Treatment with polyamine oxidase inhibitor reduces microglial activation and limits vascular injury in ischemic retinopathy.

Retinal vascular injury is a major cause of vision impairment in ischemic retinopathies. Insults such as hyperoxia, oxidative stress and inflammation contribute to this pathology. Previously, we showed that hyperoxia-induced retinal neurodegeneration is associated with increased polyamine oxidation. Here, we are studying the involvement of polyamine oxidases in hyperoxia-induced injury and death of retinal vascular endothelial cells. New-born C57BL6/J mice were exposed to hyperoxia (70% O2) from postnatal day (P) 7 to 12 and were treated with the polyamine oxidase inhibitor MDL 72527 or vehicle starting at P6. Mice were sacrificed after different durations of hyperoxia and their retinas were analyzed to determine the effects on vascular injury, microglial cell activation, and inflammatory cytokine profiling. The results of this analysis showed that MDL 72527 treatment significantly reduced hyperoxia-induced retinal vascular injury and enhanced vascular sprouting as compared with the vehicle controls. These protective effects were correlated with significant decreases in microglial activation as well as levels of inflammatory cytokines and chemokines. In order to model the effects of polyamine oxidation in causing microglial activation in vitro, studies were performed using rat brain microvascular endothelial cells treated with conditioned-medium from rat retinal microglia stimulated with hydrogen peroxide. Conditioned-medium from activated microglial cultures induced cell stress signals and cell death in microvascular endothelial cells. These studies demonstrate the involvement of polyamine oxidases in hyperoxia-induced retinal vascular injury and retinal inflammation in ischemic retinopathy, through mechanisms involving cross-talk between endothelial cells and resident retinal microglia.

Endoplasmic reticulum stress-regulated CXCR3 pathway mediates inflammation and neuronal injury in acute glaucoma.

Acute glaucoma is a leading cause of irreversible blindness in East Asia. The mechanisms underlying retinal neuronal injury induced by a sudden rise in intraocular pressure (IOP) remain obscure. Here we demonstrate that the activation of CXCL10/CXCR3 axis, which mediates the recruitment and activation of inflammatory cells, has a critical role in a mouse model of acute glaucoma. The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. Blockade of the CXCR3 pathway by deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1ß and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia.

Arginase 2 deficiency reduces hyperoxia-mediated retinal neurodegeneration through the regulation of polyamine metabolism.

Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N'-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism.

The role of arginase I in diabetes-induced retinal vascular dysfunction in mouse and rat models of diabetes.

AIMS/HYPOTHESIS: A reduction in retinal blood flow occurs early in diabetes and is likely to be involved in the development of diabetic retinopathy. We hypothesise that activation of the arginase pathway could have a role in the vascular dysfunction of diabetic retinopathy. METHODS: Experiments were performed using a mouse and rat model of streptozotocin (STZ)-induced diabetes for in vivo and ex vivo analysis of retinal vascular function. For in vivo studies, mice were infused with the endothelial-dependent vasodilator acetylcholine (ACh) or the endothelial-independent vasodilator sodium nitroprusside (SNP), and vasodilation was assessed using a fundus microscope. Ex vivo assays included pressurised vessel myography, western blotting and arginase activity measurements. RESULTS: ACh-induced retinal vasodilation was markedly impaired in diabetic mice (40% of control values), whereas SNP-induced dilation was not altered. The diabetes-induced vascular dysfunction was markedly blunted in mice lacking one copy of the gene encoding arginase I and in mice treated with the arginase inhibitor 2(S)-amino-6-boronohexanoic acid. Ex vivo studies performed using pressure myography and central retinal arteries isolated from rats with STZ-induced diabetes showed a similar impairment of endothelial-dependent vasodilation that was partially blunted by pretreatment of the isolated vessels with another arginase inhibitor, (S)-2-boronoethyl-L-cysteine. The diabetes-induced vascular alterations were associated with significant increases in both arginase I protein levels and total arginase activity. CONCLUSIONS/INTERPRETATION: These results indicate that, in the mouse and rat model, diabetes-induced increases in arginase I were involved in the diabetes-induced impairment of retinal blood flow by a mechanism involving vascular endothelial cell dysfunction.

Oxidative species increase arginase activity in endothelial cells through the RhoA/Rho kinase pathway.

