were observed as early as 15 min after insulin treatment; by 20 min, a new 5′-splice site variant of PKCβII was also observed. After 30 min, the longer 5′-splice site variant became the predominate species through activation of a downstream 5′ splice site. Similar results were obtained using IGF-I. Although the role of this new PKCβII mRNA species is presently unknown, inclusion of either PKCβII-specific exon results in the same PKCβII protein.

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.

Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCβII mRNA in vascular smooth muscle cells

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCβ gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCβII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCβI levels were unaltered. PKCβ mRNA levels were depleted by 60–75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCβ expression via the common promoter for PKCβI and PKCβII, deletion constructs of the PKCβ promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (−411 to + 179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5′ promoter region of the PKCβ gene. In actinomycin D‐treated A10 cells, a 60% decrease in PKCβ mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose‐induced posttranscriptional destabilization of PKCβII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to post‐transcriptionally destabilize PKCβII mRNA, but not the PKCβI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.—Patel, N. A., Chalfant, C. E., Yamamoto, M., Watson, J. E., Eichler, D. C., Cooper, D. R. Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCβII mRNA in vascular smooth muscle cells. FASEB J. 13, 103–113 (1999)

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.

Nucleotide-specific antibodies as potential blocking agents in the structural analysis of nucleic acids

Abstract Anti-nucleotide antibodies might be useful blocking agents in directing the nuclease catalyzed hydrolysis of RNA for sequence analysis, but only if the antibodies recognized a nucleotide or sequence with sufficient specificity and affinity to compete with a nuclease at only specific sites in an RNA. As a test, antibodies directed against mononucleotide haptens were induced with 5′-nucleotide (oxidized with periodate) coupled to bovine serum albumin. Nucleotide-specific antibodies, isolated by affinity chromatography on nucleotide-agarose columns, were at least 95 % pure 7-S γ-globulin. The antibodies precipitated RNA, homologous nucleotide-rabbit albumin conjugates, and to a lesser extent, heterologous conjugates. Antibodies further purified by passage through an affinity column of heterologous nucleotides showed specificity for the single homologous nucleotide. Equilibrium dialysis measurements gave dissociation constants for the antibody-homologous nucleotide complex of about 10 −5 M, while binding of a heterologous nucleotide was insufficient to be measured. The 5′-phosphate group, the base, and the ribose derivative in the conjugate were all elements of structure recognized by the antibodies. Hydrolysis of antibodies with insolubilized papain produced monovalent Fab fragments with dissociation constants essentially as determined for intact antibodies. Antibodies were found to compete with pancreatic ribonuclease for dinucleoside phosphate substrates in a manner consistent with the binding results. Anti-nucleotide Fab fragments inhibited the nuclease hydrolysis of f2 RNA, but no directed cleavage to give altered RNA fragmentation patterns was observed.

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.

Leptin and the adipocyte endocrine system

Although adipose tissue has long been considered to be metabolically passive and primarily responsible for energy storage, recent scientific advances have dramatically altered our understanding of the function of this ubiquitous tissue. The fat cell is a transducer of energy supply for the changing metabolic needs of the body, modulating glucose homeostasis, hypothalamic function, sympathetic output, vascular tone, immune response, and reproduction. Through endocrine/autocrine and paracrine actions, adipocyte-derived molecules defend the body during periods of energy deficit and stress. With the development of obesity, maladaptive responses to adipose excess result in pathologic states of inflammation, coagulopathy, and altered insulin sensitivity.

Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues.