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A I Smith

Publications and source records attributed to A I Smith.

At least 19 recordsLinked to original sources
PubMed2006

Developmental expression of ACE2 in the SHR kidney: a role in hypertension?

The abnormal development of the intrarenal renin-angiotensin system (RAS) is thought contribute to adult-onset hypertension in the spontaneously hypertensive rat (SHR). Angiotensin-converting enzyme 2 (ACE2) is a novel enzyme with complementary actions to that of ACE. Recent studies have shown that ACE2 expression is reduced in the adult SHR. However, its regulation in pre-hypertensive animals is unknown. In this study, we examine the developmental expression of ACE2 in the rodent kidney and its temporal expression, as it relates to the development of hypertension in the SHR model. Kidneys from SHR and normotensive Wistar Kyoto (WKY) rats (n=8-12/group) at birth, 6 weeks of age, and adulthood (80 days) were examined. Gene expression and activity of ACE2 were determined by real-time reverse transcription-polymerase chain reaction and quenched fluorescence assays, respectively. Renal expression was localized by in situ hybridization and immunohistochemistry. The expression and ACE2 activity are significantly increased in the SHR kidney at birth. With the onset of hypertension, the tubular expression of ACE2 falls in SHR compared to WKY and remains reduced in the adult SHR kidney. Glomerular expression is paradoxically increased in the SHR glomerulus. The overall developmental pattern of ACE2 expression in the SHR kidney is also modified, with declining expression over the course of renal development. The developmental pattern of ACE2 expression in the SHR kidney is altered before the onset of hypertension, consistent with the key role of the RAS in the pathogenesis of adult-onset hypertension. Further research is required to distinguish the contribution of these changes to the development and progression of hypertension in this model.

Angiotensin-Converting Enzyme 2↗
PubMed2005

Chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2.

BACKGROUND: Angiotensin converting enzyme (ACE) 2 is a recently identified homologue of ACE that may counterregulate the actions of angiotensin (Ang) II by facilitating its breakdown to Ang 1-7. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of cirrhosis but the role of ACE2 in liver disease is not known. AIMS: This study examined the effects of liver injury on ACE2 expression and activity in experimental hepatic fibrosis and human cirrhosis, and the effects of Ang 1-7 on vascular tone in cirrhotic rat aorta. METHODS: In sham operated and bile duct ligated (BDL) rats, quantitative reverse transcriptase-polymerase chain reaction was used to assess hepatic ACE2 mRNA, and western blotting and immunohistochemistry to quantify and localise ACE2 protein. ACE2 activity was quantified by quenched fluorescent substrate assay. Similar studies were performed in normal human liver and in hepatitis C cirrhosis. RESULTS: ACE2 mRNA was detectable at low levels in rat liver and increased following BDL (363-fold; p < 0.01). ACE2 protein increased after BDL (23.5-fold; p < 0.05) as did ACE2 activity (fourfold; p < 0.05). In human cirrhotic liver, gene (>30-fold), protein expression (97-fold), and activity of ACE2 (2.4 fold) were increased compared with controls (all p < 0.01). In healthy livers, ACE2 was confined to endothelial cells, occasional bile ducts, and perivenular hepatocytes but in both BDL and human cirrhosis there was widespread parenchymal expression of ACE2 protein. Exposure of cultured human hepatocytes to hypoxia led to increased ACE2 expression. In preconstricted rat aorta, Ang 1-7 alone did not affect vascular tone but it significantly enhanced acetylcholine mediated vasodilatation in cirrhotic vessels. CONCLUSIONS: ACE2 expression is significantly increased in liver injury in both humans and rat, possibly in response to increasing hepatocellular hypoxia, and may modulate RAS activity in cirrhosis.

Angiotensin I↗
PubMed2004

Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.

Biomarkers, Tumor↗
PubMed2004

Angiotensin-converting enzyme-2: a molecular and cellular perspective.

Angiotensin-converting enzyme-2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein which functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the C-terminus of its substrates. ACE2 efficiently hydrolyses the potent vasoconstrictor angiotensin II to angiotensin (1-7). It is a consequence of this action that ACE2 participates in the renin-angiotensin system. However, ACE2 also hydrolyses dynorphin A (1-13), apelin-13 and des-Arg(9) bradykinin. The role of ACE2 in these peptide systems has yet to be revealed. A physiological role for ACE2 has been implicated in hypertension, cardiac function, heart function and diabetes, and as a receptor of the severe acute respiratory syndrome coronavirus. This paper reviews the biochemistry of ACE2 and discusses key findings such as the elucidation of crystal structures for ACE2 and testicular ACE and the development of ACE2 inhibitors that have now provided a basis for future research on this enzyme.

Angiotensin-Converting Enzyme 2↗
PubMed2004

Identification of angiotensin converting enzyme 2 in the rodent retina.

PURPOSE: An active renin-angiotensin system has been found in the retina of rats and humans. Angiotensin-converting enzyme 2 (ACE2) is a recently discovered enzymatic homologue of Angiotensin-converting enzyme (ACE) that may be an important new component of the renin-angiotensin system (RAS). This study assesses the involvement of ACE2 in the normal and diabetic rodent retina and its modulation by ACE inhibition. METHODS: Sprague-Dawley rats were randomised into three groups, control, diabetes, and diabetes plus ramipril, with diabetes induced with the cell toxin streptozocin and the study run for 24 weeks. ACE2 and ACE gene levels were measured using quantitative real-time polymerase chain reaction (QRT-PCR), ACE2 protein expression was confirmed by Western blotting, and ACE and ACE2 catalytic activity were measured using specific activity assays in the rat retina. Localisation of ACE2 mRNA and protein were determined by in situ hybridisation and immunohistochemistry, respectively. RESULTS: ACE mRNA levels were decreased to approximately 50% in the diabetic retina, but ACE2 mRNA levels were not significantly changed. ACE but not ACE2 gene expression was influenced by ramipril treatment. Following immunostaining, both ACE2 and ACE protein were localised predominantly to the inner nuclear layer (INL) but also to photoreceptors. In the diabetic retina, ACE enzyme activity was decreased, whereas ACE2 enzyme activity was increased. CONCLUSIONS: This study has identified ACE2 gene and catalytically active protein in the rodent retina. In diabetes, the major changes were a decrease in ACE but an increase in ACE2 enzymatic activity. The ACE inhibitor ramipril did not reduce ACE2 enzymatic activity.

Angiotensin-Converting Enzyme 2↗
PubMed2002

Biliary surgery in sickle cell disease: the Jamaican experience.

Of 62 patients with homozygous sickle cell disease subjected to open cholecystectomy for symptomatic gallstones over a 12-year period at the University Hospital of the West Indies, 25 were males and 37 were females. Of these, 27 were paediatric patients aged 18 years or less, and 35 were adults. Preoperative transfusion was selectively administered. All cases presented with right upper quadrant pain and 15 of 62 with obstructive jaundice. Mucocoele of the gallbladder, empyema of the gallbladder and common bile duct stones were detected in 2, 2, and 23 patients, respectively. Exploration of the common bile duct was necessary in 31 cases and a T-tube sited in 15 cases. Twelve of the 62 patients developed acute chest syndrome post operatively (20%). There were 2 deaths, both occurring in patients who had developed acute chest syndrome; in a 34 year old and a 10 year old patient. Common bile duct related morbidity was proportionately more common in paediatric patients than adults, represented by ductal dilation (48% vs 37%), ductal calculi (44% vs 31%) and retained stones (7% vs 3%).

Adolescent↗
PubMed2001

A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI.

The interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin and alboaggregin-A activate platelets by interacting with GPVI. In this study, we isolated from white-lipped tree viper (Trimeresurus albolabris) venom, alborhagin, which is functionally related to convulxin because it activates platelets but is structurally different and related to venom metalloproteinases. Alborhagin-induced platelet aggregation (EC50, <7.5 microg/ml) was inhibitable by an anti-alphaIIbbeta3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to alphaIIbbeta3-dependent aggregation. Additional evidence suggested that, like convulxin, alborhagin activated platelets by a mechanism involving GPVI. First, alborhagin- and convulxin-treated platelets showed a similar tyrosine phosphorylation pattern, including a similar level of phospholipase Cgamma2 phosphorylation. Second, alborhagin induced GPVI-dependent responses in GPVI-transfected K562 and Jurkat cells. Third, alborhagin-dependent aggregation of mouse platelets was inhibited by the anti-GPVI monoclonal antibody JAQ1. Alborhagin had minimal effect on convulxin binding to GPVI-expressing cells, indicating that these venom proteins may recognize distinct binding sites. Characterization of alborhagin as a GPVI agonist that is structurally distinct from convulxin demonstrates the versatility of snake venom toxins and provides a novel probe for GPVI-dependent platelet activation.

Amino Acid Sequence↗
PubMed2001

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop.

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.

Amino Acid Sequence↗
PubMed2001

Metalloendopeptidases EC 3.4.24.15 and EC 3.4.24.16 and bradykinin B2 receptors do not play important roles in renal wrap hypertension in rabbits.

1. The aim of the present study was to determine the effects of the metalloendopeptidase (EP) 24.15 and 24.16 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA-2) on haemodynamics and renal function in conscious rabbits with two-kidney, two-wrapped hypertension. We have also examined the role of endogenous bradykinin in the maintenance phase of this form of renovascular hypertension and whether inhibition of bradykinin degradation contributes to any potential effects of JA-2. 2. In two preliminary operations, rabbits were equipped with transit-time ultrasound flow probes for measuring cardiac output (CO) and renal blood flow (RBF) and had both kidneys wrapped in cellophane. Starting 4 weeks after the last operation, rabbits underwent four studies (3-5 days apart), during which they were treated with combinations of the bradykinin B2 receptor antagonist icatibant or its vehicle (1 mL/kg bodyweight 0.9% w/v NaCl) and JA-2 or its vehicle (1 mL/kg of a 5% w/v 2-hydroxypropyl-beta-cyclodextrin, 2.5% v/v dimethylsulphoxide solution). Renal function was monitored using standard renal clearance methods. 3. Icatibant (10 microg/kg) had no significant effects on systemic haemodynamic variables (mean arterial pressure, heart rate or CO), renal haemodynamic variables (RBF or glomerular filtration rate), urine flow or sodium excretion. At 5 mg/kg plus 3 mg/kg per h, JA-2 also did not affect any of these variables, either after icatibant vehicle treatment or after icatibant treatment. 4. Our data do not support major roles for endogenous bradykinin or bradykinin degradation by EP 24.15/24.16 in the control of systemic and renal haemodynamics or renal excretory function in two-kidney, two-wrapped hypertension in rabbits.

Adrenergic beta-Antagonists↗
PubMed2001

Immuno-unreactive albumin excretion increases in streptozotocin diabetic rats.

We previously showed that albumin is fragmented (>90%) during renal passage to low-molecular-weight (<10 kd) peptides. The aim of the present study was to document the renal handling of albumin in experimental diabetes. Tritium-labeled albumin was infused into control and streptozotocin (STZ) diabetic rats during 7 days. Urinary radioactivity, assessed by size exclusion chromatography, revealed a major peak corresponding to low-molecular-weight, albumin-derived fragments and a minor peak corresponding to intact albumin or high-molecular-weight, albumin-derived protein. The fractional clearance of albumin, calculated from total radioactivity measurements, was at least 100-fold greater than the fractional clearance of albumin determined by radioimmunoassay (RIA) for control and diabetic rats. This result was mainly because low-molecular-weight, albumin-derived fragments were not detected by RIA. The fractional clearance of high-molecular-weight, albumin-derived protein was 2- to 10-fold greater than the fractional clearance determined by RIA. The immuno-unreactive high-molecular-weight, albumin-derived protein (called ghost albumin), characterized by size exclusion chromatography and high-performance liquid chromatography, was present in control and diabetic rat urine. Ghost albumin excretion rate was enhanced 11-fold after 8 weeks of STZ diabetes as compared with aged-matched controls. This study shows that renal modification resulting in low-molecular-weight and high-molecular-weight components of albumin is a major contributor to the renal handling of albumin. The results indicate that excretion of modified albumin is increased in STZ rats as compared with albumin detected by conventional RIA. Long-term studies are necessary to evaluate the potential of ghost albumin as a new marker for the assessment of urinary albumin in diabetes.

