Rat Angiotensin-converting Enzyme Research Articles

Cloning and characterization of a single-chain fragment of monoclonal antibody to ACE suitable for lung endothelial targeting

Monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) demonstrates selective accumulation in lung tissue of the rat, hamster, cat, monkey and human after systemic injection. It has also been demonstrated that mAb 9B9 is the useful tool for targeting therapeutic agents or genes to lung endothelium. In this study, we describe the generation and characterization of a single-chain derivative (scFv) of mAb9B9 (scFv 9B9). In vitro, scFv9B9 retains the ability of the parental antibody to recognize human and rat ACE when expressed both on the surface of phage and as a soluble protein in prokaryotic and eukaryotic expression systems. The ability of scFv 9B9 presented by phage or the soluble protein labeled with I 125 to recognize ACE in the pulmonary circulation was also confirmed in an in vivo rat model. Sequence analysis revealed a putative glycosylation site in close proximity to the complementarity determining region 2 (CDR2) of the scFv 9B9 heavy chain. Mutation of Asn68 to Gln in the heavy chain of scFv 9B9 eliminated the glycosylation site and significantly improved the binding affinity of scFv 9B9 to human ACE as determined by cell ELISA and Western Blot. Moreover, Asn68Gln scFv 9B9 showed a greater rate of secretion at 30 °C than wild type scFv 9B9, but had a decreased thermal stability at 37 °C. The development of a stable and functional single-chain format of mAb 9B9 which specifically recognizes human and rat ACE represents a novel antibody-based reagent suitable for targeted delivery of drugs/genes to the pulmonary circulation.

Amino Acid Substitutions in the S2 Region Enhance Severe Acute Respiratory Syndrome Coronavirus Infectivity in Rat Angiotensin-Converting Enzyme 2-Expressing Cells

To clarify the molecular basis of severe acute respiratory syndrome coronavirus (SARS-CoV) adaptation to different host species, we serially passaged SARS-CoV in rat angiotensin-converting enzyme 2 (ACE2)-expressing cells. After 15 passages, the virus (Rat-P15) came to replicate effectively in rat ACE2-expressing cells. Two amino acid substitutions in the S2 region were found on the Rat-P15 S gene. Analyses of the infectivity of the pseudotype-bearing S protein indicated that the two substitutions in the S2 region, especially the S950F substitution, were responsible for efficient infection. Therefore, virus adaptation to different host species can be induced by amino acid substitutions in the S2 region.

Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild‐type angiotensin‐converting enzyme

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.

Selective rat lung endothelial targeting with a new set of monoclonal antibodies to angiotensin I-converting enzyme

We demonstrated previously that monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) accumulates selectively in the rat lung after systemic injection and thus is a powerful tool for immunotargeting therapeutic agents/genes to the lung microvasculature. Bearing in mind the tremendous research and therapeutic potential of lung immunotargeting via ACE, we generated a novel set of mAbs to rat ACE in order to enhance the repertoire of mAbs suitable for targeting drugs/genes to the rat lung. Five new mAbs recognizing different epitopes on rat ACE were examined for their efficacy to bind rat ACE both in vitro and in vivo. Gene delivery into cultured rat lung endothelial cells increased 30–50-fold after coating modified adenoviruses (containing Ig-binding domain) with mAbs to rat ACE. Radiolabeled mAbs specifically accumulated in the lung after systemic injection. mAb 1A2, 4H3 and 2E1 demonstrated the highest efficacy of lung uptake-around 50% of injected dose per gram of tissue; for mAb 1A2, the selectivity of lung uptake (ratio of lung to blood radioactivity) was 205. The effect of the mAbs on ACE shedding was epitope-specific: injection of mAb 1A2 and 4H3 did not change lung ACE activity, whereas injection of mAb 2E1 and 9B9 decreased rat lung ACE activity by 20%. None of the tested mAbs inhibited ACE activity in vitro. A new set of mAbs to rat ACE demonstrated highly efficient and selective lung accumulation and thus have the potential for targeting drugs/genes to the pulmonary vasculature in different rat models of lung diseases.