BACKGROUND AND PURPOSE: NO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available l-arginine, reducing NO and increasing superoxide (O(2) (.-)) production via NOS uncoupling. Elevated O(2) (.-) combines with NO to form peroxynitrite (ONOO(-)), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide-induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase (ROCK) pathway. EXPERIMENTAL APPROACH: Arginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO(-) generator (SIN-1) or H(2) O(2). Pretreatment with inhibitors of Rho kinase (Y-27632) or PKC (Gö6976) was used to investigate the mechanism involved in arginase activation. KEY RESULTS: Exposure to SIN-1 (25 µM, 24 h) or H(2) O(2) (25 µM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y-27632, PKC inhibitor, Gö6976 or siRNA to p115-Rho GEF. There was an early activation of p115-Rho GEF (SIN-1, 2 h; H(2) O(2), 1 h) and Rho A (SIN-1, 4 h; H(2) O(2), 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN-1 and H(2) O(2 ) also reduced NOS activity, which was blocked by pretreatment with p115-RhoGEF siRNA. CONCLUSIONS AND IMPLICATIONS: Our data indicate that the oxidative species ONOO(-) and H(2) O(2) increase arginase activity/expression through PKC-mediated activation of RhoA/Rho kinase pathway.

Acute activation of eNOS by statins involves scavenger receptor-B1, G protein subunit Gi, phospholipase C and calcium influx.

BACKGROUND AND PURPOSE: Statins (HMG CoA reductase inhibitors) have beneficial effects independent of reducing cholesterol synthesis and this includes their ability to acutely activate endothelial nitric oxide synthase (eNOS). The mechanism by which this occurs is largely unknown and thus we characterized the pathways by which statins activate NOS, including involvement of scavenger receptor-B1 (SR-B1), which is expressed in endothelial cells and maintains cholesterol concentrations. EXPERIMENTAL APPROACH: Nitric oxide production was monitored in bovine aortic endothelial cells (BAECs) exposed to lovastatin (LOV) or pravastatin (PRA) for 10-20 min, alone or following pre-exposure to the end product of HMG-CoA reductase (mevalonate), G protein inhibitors (pertussis/cholera toxins), phospholipase C (PLC) inhibitor (U-73122), or intracellular and extracellular calcium chelators - BAPTA-AM and EGTA (respectively), or a function blocking antibody to SR-B1. KEY RESULTS: Both statins increased NO production in a rapid, dose-dependent and HMG-CoA reductase-independent manner. Inhibiting Gi protein or PLC almost completely blocked statin-induced NO generation. Additionally, removing extracellular calcium inhibited statin-induced NO production. COS-7 cells co-transfected with eNOS and SR-B1 increased NO production when exposed to LOV or high-density lipoprotein (HDL), an agonist of SR-B1. These effects were not observed in COS-7 cells with eNOS alone or co-transfected with bradykinin receptor 2, indicating specificity for SR-B1. Further, pretreatment of BAEC with blocking antibody for SR-B1 blocked NO responses to statins and HDL. CONCLUSIONS AND IMPLICATIONS: LOV and PRA acutely activate eNOS through pathways that include the cell surface receptor SR-B1, Gi protein, phosholipase C and entry of extracellular calcium into endothelial cells.

Vascular endothelial growth factor in eye disease.

Collectively, angiogenic ocular conditions represent the leading cause of irreversible vision loss in developed countries. In the US, for example, retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration are the principal causes of blindness in the infant, working age and elderly populations, respectively. Evidence suggests that vascular endothelial growth factor (VEGF), a 40kDa dimeric glycoprotein, promotes angiogenesis in each of these conditions, making it a highly significant therapeutic target. However, VEGF is pleiotropic, affecting a broad spectrum of endothelial, neuronal and glial behaviors, and confounding the validity of anti-VEGF strategies, particularly under chronic disease conditions. In fact, among other functions VEGF can influence cell proliferation, cell migration, proteolysis, cell survival and vessel permeability in a wide variety of biological contexts. This article will describe the roles played by VEGF in the pathogenesis of retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the rationales for targeting other VEGF-related modulators of angiogenesis.

