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S S Gross

Publications and source records attributed to S S Gross.

At least 19 recordsLinked to original sources
PubMed2000

The preferred source of arginine for high-output nitric oxide synthesis in blood vessels.

L-arginine is the substrate for nitric oxide (NO) production by each of the 3 NO synthase (NOS) isoforms encoded by the mammalian genome. Despite the pivotal roles of NO in mammalian physiology and pathophysiology, the source of arginine for NO synthesis is not clearly defined. In this context, it is notable that cell types that do not have a complete urea cycle often possess the urea cycle enzymes argininosuccinate synthase and argininosuccinate lyase; together, these enzymes confer the ability to regenerate arginine from the NOS product, L-citrulline. Herein, the authors summarize evidence to support the view that argininosuccinate synthase and argininosuccinate lyase function in an arginine-citrulline cycle, providing a ready source of arginine for high-output NO synthesis. The arginine-citrulline cycle is induced in vascular cells by the same cytokines that trigger iNOS expression and provides the preferred source of substrate for NO production. Evidence suggests that argininosuccinate synthase activity is rate-limiting to high-output NO synthesis and, hence, represents a novel target for the treatment of pathophysiological conditions arising from NO overproduction.

Animals↗
PubMed2000

The autoinhibitory control element and calmodulin conspire to provide physiological modulation of endothelial and neuronal nitric oxide synthase activity.

NO production by the endothelial and neuronal isoforms of nitric oxide synthase (cNOS) is regulated on a moment-to-moment basis by calmodulin binding, triggered by transient elevations in intracellular-free calcium levels. Nonetheless, additional modes of cNOS regulation are implicit in the discoveries of stimuli that elicit a sustained increase in cNOS activity despite undetectable or transient increases in intracellular Ca2+ in endothelial cells; such stimuli include shear-stress, oestrogen, insulin or insulin-like growth factor treatment of endothelial cells. Recently, we identified a peptide insertion within the FMN-binding domain of mammalian NOSs that is unique to calcium-dependent isoforms, and not shared with inducible NOS or ancestral flavoproteins. Evidence suggests that this insertion serves as a fundamental control element, analogous to intrinsic autoinhibitory peptides that have been demonstrated to regulate activity of other calmodulin-dependent enzymes. Thus, the peptide insertion of cNOSs appears to function as structural element that is displaced upon calmodulin binding, resulting in dysinhibition of NO synthesis. Once displaced, the peptide may also be subject to transient chemical modifications and protein-protein interactions that modulate autoinhibitory function. Herein we summarize our present knowledge and speculate on mechanisms by which calmodulin and the autoinhibitory peptide conspire to regulate cNOS activity.

Animals↗
PubMed1999

Glycated serum albumin-induced nitric oxide production in vascular smooth muscle cells by nuclear factor kappaB-dependent transcriptional activation of inducible nitric oxide synthase.

Glycated proteins, including serum albumin, may be involved in the pathogenesis of diabetic vasculopathy. Recent evidence suggests that expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMC) may, in part, promote atherosclerosis by increasing local oxidative stress. We therefore investigated whether VSMC exposed to glycated human serum albumin (GHSA) produce nitric oxide (NO) by increasing iNOS expression through transcriptional activation of the iNOS gene and whether this process is dependent on nuclear factor kappaB (NF-kappaB) activation. Treatment of VSMC with GHSA causes activation of NF-kappaB and the iNOS promoter. Induction of NF-kappaB and the iNOS promoter by GHSA exhibited dose-dependent kinetics at concentrations ranging from 3 to 1000 microgram/ml. GHSA alone was a weak inducer of NO production in VSMC as measured by determining nitrite levels, and interferon-gamma alone was totally ineffective, whereas the combination of GHSA and interferon-gamma was a strong stimulus. This synergy for NO production corresponded to Northern blot analyses of iNOS mRNA expression. Thus, GHSA may promote atherosclerosis in part by activation of NF-kappaB and upregulation of iNOS, thereby fostering local inflammation and oxidative stress.

Animals↗
PubMed1999

Adrenomedullin augments nitric oxide and tetrahydrobioptein synthesis in cytokine-stimulated vascular smooth muscle cells.