Albumins↗
PubMed2001

Synapsin I identified as a novel brain-specific autoantigen.

BACKGROUND: We report the identification and characterization of a novel 74-kd brain-specific autoantigen that is reactive with serum from a patient with discoid lupus erythematosus and chronic lymphocytic leukemia. METHODS: We determined the molecular weight, tissue distribution and subcellular distribution of the autoantigen and obtained limited amino acid sequence after purification by ion-exchange chromatography and trypsin digestion. RESULTS: We identified the 74-kd autoantigen as synapsin I on the basis of the following observations. First, the autoantigen has properties consistent with synapsin I: molecular weight of approximately equals 74 kd, brain-specific distribution, presence in cytosol and on synaptosomes, and association with taxol-stabilized microtubules. Second, limited amino acid sequence determination after trypsin digestion of the autoantigen shows identity with synapsin I. Third, the autoimmune serum immunoblots fusion proteins that incorporate rat synapsin Ia. The autoantibodies reactive to synapsin Ia are of immunoglobulin (Ig) G and IgM class. CONCLUSIONS: This is the first report of autoantibodies that are reactive to synapsin Ia. Autoantibodies that are reactive to synapsin Ia are not restricted to discoid lupus erythematosus patients, because we found identical reactivity in two of 18 sera from dsDNA-positive systemic lupus erythematosus patients and in two of 14 rheumatoid factor-positive sera. Whether autoantibodies to synapsin I are associated with neuropsychiatric manifestations is currently unknown.

Animals↗
PubMed2000

The neuropeptide processing enzyme EC 3.4.24.15 is modulated by protein kinase A phosphorylation.

The metalloendopeptidase EC (EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH(1-9), bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 7-fold increase in both K(m) and k(cat) for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.

Amino Acid Sequence↗
PubMed2000

Soluble neutral metallopeptidases: physiological regulators of peptide action.

Classically, the pre- and post-secretory processing of peptide signals appears to be mediated primarily by subtilisin-like peptidases in secretory vesicles and/or membrane-associated neutral endopeptidases in the extracellular environment. This article presents both biochemical and physiological evidence to support a role for soluble neutral metallopeptidases in the mediation of cell-to-cell communication by the selective generation and termination of peptide signals. These soluble peptidases have been implicated in the normal and disease-state processing of peptides involved in neurological, endocrine and cardiovascular functions. In this context, specific inhibitors of these enzymes could selectively modulate peptide levels and thus have considerable therapeutic potential. The aim of this review is to discuss the design and development of specific inhibitors of soluble neutral metallopeptidases that have been instrumental in identifying the roles of these enzymes. It will also review the evidence and present a case for the involvement of soluble neutral metallopeptidases in the regulation of peptide signalling in both central nervous system (CNS) and peripheral tissues.

Amino Acid Sequence↗
PubMed2000

Bradykinin analogues with beta-amino acid substitutions reveal subtle differences in substrate specificity between the endopeptidases EC 3.4.24.15 and EC 3.4.24.16.