Homocysteine induces endothelial dysfunction via inhibition of arginine transport.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. High levels of plasma homocysteine (HCY) increase oxidative stress and reduce endothelial-dependent relaxation. We determined whether hyperhomocysteinemia-induced endothelial dysfunction is mediated through inhibition of cellular transport of L-arginine. In endothelial cells, HCY had a biphasic effect on arginine transport. HCY treatment for 6 hr increased L-arginine uptake by 34%; however, uptake was decreased by 25% after 24 h. HCY caused membrane hyperpolarization during both 6 and 24 h incubation periods, indicating that the negative charge facilitating arginine uptake was maintained. HCY significantly reduced expression of cellular arginine transporter protein (CAT-1) after 24 h treatment; whereas endothelial nitric oxide synthase (eNOS) protein levels and basal eNOS activity were not altered. Nevertheless, nitric oxide (NO) formation was significantly decreased. The antioxidant ascorbic acid prevented the effect of HCY on arginine transport. HCY induced formation of the peroxynitrite biomarker nitrotyrosine, which was blocked by supplemental L-arginine. HCY treatment of aortic rings caused decreased vasorelaxation to acetylcholine, which was prevented by supplemental arginine. In conclusion, HCY decreased NO formation and induced endothelial dysfunction without altering protein level or basal activity of eNOS, but through decreases in function and protein expression of the CAT-1 transporter. Reduced arginine supply may lead to eNOS uncoupling and generation of superoxide, contributing to HCY-induced oxidative stress.

Roles of superoxide, peroxynitrite, and protein kinase C in the development of tolerance to nitroglycerin.

A current hypothesis states that tolerance to nitroglycerin (GTN) involves increased formation of superoxide (O2*-). Studies showing that inhibitors of protein kinase C (PKC) prevent tolerance to GTN suggest the involvement of PKC activation, which can also increase O2*-. We examined the roles of O2*-, peroxynitrite (ONOO-), and PKC activation in GTN tolerance. Pre-exposure of rat aortic rings to GTN (5 x 10(-4) M) for 2 h caused tolerance to the vasodilating effect of GTN, as evidenced by a substantial rightward shift of GTN concentration-relaxation curves. This shift was reduced by treatment of the rings with the antioxidants uric acid, vitamin C, or tempol or the PKC inhibitor chelerythrine. We also found that O2*- generation via xanthine/xanthine oxidase in the bath induced tolerance to GTN. However, responses to nitroprusside were not affected. In vivo tolerance produced in rats by 3-day i.v. infusion of GTN was also almost completely prevented by coinfusion of tempol. In bovine aortic endothelial cells (EC), addition of GTN produced a marked increase in tyrosine nitrosylation, indicating increased ONOO- formation. This action was blocked by prior treatment with uric acid, superoxide dismutase, NG-nitro-L-arginine methyl ester, or chelerythrine. We also demonstrated that GTN translocates the alpha- and epsilonPKC isoforms in EC. However, PKCzeta was not affected by GTN treatment. In conclusion, tolerance to GTN involves enhanced production of O2*- and ONOO- and activation of NO synthase. Furthermore, sustained activation of alpha- and epsilonPKC isozymes in EC by GTN may play a role in development of tolerance.

Evaluation of Sampling Strategies for Determining Incidence of Cranberry Fruit Rot and Fruit Rot Fungi.

Two sampling strategies were compared and sources of variability in the sampling protocols analyzed to optimize sampling methods for studies of cranberry fruit rot that occurs in the field (i.e., field rot). For the first method, fruit were dry picked by hand from randomly assigned quadrats; for the second method, fruit were scooped from harvest floodwaters. Rot incidence, which ranged from 1.8 to 9.7%, did not differ significantly between upland and lowland sites or, in general, between dry-picked and wet-harvested samples. There were no consistent differences between upland and lowland sites in the frequency of isolation of any fungus from either rotten or sound fruit. The incidence of certain saprophytic and soilborne fungi was greater in wet-harvested compared with dry-picked fruit. In general, rot incidence and incidence of various fungal taxa isolated from fruit varied more among samples within sites than among sites. Site type (i.e., upland or lowland) was never a major source of variability. These findings suggest that if the goal were to assess the occurrence of cranberry fruit rot within a region, intensive within-site sampling would be necessary, but site type would not be an important consideration, at least in Wisconsin, where this study was conducted.

Effects of Calcium Salts on the Cranberry Fruit Rot Disease Complex.