Potency and selectivity of RXP407 on human, rat, and mouse angiotensin-converting enzyme

By screening phosphinic peptide libraries, we recently reported the discovery of RXP407 (Ac-Asp-PheY(PO 2-CH 2) LAla-Ala-NH 2), a potent N-domain-selective inhibitor of recombinant human angiotensin-converting enzyme (ACE). Preliminary studies to evaluate the in vivo activity of RXP407 in rat led us to suspect possible differences in the binding property of RXP407 between human and rat ACE. The aim of the present study was thus to determine the potency of RXP407 toward rat and mouse ACEs, as compared to non-recombinant human ACE, and to assess the efficacy of this inhibitor in discriminating between the N- and C-domains of these ACE enzymes. By comparing the ability of RXP407 to block purified somatic and germinal ACE from mice, RXP407 was shown to be a potent N-domain-selective inhibitor of mouse somatic ACE, a behavior similar to that observed with human somatic ACE. In contrast, RXP407 appeared less potent toward purified ACE from rat and furthermore was unable to block ACE activity present in crude rat plasma. This study demonstrated that for further evaluation of the in vivo efficacy of RXP407, mice rather than rats should be used as the animal model. Thus, following the change in the Ac-S-D-K-P plasmatic levels, after i.v. injection of RXP407 to mice, will permit the potency and selectivity of this novel ACE inhibitor to be assessed.

Heart Protection Using a New Approach - Antisense to Angiotensin Converting Enzyme Mrna

P200 Introduction. Angiotensin converting enzyme (ACE), plays a major role in many cardiovascular pathophysiological conditions, such as hypertension, heart failure and myocardial ischemia. Currently used ACE inhibitors have some adverse effects and non-compliance problem. Therefore, our lab has been developing an antisense approach to inhibit the renin-angiotensin system at the gene expression level. The present study had three aims: 1. Design antisense oligodeoxynucleotide (AS-ODN) directed at ACE mRNA 2. Check tissue distribution of AS after iv injection 3. Test AS for heart protection in ischemia- reperfusion injury Methods. Epifluorescent and confocal laser-scanning microscopy, immunohistochemistry, Western blotting. Animals : mice, males, 20 g and Sprague-Dawley rats, males, 200-250 g. AS 5’ -ATTTCGTGGTGGG-3’ , inverted control (INV) 5’ -GGGTGGTGCTTTA-3’, phosphorothioates mixed with liposomes and injected iv 0.04 mg/mouse and 0.2 mg/rat. Captopril dose was 5 mg/kg. Isolated perfused heart function recordings : coronary perfusion pressure (CPP), left ventricular end-diastolic (LVEDP) and systolic pressures (LVSP). Results. Based on the rat ACE cDNA sequence, the AS was designed as 13-mer directed to the second zinc-binding domain (2998-3010 bp). Tissue distribution of phosphorothioated, fluorescein isothiocyanate (FITC)-labeled AS, 4h after iv injection, showed its presence in the lung, spleen, liver and heart, but absence in the brain. Immunostaining of the parallel heart slices with anti sarcomeric alpha-actinin antibody indicated AS localization within cardiomyocytes. ACE AS injected iv to the rats 24 h before excising the heart, which was then subjected to 25 min ischemia and 30 min reperfusion, preserved heart function. Increase in CPP and LVEDP during reperfusion was attenuated by AS as compared to INV or saline treatment (n=8 in each group, p

Heart Protection Using a New Approach - Antisense to Angiotensin Converting Enzyme Mrna