OBJECTIVE: Immunostimulants increase nitric oxide (NO) and tetrahydrobiopterin (BH4) synthesis in vascular smooth muscle cells (VSMC) by coinducing expression of an isoform of NO synthase (iNOS) and GTP cyclohydrolase I (GTPCH). GTPCH is the first and rate-limiting enzyme in the synthesis of BH4, a cofactor of NO synthases. Given the adrenomedullin (AM) increases NO production, this effect of AM may involve modulation of BH4 synthesis in cytokine-stimulated VSMC. METHODS: We investigated the effects of AM on the synthesis of NO and BH4, the expression of iNOS and GTPCH mRNA, and the promoter activity of iNOS and GTPCH genes in rat VSMC stimulated with interleukin-1 (IL-1). RESULTS: IL-1 increased both NO and BH4 synthesis as well as the abundance of iNOS and GTPCH mRNA. AM significantly increased both NO and BH4 synthesis caused by IL-1 stimulation. AM also augmented the IL-1-induced increase in the abundance of iNOS and GTPCH mRNA. IL-1 activated the iNOS promoter activity as well as the GTPCH promoter activity in VSMC. AM alone had no effect on the activity of either the iNOS or the GTPCH promoter, nor did it potentiate the activation by IL-1 of either of these promoters. CONCLUSION: These results suggest that AM increases IL-1-induced NO and BH4 synthesis by enhancing the expression of iNOS and GTPCH genes at the post-transcriptional level. Thus, the potentiating effect of AM on NO synthesis appears to be associated with an increased expression of both genes necessary for cellular NO synthesis in VSMC.

Adrenomedullin↗
PubMed1999

Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats.

Immunostimulants trigger vascular smooth muscle cells (VSMC) to express the inducible isoform of NO synthase (iNOS) and increased arginine transport activity. Although arginine transport in VSMC is considered to be mediated via the y+ system, we show here that rat VSMC in culture express the cat-1 gene transcript as well as an alternatively spliced transcript of the cat-2 gene. An RT-PCR cloning sequence strategy was used to identify a 141-base nucleotide sequence encoding the low-affinity domain of alternatively spliced CAT-2A and a 138-base nucleotide sequence encoding the high-affinity domain of CAT-2B in VSMC activated with lipopolysaccharide (LPS) in combination with interferon-gamma (IFN). With this sequence as a probe, Northern analyses showed that CAT-1 mRNA and CAT-2B mRNA are constitutively present in VSMC, and the expression of both mRNAs was rapidly stimulated by treatment with LPS-IFN, peaked within 4 h, and decayed to basal levels within 6 h after LPS-IFN. CAT-2A mRNA was not detectable in unstimulated or stimulated VSMC. Arginine transporter activity significantly increased 4-10 h after LPS-IFN. iNOS activity was reduced to almost zero in the absence of extracellular arginine uptake via system y+. Induction of arginine transport seems to be a prerequisite to the enhanced synthesis of NO in VSMC. Moreover, this work demonstrates tissue expression of CAT mRNAs with use of a model of LPS injection in rats. RT-PCR shows that the expression of CAT-1 and CAT-2B mRNA in the lung, heart, and kidney is increased by LPS administration to rats, whereas CAT-2A mRNA is abundantly expressed in the liver independent of LPS treatment. These findings suggest that together CAT-1 and CAT-2B play an important role in providing substrate for high-output NO synthesis in vitro as well as in vivo and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane arginine transport.

Amino Acid Sequence↗
PubMed1999

Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS.

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 microM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on L-arginine but not D-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 microM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

Acetylcholine↗
PubMed1999

Nocturnal masseter EMG activity of healthy subjects in a natural environment.

Facial pain of patients with craniomandibular disorders might be caused by muscle overload. However, the activity of masticatory muscles of healthy individuals is still unknown. The aim of this study was therefore a first attempt to clarify this question by recording the masseter muscle activity of healthy subjects during sleep by means of portable recorders. The study was performed on 21 healthy subjects selected after telephone and questionnaire screenings and clinical examination from among randomly selected inhabitants of Zürich. The masseter EMG was recorded during seven nights in each subject's natural environment with the electrodes in reproducible position. The signal was analyzed for number, amplitude, and duration of contraction periods defined as signal portions above a threshold which could contain sub-threshold signal portions shorter than the standby time of 5 sec. The signal amplitude was expressed in percent of the amplitude recorded during maximum voluntary clenches (%MVC). An average of 71.9 +/- 28.7 contraction episodes per night (men, 74.7 +/- 30.1; women, 65.0 +/- 23.8; p = 0.043), i.e., of 10.5 +/- 3.8 per hour (men, 11.0 +/- 4.0; women, 9.3 +/- 3.0; p = 0.005), was found. The average mean amplitude was 26.2 +/- 6.4% MVC (men, 27.0 +/- 6.8; women, 24.4 +/- 4.5; p = 0.009). The duration of the episodes had a mode of 0.5 sec, and the group mean of the integral of the amplitude over time was 123.7 +/- 157.9% MVC (men, 138.9 +/- 184.0; women, 85.9 +/- 28.2; p = 0.005). Healthy subjects showed intermittent periods of masseter activity during sleep which, on average, were of rather low intensity and short duration.