Calcium salts were applied during the growing season to fresh-fruit cranberry beds to test their effects on cranberry fruit rot incidence and the incidence of specific fungi isolated from rotten and sound cranberry fruit at the time of harvest and after storage. Calcium salts did not affect fruit rot incidence, nor did they affect the recovery of specific fungi from berries. The field treatments did not result in higher calcium content in mature berries, nor did they affect the force required to penetrate the berry epidermis. Calcium propionate inhibited growth in vitro of Allantophomopsis cytisporea, A. lycopodina, Coleophoma empetri, Fusicoccum putrefaciens, and Physalospora vaccinii. Calcium chloride and calcium nitrate inhibited growth of Coleophoma empetri and Fusicoccum putrefaciens, but these salts enhanced growth of Physalospora vaccinii. P. vaccinii was the fungus most frequently isolated from rotten berries at the time of harvest. The fungi most frequently isolated from rotten berries after several weeks in storage varied among sites. P. vaccinii, which was common in sound fruit at harvest, persisted in sound fruit in storage but was also isolated frequently from rotten berries after storage. A. lycopodina and F. putrefaciens, which were isolated infrequently from sound berries at the time of harvest, were isolated frequently from rotten berries after storage. In two of four trials, no fungi were isolated from a large proportion of fruit that decayed in storage.

Endothelial cell dysfunction in a model of oxidative stress.

BACKGROUND: We have investigated the role of L-arginine in hyperhomocysteinemia (HHCY). L-arginine is the substrate required for NO production by endothelial NOS (eNOS). When L-arginine is limited, NOS acts principally upon O2 to form superoxide (O2.-). Because HHCY causes formation of reactive oxygen species and reduced endothelial-dependent vasodilation, we hypothesized that HHCY decreases NO formation by limiting the cellular supply of L-arginine. MATERIAL AND METHODS: Studies with cultured bovine aortic endothelial cells (ECs) determined effects of HCY on transport of [3H] L-arginine. Effects on L-arginine transporter protein CAT-1 and eNOS protein were assessed by immunoblotting. Peroxynitrite formation was evaluated by an immunoassay for nitrotyrosine levels. eNOS activity in forming NO was determined by assay for 3H-L-arginine to 3H-citrulline conversion. RESULTS: HCY had a depressive effect on arginine transport in ECs. HCY treatment for a 24 hr period decreased arginine uptake by 27%. HCY treatment for 24 hr significantly reduced cellular levels of the CAT-1 arginine transporter protein ( approximately 30%) and increased nitrotyrosine formation, whereas levels of eNOS protein and basal NOS activity were not altered. Nevertheless, total NO production as indicated by citrulline conversion was significantly decreased. Treatment with the antioxidant N-acetylcysteine reversed the HCY effect on arginine transport, suggesting that transporter oxidation may contribute to the endothelial dysfunction. CONCLUSIONS: The association of HCY-induced decreases in NO formation with decreases in function and expression of the arginine transporter in the absence of alterations in eNOS expression or activity suggests a primary role for arginine transport alterations in HHCY. The action of HCY to reduce arginine uptake may accentuate endothelial dysfunction due to generation of O2.- and peroxynitrite formation, which may cause further oxidative injury.

Endothelial nitric oxide synthase is a site of superoxide synthesis in endothelial cells treated with glyceryl trinitrate.

Tolerance to glyceryl trinitrate (GTN) involves superoxide (O(2)(*-)) production by endothelial cells. Nitric oxide synthase (NOS) produces O(2)(*-) when L-arginine (L-arg) is limited. The purpose of this study was to test the hypothesis that GTN stimulates NOS to increase O(2)(*-) synthesis in endothelial cells when L-arg is limited. Production of O(2)(*-) by bovine aortic endothelial cells (BAEC, passages 3 - 5) was determined by spectrophotometrically measuring superoxide dismutase-inhibited reduction of ferricytochrome C to ferrocytochrome C. Cells were incubated in buffer without L-arg. O(2)(*-) production was measured using BAEC either untreated or treated with L-NAME or L-arg alone or following treatment with GTN (10(-9) to 10(-6) M) for 30 min or DPTA NONOate (10(-7) and 10(-6) M) alone or with GTN or DPTA NONOate after pretreatment with nitro-L-arginine methyl ester (L-NAME), L-arg or their inactive enantiomers, D-NAME or D-arg (all 5 x 10(-4) M) (n=6 - 7/group). L-NAME alone produced a 69% reduction in O(2)(*-) levels. Treatment with L-arg alone had no effect. Cells treated with GTN alone exhibited an increase in O(2)(*-). This effect was prevented by pretreatment with either L-NAME or L-arg, and was unaffected by D-NAME or D-arg. We observed a dose-response relationship in O(2)(*-) production to GTN over a range of 10(-9) to 10(-7) M. The NO donor, DPTA-NONOate, unlike GTN, did not have a significant effect on O(2)(*-) production. In conclusion, endothelial NOS is a site of O(2)(*-) synthesis in endothelial cells activated by GTN.