P200 Introduction. Angiotensin converting enzyme (ACE), plays a major role in many cardiovascular pathophysiological conditions, such as hypertension, heart failure and myocardial ischemia. Currently used ACE inhibitors have some adverse effects and non-compliance problem. Therefore, our lab has been developing an antisense approach to inhibit the renin-angiotensin system at the gene expression level. The present study had three aims: 1. Design antisense oligodeoxynucleotide (AS-ODN) directed at ACE mRNA 2. Check tissue distribution of AS after iv injection 3. Test AS for heart protection in ischemia- reperfusion injury Methods. Epifluorescent and confocal laser-scanning microscopy, immunohistochemistry, Western blotting. Animals : mice, males, 20 g and Sprague-Dawley rats, males, 200-250 g. AS 5’ -ATTTCGTGGTGGG-3’ , inverted control (INV) 5’ -GGGTGGTGCTTTA-3’, phosphorothioates mixed with liposomes and injected iv 0.04 mg/mouse and 0.2 mg/rat. Captopril dose was 5 mg/kg. Isolated perfused heart function recordings : coronary perfusion pressure (CPP), left ventricular end-diastolic (LVEDP) and systolic pressures (LVSP). Results. Based on the rat ACE cDNA sequence, the AS was designed as 13-mer directed to the second zinc-binding domain (2998-3010 bp). Tissue distribution of phosphorothioated, fluorescein isothiocyanate (FITC)-labeled AS, 4h after iv injection, showed its presence in the lung, spleen, liver and heart, but absence in the brain. Immunostaining of the parallel heart slices with anti sarcomeric alpha-actinin antibody indicated AS localization within cardiomyocytes. ACE AS injected iv to the rats 24 h before excising the heart, which was then subjected to 25 min ischemia and 30 min reperfusion, preserved heart function. Increase in CPP and LVEDP during reperfusion was attenuated by AS as compared to INV or saline treatment (n=8 in each group, p<0.05). Moreover, ACE protein was not upregulated, as it was in the captopril-treated group. Conclusion. ACE AS is taken up by the heart tissue and provides cardioprotection by effectively preventing myocardial dysfunction caused by ischemia-reperfusion injury.

Angiotensin-converting enzyme inhibition and AT1 receptor blockade modify the pressure-natriuresis relationship by additive mechanisms in rats with human renin and angiotensinogen genes.

The intrarenal factors responsible for hypertension in double-transgenic rats (dTGR) harboring human renin and human angiotensinogen genes are unclear. The pressure-natriuresis and -diuresis relationships in response to chronic angiotensin-converting enzyme (ACE) inhibition and AT1 receptor blockade were evaluated. Renal renin-angiotensin and nitric oxide (NO) system gene expression was also investigated. Six-week-old dTGR were treated for 3 wk with submaximal doses of cilazapril (10 mg/kg, orally) or losartan (10 mg/kg, orally) or with the drug combination. In untreated dTGR, pressure-natriuresis relationships were maximally shifted rightward by approximately 70 to 80 mmHg, and both renal blood flow (RBF) and GFR were markedly decreased. Submaximal cilazapril and losartan dosages both decreased systolic BP by 30 mmHg and shifted the pressure-natriuresis curves leftward by 25 to 30 mmHg. Cilazapril increased RBF and GFR to values observed in normotensive control animals but did not significantly affect fractional sodium excretion (FENa) or fractional water excretion (FEH2O) curves. In contrast, losartan had no significant effect on RBF or GFR but shifted the FENa and FEH2O curves leftward. The cilazapril and losartan combination completely normalized BP and shifted the pressure-natriuresis curves leftward more than did either drug alone. When cilazapril and losartan were administered at higher doses (30 mg/kg, orally), the two drugs equally shifted the pressure-natriuresis curves leftward, by 50 mmHg. Both drugs increased RBF and GFR; however, only losartan shifted FENa and FEH2O curves leftward. Human and rat renin and angiotensinogen genes were downregulated in dTGR and were increased by losartan and cilazapril treatments, whereas no changes in the expression of rat ACE and AT1A receptor genes were observed. Endothelial NO synthase expression was increased by cilazapril but not by losartan. Neither inducible NO synthase nor neural NO synthase gene expression was affected by drug treatments. Therefore, submaximal ACE inhibition enhanced sodium excretion mainly by increasing RBF and GFR, whereas submaximal AT1 receptor blockade decreased tubular sodium and water reabsorption. The combination of the two drugs produced an additive effect. The ACE inhibitor effects may involve increased endothelial NO synthase expression, perhaps related to the inhibition of bradykinin degradation.

Rat angiotensin-converting enzyme promoter regulation by beta-adrenergics and cAMP in endothelium.