Adult↗
PubMed1999

Cell signaling by nitric oxide.

Appreciation of the role of nitric oxide (NO) in mammalian cell biology has toppled the paradigm that biological signaling is initiated exclusively by noncovalent, lock-and-key-type interactions with receptor proteins. Remarkably, nitric oxide is a free radical that signals by chemical reaction with its protein targets, resulting in covalent modifications and a stable alteration in protein structure and function. Although most proteins may be coerced to react with NO in vitro, the specific proteins that are functionally modified by NO within cells will depend on the concentration of NO and the composition of the intracellular milieu. A further level of complexity is introduced into NO signaling by the fact that reactions can occur with NO directly, or secondarily with NO-derived species. Much to the surprise of those who thought that reactive molecules are generated and act only under pathophysiological conditions (e.g., ischemia-reperfusion injury), NO has emerged as a prototype molecule that signals by chemistry in normal physiology. The unique attributes and importance of NO were recently recognized by the Nobel Prize Committee, with their decision to award the 1998 Prize in Medicine to Drs Furchgott, Ignarro, and Murad, pioneers in NO biology. This review surveys what we believe to be the most important mechanisms and targets of signaling by NO.

Animals↗
PubMed1998

The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding.

It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [3H]Nomega-nitro-L-arginine or [3H]BH4, but [3H]BH4 binding was reinstated after extended incubation with excess L-arginine. On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2, 6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated Kd for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 microM versus 2-3 microM). Here we propose that Cys331 plays an important role in stabilizing L-arginine binding to nNOS. Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH4 binding and nitric oxide synthetic activity.

Animals↗
PubMed1998

GTP cyclohydrolase I inhibition by the prototypic inhibitor 2, 4-diamino-6-hydroxypyrimidine. Mechanisms and unanticipated role of GTP cyclohydrolase I feedback regulatory protein.

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered to be a selective and direct-acting inhibitor of GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the pathway for synthesis of tetrahydrobiopterin (BH4). Accordingly, DAHP has been widely employed to distinguish whether de novo BH4 synthesis is required in a given biological system. Although it has been assumed that DAHP inhibits GTPCH by direct competition with substrate GTP, this has never been formally demonstrated. In view of apparent structural homology between DAHP and BH4, we questioned whether DAHP may mimic BH4 in its inhibition of GTPCH by an indirect mechanism, involving interaction with a recently cloned 9.5-kDa protein termed GTPCH Feedback Regulatory Protein (GFRP). We show by reverse transcription-polymerase chain reaction that GFRP mRNA is constitutively expressed in rat aortic smooth muscle cells and further induced by treatment with immunostimulants. Moreover, functional GFRP is expressed and immunostimulant-induced BH4 accumulates in sufficient quantity to trigger feedback inhibition of GTPCH. Studies with DAHP reveal that GFRP is also essential to achieve potent inhibition of GTPCH. Indeed, DAHP inhibits GTPCH by dual mechanisms. At a relatively low concentration, DAHP emulates BH4 and engages the GFRP-dependent feedback inhibitory system; at higher concentrations, DAHP competes directly for binding with GTP substrate. This knowledge predicts that DAHP would preferably target GTPCH in tissues with abundant GFRP.

Animals↗
PubMed1997

An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase.

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]NG-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

Amino Acid Sequence↗
PubMed1997

Argininosuccinate synthetase overexpression in vascular smooth muscle cells potentiates immunostimulant-induced NO production.