The closely related zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) cleave many common substrates, including bradykinin (BK). As such, there are few substrate-based inhibitors which are sufficiently selective to distinguish their activities. We have used BK analogues with either alanine or beta-amino acid (containing an additional carbon within the peptide backbone) substitutions to elucidate subtle differences in substrate specificity between the enzymes. The cleavage of the analogues by recombinant EP24.15 and EP24.16 was assessed, as well as their ability to inhibit the two enzymes. Alanine-substituted analogues were generally better substrates than BK itself, although differences between the peptidases were observed. Similarly, substitution of the four N-terminal residues with beta-glycine enhanced cleavage in some cases, but not others. beta-Glycine substitution at or near the scissile bond (Phe5-Ser6) completely prevented cleavage by either enzyme: interestingly, these analogues still acted as inhibitors, although with very different affinities for the two enzymes. Also of interest, beta-Gly8-BK was neither a substrate nor an inhibitor of EP24.15, yet could still interact with EP24.16. Finally, while both enzymes could be similarly inhibited by the D-stereoisomer of beta-C3-Phe5-BK (IC50 approximately 20 microM, compared to 8 microM for BK), EP24.16 was relatively insensitive to the L-isomer (IC50 12 approximately microM for EP24.15, >40 microM for EP24.16). These studies indicate subtle differences in substrate specificity between EP24.15 and EP24.16, and suggest that beta-amino acid analogues may be useful as templates for the design of selective inhibitors.

Alanine↗
PubMed2000

Design and synthesis of inhibitors incorporating beta -amino acids of metalloendopeptidase EC 3.4.24.15.

Endopeptidase EC 3.4.24.15 (EP 24.15) is a thermolysin-like metalloendopeptidase which is expressed widely throughout the body, with the highest concentrations in the brain, pituitary and testis. While the precise role of EP 24.15 remains unknown, it is thought to participate in the regulated metabolism of a number of specific neuropeptides. Of the limited number of inhibitors described for EP 24.15, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-amino benzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but is unstable in vivo due to its cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). The cpp-Ala-Ala N-terminal product of this cleavage is a potent inhibitor of angiotensin converting enzyme, which further limits the use of CFP in vivo. To generate specific inhibitors of EP 24.15 that are resistant to in vivo proteolysis by EP 24.11, beta-amino acids have been incorporated into the structure of CFP. We have prepared racemic mixtures of beta-amino acids containing proteogenic side chains, which are 9-fluorenylmethoxycarbonyl (Fmoc)-protected, and several analogues of CFP containing beta-amino acids have been synthesized by solid phase peptide synthesis. The results of stability and inhibitory studies of these new analogues show that the incorporation of beta-amino acids adjacent to the scissile bond can indeed stabilize the peptides against cleavage by EP 24.11 and still inhibit EP 24.15. The results obtained in these studies demonstrate the potential of these amino acids in the synthesis of peptidomimetics and in the design of new stable and specific therapeutics.

Alanine↗
PubMed2000

Nitric oxide mediated modulation of norepinephrine transport: identification of a potential target for S-nitrosylation.

1. Carrier mediated uptake (uptake-1) transport of norepinephrine (NE) plays a key role in the regulation of sympathetic neurotransmission. Recent investigations indicate that nitric oxide (NO) may modulate uptake-1 activity, possibly in a cyclic GMP independent manner. 2. Carrier mediated transport of [(3)H-NE] and [(3)H-dopamine, DA] was examined in CHO cells transfected with cDNA for the NE and DA transporters (NET, DAT) respectively. 3. While exposure to the NO donor S-nitroso-N-acetylpenicillamine (100 microM, SNAP) significantly reduced [(3)H-NE] uptake (P<0.001), no effect on [(3)H-DA] transport was apparent. 4. Comparison of the amino acid sequences for NET and DAT identified cysteine residue 351 in NET, which was not present in DAT. Site-directed mutagenesis of Cys 351 to Ser produced a functional NET that was resistant to the inhibitory effects of SNAP. 5. The presence of SNAP mediated nitrosylation of the cysteine residue in an 8-mer model peptide based around Cys 351 in NET was confirmed by both biochemical and mass spectroscopic means. 6. These data indicate the potential regulatory role for NO in modulating sympathetic neurotransmission, and further confirm the importance of non-cyclic GMP dependent mechanisms in mediating the actions of NO.

Amino Acid Sequence↗