To shed light on mechanisms of angiotensin-converting enzyme (ACE) upregulation, we used a rabbit endothelial cell model to characterize intracellular pathways of beta-adrenergic stimulation. In these cells, ACE activity is increased by isoproterenol (ISO). The stably transfected 1273-bp ACE promoter is stimulated by ISO in the presence of isobutyl methylxanthine. This effect is abolished by propranolol. Promoter stimulation is mimicked by cholera toxin, forskolin, and 8BrcAMP, but not by 8BrcGMP. Promoter stimulation by ISO and isobutyl methylxanthine is blocked by protein kinase A inhibitors, indicating that beta-adrenergic stimulation of the ACE gene depends on phosphorylation of protein kinase A targets. Activation by cAMP, resistance to phorbol ester, and lack of synergism between cAMP and phorbol ester suggest that promoter regulation is due to cAMP responsive element rather than to activating protein-2 sequences. Okadaic acid potentiation of 8BrcAMP induction indicated that promoter activation by cAMP is regulated by phosphatases controlling activation of typical cAMP responsive element regulated genes. In summary, beta-adrenergic activation of rat ACE promoter is specific; uses G(s) proteins, adenylyl cyclase, protein kinase A; and probably includes cAMP responsive element-like sequences.

The renin–angiotensin system in hybrid NG108-15 cells: Renin gene is from mouse neuroblastoma, angiotensinogen and angiotensin-converting enzyme genes are of rat glioma origin

Angiotensin II (Ang II) increases the level of tyrosine phosphorylation of several proteins in nondifferentiated NG108-15 cells, a hybrid derived from the fusion of mouse neuroblastoma and rat glioma cells. Conversely, incubation of NG108-15 cells with an angiotensin-converting enzyme (ACE) inhibitor decreased the basal level of tyrosine phosphorylation of proteins, suggesting that locally secreted Ang II may act as an autocrine regulator. By RT-PCR, we found that nondifferentiated NG108-15 cells contained the mRNA transcript of the rat angiotensinogen, mouse renin and rat ACE genes, thus confirming that NG108-15 cells contain all the elements of a local renin–angiotensin system.

Suppression of angiotensin-converting enzyme expression and activity by shear stress.

Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm2 for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm2) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods > 8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo.

Cardiac angiotensin converting enzyme overproduction indicates interstitial activation in renovascular hypertension

Angiotensin converting enzyme (ACE) activity in the plasma does not change significantly with hypertension in two-kidney, one-clip hypertensive (2K-1C) rats. However, heart ACE activity and mRNA increase with hypertension. We measured the ACE activity and mRNA in hypertrophied hearts at different times after clipping, and determined the cellular distribution of its increase in the left ventricle of 2K-1C hypertensive rats. Cardiac ACE activity was quantified in left and right ventricles using a radiolabeled synthetic ACE substrate, and ACE mRNA steady-state level was quantified by ribonuclease protection assay. Tissue localization of ACE in normal and hypertrophied hearts was determined by measuring ACE activity in isolated ventricular cells. In situ hybridization with a rat ACE cDNA and immunohistochemistry with a monoclonal anti-ACE antibody were used to identify tissue compartments producing ACE mRNA and protein. The left ventricle was hypertrophied 2 weeks after clipping and remained hypertrophied at 12 weeks. Left ventricular ACE activity was significantly increased 2 and 4 weeks (3.2 +/- 0.3 in 2K-1C vs. 1.7 +/- 0.1 pmol/mg prot/min in sham-operated rat) after renal artery clipping, but not at 12 weeks. The right ventricle was slightly hypertrophied 4 weeks after clipping and remained hypertrophied at 12 weeks. Right ventricular ACE activity was significantly increased at 4 (6.7 +/- 0.6 in 2K-1C vs. 3.1 +/- 0.3 pmol/mg prot/min in sham-operated rat) and 12 weeks. ACE activity was not detectable in cardiomyocytes isolated by Percoll gradient. Neither was ACE mRNA detected in isolated cardiomyocytes, even after ACE mRNA amplification by RT-PCR. In contrast, ACE activity and mRNA were detected in pooled non-cardiomyocytic cells. Thus the increase in cardiac ACE activity associated with hypertension must be due to an increase in ACE expression by non-cardiomyocytic cells. In situ hybridization showed an autoradiographic signal for ACE mRNA over the endothelial cells of coronary arteries and over the interstitial spaces including pericoronary and fibrosis areas. Immunohistochemistry confirmed these data, showing ACE on endothelial cells and in pericoronary spaces with an increased signal in pericoronary and fibrosed areas in hypertensive hypertrophied left ventricle. Besides its usual endothelial expression, ACE is absent from cardiomyocytes and present in interstitial tissue, in the pericoronary spaces in normal tissue and more markedly in hypertrophied ventricles.