Immunostimulants trigger vascular smooth muscle cells (VSMC) to express both the inducible isoform of NO synthase (iNOS) and argininosuccinate synthetase (AS). With constitutively expressed argininosuccinate lyase (AL), AS confers cells with an Arg/Cit cycle that can sustain NO production via continuous regeneration of the NOS substrate, L-arginine (Arg), from the NOS coproduct, L-citrulline (Cit). To assess whether NO synthesis can be rate-limited by Arg recycling, we tested whether AS-overexpressing cells have an enhanced capacity for immununostimulant-induced NO synthesis. Rat VSMC were stably transfected with human AS cDNA in a eukaryotic cell expression vector, driven by a strong viral promoter. AS activity in transfected VSMC exceeded that induced in untransfected cells treated for 24 h with a combination of bacterial lipopolysaccharide and interferon-gamma (LPS/IFN). AS activity was predominantly associated with membranes but was also found in cytosol. Recombinant AS was purified from cytosol and possessed a specific activity exceeding that reported for native AS. Western blotting verified the basal expression of AS antigen in membranes from untreated AS-transfected VSMC and from untransfected VSMC after 24 h exposure to LPS/IFN. Epifluorescence histochemistry revealed a punctate distribution of AS antigen in transfected cells, consistent with a predominant membrane localization. Remarkably, on a per cell basis, LPS/IFN-induced NO production was 3-4-fold greater in AS-transfected cells than untransfected VSMC. In untransfected VSMC, maximal NO production during 48 h required millimolar Arg; notably, Cit was needed at approximately 3-fold higher concentrations than Arg for a comparable NO synthesis rate. In contrast, AS-transfected VSMC utilized Arg and Cit equi-effectively and at much lower concentrations; 100 microM of either precursor supported a maximal rate of NO synthesis for 48 h. The enhanced ability of AS-transfected cells to produce NO, compared with untransfected cells, could not be ascribed to differences in iNOS protein content or LPS/IFN potency for immunoactivation. We conclude that transfection with AS provides a continuous flux of Arg which drives NO synthesis in immunoactivated VSMC. Arg regeneration by AS is rate-limiting to NO synthesis and apparently provides iNOS with a preferred cellular source of Arg. In accord with the reported "channeling" of substrates by urea cycle enzymes, we hypothesize that the Arg/Cit cycle sequesters a discrete pool of recyclable substrate that sustains high-output NO synthesis.

Adjuvants, Immunologic↗
PubMed1997

Nitric oxide, an autocrine regulator of wound fibroblast synthetic function.

Nitric oxide (NO) is synthesized in wounds, but its exact role and cellular source are not known. Wound fibroblasts (WF) are phenotypically characterized by increased collagen synthesis and contractility. We hypothesized that WF may be also phenotypically altered during wound healing to synthesize NO. WF were isolated from polyvinyl alcohol sponges implanted in male Lewis rats and harvested 10 days later. Proliferation in response to 10% fetal bovine serum was assessed by [3H]thymidine incorporation in a microculture system. A fibroblast-populated collagen lattice was used for assaying contractility. Collagen synthesis was determined by measuring the collagenase-sensitive fraction of protein-incorporated [3H]proline. Fibroblasts were incubated in the presence or the absence of 0.5 mM S-methyl-isothio-uronium or 0.5 mM N-monomethyl-L-arginine, both competitive inhibitors of NO synthase. WF spontaneously synthesize and release NO (4.60 +/- 0.29 nmol nitrite/microg DNA/48 h). Normal dermal fibroblasts do not synthesize NO. WF NO synthesis was limited to the first and second passages postharvest and was inhibitable by S-methyl-isothio-uronium (96%) and N-monomethyl-L-arginine (84%). In vivo iNOS expression by WF was confirmed by in situ hybridization and immunohistochemistry. Inhibition of endogenous NO synthesis had no effect on fibroblast proliferation. However, fibroblast-mediated collagen contraction was enhanced (p < 0.01), and collagen synthesis was significantly decreased (p < 0.05) by inhibiting NO synthase. The data show that WF are phenotypically altered during the healing process to synthesize NO, which, in turn, regulates their collagen synthetic and contractile activities.

Animals↗
PubMed1997

Evidence for nitric oxide participation in down-regulation of CYP2B1/2 gene expression at the pretranslational level.

Septic or inflammatory stimuli suppress drug metabolism by cytochrome P-450 in the liver, presumably at the pretranslational level. We have shown previously that nitric oxide is responsible at least in part for the inhibition by bacterial lipopolysaccharide of phenobarbital-induced CYP2B1/2 activity in vivo. This was attributed to the interaction of nitric oxide with heme in the active-center of cytochrome P450, leading to enzyme inactivation. Here, we report that endogeneous nitric oxide also contributes to LPS-induced suppression of CYP2B1/2 in vivo by down-regulating the expression of CYP2B1/2 protein and mRNA.

Animals↗
PubMed1997

Induction of tetrahydrobiopterin synthesis in rat cardiac myocytes: impact on cytokine-induced NO generation.