In situ localization of cholesterol in skeletal muscle by use of a monoclonal antibody.

A common perception is that cholesterol, the major structural lipid found in mammalian membranes, is localized nearly exclusively to the plasma membrane of living cells and that it is found in much smaller quantities in internal membranes. This perception is based almost exclusively on cell fractionation studies, in which density gradient centrifugation is used for purification of discrete subcellular membrane fractions. Here we describe a monoclonal antibody, MAb 2C5-6, previously reported to detect purified cholesterol in synthetic membranes (Swartz GM Jr, Gentry MK, Amende LM, Blanchette-Mackie EJ, and Alving CR. Proc Natl Acad Sci USA 85: 1902-1906, 1988), that is capable of detecting cholesterol in situ in the membranes of skeletal muscle sections. Localization of cholesterol, the dihydropyridine receptor of the T tubule, and the Ca(2+)-ATPase of the sarcoplasmic reticulum (SERCA2) by means of double and triple immunostaining protocols clearly demonstrates that cholesterol is primarily localized to the sarcoplasmic reticulum membranes of skeletal muscle rather than the sarcolemmal or T tubule membranes. The availability of this reagent and its ability to spatially localize cholesterol in situ may provide a greater understanding of the relationship between membrane cholesterol content and transmembrane signaling in skeletal muscle.

Vascular and endothelial actions of inhibitors of substance P amidation.

Formation of mature active neuropeptides such as substance P (SP) from their glycine extended precursors entails alpha-amidation of peptide precursors by the sequential enzymatic action of peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL). We reported that these two enzymes that can produce mature active neuropeptides are present in cultured bovine aortic endothelial cells (BAECs). We hypothesize that alpha-amidation of peptides occurs in endothelial cells and that these peptides are critically involved in the overall regulation of cardiovascular function. In this study, this hypothesis was tested using specific amidation inhibitors to determine their effects on the actions of SP and its glycine-extended precursor (SP-Gly). We have found that SP and SP-Gly are equipotent in stimulating nitric oxide (NO) release by BAECs. At 10(-5) M, the specific inhibitors of PAM (4-phenyl-3-butenoic acid; PBA) and PGL (5-acetamido-2,4-diketo-6-phenyl-hexanoic acid and its methyl ester) reduced NO basal release by 40, 34, and 45%, respectively. They also reduced the production of NO induced by SP-Gly by 63, 68, and 69%, respectively, but had no effect on NO production in response to either SP or acetylcholine. SP and SP-Gly also were equipotent in relaxing rat aortic segments. The vasorelaxation to SP-Gly was endothelium dependent and inhibited by the NOS antagonist L-nitroarginine methyl ester (L-NAME), but it was not affected by inhibition of prostaglandin synthesis. Inhibitors of both PAM and PGL significantly reduced the vasorelaxing actions of SP-Gly, whereas responses to SP were not affected. A cumulative infusion of PBA into the femoral artery of rabbits, at final concentrations of 2.4, 24, and 240 microM for 20 min each, increased the vascular resistance (VR), indicating the tonic production of vasodilating amidated peptide(s). This effect was maximum at 60 min after infusion (20.5 +/- 4.7 vs. 8.2 +/- 0.7 mm Hg/ml/min; p < 0.05). These results suggest that endothelial cells can produce mature SP from its SP-Gly precursor and that a product of peptide alpha-amidation tonically stimulates endothelial cell NO release to control vascular tone.

Role of L-arginine in the vascular actions and development of tolerance to nitroglycerin.

The goal of this work was to test the role of nitric oxide synthase (NOS) and its substrate L-arginine in development of tolerance to nitroglycerin's (GTN) vasodilator actions. GTN's effects on NOS activity and NO formation were tested in cultured bovine aortic endothelial cells (BAECs). The arginine to citrulline conversion assay showed that GTN stimulated NOS basal activity in BAECs by approximately 40%, comparable with acetylcholine (ACh)-treated controls. Both effects were blocked by L-NMMA. Photometric assays showed that both GTN and ACh-stimulated NO formation. Both effects were potentiated by L-arginine and inhibited by L-NAME. L-NAME inhibited ACh responses approximately 80% compared with approximately 40% for GTN responses. The aortic ring assay showed that 2 h pretreatment with GTN caused substantial tolerance to GTN's vasodilating effects as evidenced by a 38 fold rightward shift of the concentration-relaxation curve. In contrast to D-arginine, addition of L-arginine substantially inhibited this effect, reducing the rightward shift to 4.4 fold of control values. GTN tolerance was associated with a 40% reduction in L-arginine tissue levels. GTN had a biphasic effect on BAEC uptake of L-arginine, stimulating uptake at 5 and 15 min, and suppressing uptake after 1 and 4 h In summary, acute GTN treatment stimulates endothelial NOS activity in producing NO and increases cellular uptake of L-arginine. Prolonged GTN exposure reduces GTN's vasodilator actions, decreases L-arginine tissue levels and depresses BAECs uptake of L-arginine. Supplementation of L-arginine reduces development of GTN tolerance. These data indicate that GTN tolerance depends in part on activation of the NOS pathway.

Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production.

It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, L-Arg and O(2). The Michaelis-Menten constant (K(m)) of NOS for L-Arg is in the micromolar range; cellular levels of L-Arg are much higher. However, evidence strongly suggests that cellular supply of L-Arg may become limiting and lead to reduced NO and increased superoxide anion (O(-)(2)*) formation, promoting cardiovascular dysfunction. Uptake of L-Arg into cells occurs primarily (approximately 85%) through the actions of a Na(+)-independent, carrier-mediated transporter (system y(+)). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of L-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y(+) transporter activity. A rapidly acting NO donor initially increases L-Arg uptake; however, after longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without L-Arg to substance P and a Ca(2+) ionophore (A-23187) increased O(-)(2)* formation, which was blocked with concurrent presence of L-Arg or the NOS antagonist N(omega)-nitro-L-arginine methyl ester. We conclude that factors including NO itself control y(+) transport function and the production of NO and O(-)(2)*.

Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol-lowering actions.

OBJECTIVES: We tested the hypothesis that pravastatin (PRA) activates endothelial nitric oxide synthase (eNOS). BACKGROUND: Pravastatin has been found to have clinical benefits beyond those predicted by its actions in reducing plasma low density lipoprotein cholesterol (LDL). Both PRA and simvastatin (SIM) are equally effective in reducing LDL, but only PRA reduces platelet aggregation and is an effective vasodilator. Nitric oxide (NO) also inhibits platelet aggregation and vasodilates. METHODS: We determined PRA and SIM effects on vasorelaxation in aortic rings and NO production by cultured bovine aortic endothelial cells. Nitric oxide was measured by using a NO electrode and by an assay for conversion of hemoglobin to methemoglobin. Specificity of NOS activation was tested by using the NOS inhibitor nitro-L-arginine methyl ester (L-NAME, 1 mmol/liter) in the presence or absence of excess L-arginine (L-ARG, 1 mmol/liter). RESULTS: Endothelium-dependent vasorelaxation was maximal with acetylocholine (ACH, 100%), followed by PRA (62.8%) and then SIM (37.1%). Direct measurement of NO confirmed that vasorelaxation is due to NO release and showed that PRA and ACH had similar dose-dependent effects on NO production, while SIM was only 25% to 30% as effective. Methemoglobin assay confirmed these results and demonstrated their specificity for NOS activity. The L-NAME blunted the responses to 45% of initial values. Excess L-ARG reversed this effect and potentiated NO production to 133% of initial levels. CONCLUSIONS: Both PRA and SIM activate eNOS, but SIM is much less effective. Clinical benefits with PRA not explained by LDL reductions may be the result of an independent action of PRA on eNOS activation.

Certain norditerpenoid alkaloids and their cardiovascular action.

Thirteen new derivatives of norditerpenoid alkaloids, namely, 8-deacetyl-8-p-aminobenzoyldelphinine (1), 8-deacetyl-8-anthranoyldelphinine (2), 8-deacetyl-8-(4-hydroxy-3-methoxycinnamoyl)delphinine (3), 16-demethoxy-15,16-didehydro-8-p-anisoyl-14-benzoyldelpho nine (4), 6-acetylheteratisine N-oxide (6), 3,8-diacetylfalconerine (7), 8-stearoylfalconerine (8), 8-linolenylfalconerine (9), 13-acetylpyrodelphinine (11), 13-acetyldelphinine N-oxide (13), N-deacetyl-8,9-diacetyllappaconitine (14), 8, 9-(methylenedioxy)lappaconine (15), and 16-epipyroaconitine N-oxide (17), were prepared, and their structures were established by analysis of spectroscopic data (1D and 2D NMR, HRFABMS). The preliminary in vivo cardiovascular action (hypotensive, bradycardic, and ventricular arrhythmias) of these new compounds was tested in male Sprague-Dawley rats. The results are reported herein.