Because tetra-hydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) formation, we investigated whether BH4 synthesis is required for cytokine-induced NO production in cultured rat cardiac myocytes. The total biopterin content of untreated cardiac myocytes was below our limit of detection. However, treatment with interleukin-1 alpha (IL-1 alpha) + interferon-gamma (IFN-gamma) caused a significant rise in biopterin levels and induced NO synthesis. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I (the rate-limiting enzyme for de novo BH4 synthesis), completely abolished the elevation in biopterin levels induced by IL-1 alpha + IFN-gamma. DAHP also caused a concentration-dependent inhibition of (IL-1 alpha + IFN-gamma)-induced NO synthesis. Similarly, N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, blocked increases in biopterin levels as well as NO synthesis induced by IL-1 alpha + IFN-gamma. Sepiapterin, substrate for BH4 synthesis via the pterin salvage pathway, prevented this inhibition by DAHP or N-acetylserotonin, and this effect was blocked by methotrexate. Sepiapterin and, to a lesser extent, BH4 dose dependently enhanced (IL-1 alpha + IFN-gamma)-induced NO synthesis, suggesting that the concentration of BH4 limits the rate of NO production. Inducible NO synthase mRNA and GTP cyclohydrolase I mRNA were induced by IL-1 alpha + IFN-gamma in parallel. We thus demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by cytokines in cardiac myocytes.

Animals↗
PubMed1996

Characterization of bovine endothelial nitric oxide synthase expressed in E. coli.

Bovine endothelial constitutive nitric oxide synthase (eNOS) was expressed in E. coli as a soluble, catalytically active enzyme using the pCW expression vector coexpressed with a plasmid, pGroELS, encoding the chaperonins groEL and groES. The E. coli BL21 cultures reproducibly synthesized 6-10 mg of recombinant enzyme per liter of culture. The eNOS protein was purified using 2'5'-ADP Sepharose 4B and appeared as a single band of apparent molecular mass 135 kDa on SDS/PAGE. The recombinant resting enzyme is predominantly high spin with an absorbance maximum at 406 nm. The dithionite-reduced, CO-bound form shows an absorbance maximum at 444 nm. The spectral properties of recombinant eNOS from E. coli are identical to those observed with eNOS from stably transfected HEK 293 cells or from baculovirus expression systems. Enzymatic activity of eNOS from E. coli ranged between 68-135 nmol product formed/min/mg at 25 degrees C, using hemoglobin-NO capture or L-citrulline formation assays. The enzyme is replete with heme and flavins and both activity and [3H]-nitroarginine binding were largely dependent on tetrahydrobiopterin. The heterologous expression of eNOS offers a number of advantages over tissue sources of the protein.

Animals↗
PubMed1996

Pyrrolidine dithiocarbamate inhibits immunostimulant-induced tetrahydrobiopterin synthesis in rat vascular smooth muscle.

GTP cyclohydrolase I is the first and rate-limiting enzyme in the synthesis of tetrahydrobiopterin, a cofactor of nitric oxide (NO) synthase. Immunostimulants increase NO and tetrahydrobiopterin synthesis in vascular smooth muscle cells by coinducing NO synthase and GTP cyclohydrolase I gene expression. Given that nuclear factor kappa(B) mediates the induction of NO synthase gene expression by lipopolysaccharide (LPS), the role of nuclear factor kappa(B) in the induction of GTP cyclohydrolase I in LPS-stimulated rat vascular smooth muscle cells was assessed by examining the effects of pyrrolidine dithiocarbamate, an inhibitor of the activation of nuclear factor kappa(B), on the abundance of GTP cyclohydrolase I mRNA and biopterin synthesis. Pyrrolidine dithiocarbamate inhibited both NO and biopterin synthesis induced by LPS in a dose-dependent manner with similar half-maximal inhibitory concentrations, 12 mu M for NO and 17 mu M for biopterin, respectively. At a concentration of 25 mu M, which inhibited NO and biopterin synthesis but caused no cytotoxicity, pyrrolidine dithiocarbamate substantially reduced the LPS-induced increase in the abundance of NO synthase and GTP cyclohydrolase I mRNAs. These results suggest that pyrrolidine dithiocarbamate inhibits LPS-induced NO and biopterin synthesis by inhibiting the expression of NO synthase and GTP cyclohydrolase I genes. Thus, the induction of both genes necessary for cellular NO synthesis in vascular smooth muscle appears to be regulated, at least in part, by a common mechanism: nuclear factor kappa(B) activation.

Animals↗