Angiotensin-Converting Enzyme and Genetic Hypertension: Cloning of Rat cDNAs and Characterization of the Enzyme

Using genetic mapping approaches, a gene on chromosome 10, Bpl, has been identified in the stroke-prone spontaneously hypertensive rat (SHRSP) in the same region that contains the gene for angiotensin converting enzyme (ACE). Since ACE plays an important role in blood pressure regulation, the ACE gene is a leading candidate for Bpl. To examine the possibility that a structural abnormality of ACE exists in the SHRSP, we cloned and characterized the cDNAs for the Wistar-Kyoto rat (WKY) and SHRSP ACE. Both cDNAs encode a single polypeptide of 1,313 amino acid residues with an estimated molecular weight of 150.9 KDa. Five nucleotide differences were identified between the WKY and the SHRSP ACE cDNAs. One of these differences resulted in an amino acid substitution (Lys-207 in the WKY to Arg-207 in the SHRSP). But the enzymatic properties of partially purified ACE from the two strains were similar. Thus the data suggest that an alteration in the primary structure of rat ACE does not contribute to the hypertension in the SHRSP.

Interaction of mAb to angiotensin-converting enzyme (ACE) with antigen in vitro and in vivo: antibody targeting to the lung induces ACE antigenic modulation.

We previously described that mAb to angiotensin-converting enzyme (ACE), mAb 9B9, accumulates in the rat lungs after systemic injection. In the present work we have documented that mAb 9B9 cross-reacts with human, monkey, rat, cat and hamster ACE, while other ACE antibodies did not cross-react with the rat, cat and hamster enzyme. Anti-ACE mAb 3A5 and I2H5 inhibit human ACE in vitro, while mAb 9B9 does not inhibit ACE activity. Radiolabeled mAb 9B9, but not other antibodies, accumulates selectively in rat, cat and hamster lungs after systemic administration. No accumulation of mAb 9B9 has been observed in hamster kidney, while hamster kidney ACE activity is higher than that in the lung. mAb 9B9 does not induce complement-mediated injury to cultured endothelial cells. No pathological changes were detected in organs of animals after mAb 9B9 injection (10-100 mg/kg). However, injection of these amounts of mAb 9B9 leads to a decrease in ACE activity in the lung homogenates and an increase in serum. In cultured human endothelial cells treatment with mAb 9B9 increases ACE activity in cell medium and decreases in cell lysates. Therefore, while mAb 9B9 does not kill endothelial cells, at high dose it may induce ACE shedding from the cell. The results obtained support the potential of anti-ACE mAb 9B9 for targeting to the lung and for investigations of the pulmonary endothelium.

A Comparison of Guinea Pig Serum Angiotensin Converting Enzyme with Forms of Angiotensin Converting Enzyme from Human, Rat and Rabbit Tissues

Guinea pig serum angiotensin converting enzyme (ACE) activities exceed ACE activities of other mammalian sera by as much as two magnitudes. To examine the possibility that guinea pig ACE has a superior catalytic efficiency, we purified it to apparent homogeneity and compared it to highly purified forms of ACE from human seminal plasma, rat lungs and rabbit lungs. The first 24 amino acid residues of guinea pig and rat ACE forms were 96% identical with the sequence of human ACE. Second order rate constants (kcat/Km) for guinea pig, human and rabbit forms of ACE on reaction with benzoyl-Phe-Ala-Pro were identical (1.6E-09 M −1 min −1). Their dissociation constants on reaction with the ACE inhibitor RAC-X-65 were within a narrow range (10-16 pM). Thus, the high ACE activity of guinea pig serum is owing to high enzyme concentration and not to superior catalytic efficiency.

Pharmacology of Idrapril

Idrapril is the prototype of a new chemical class of angiotensin converting enzyme (ACE) inhibitors, the hydroxamic non-amino acid derivatives. Idrapril strongly inhibited rat and human plasma ACE and rabbit lung ACE (IC50: 7-12 nM) as well as the pressor response induced by angiotensin I in anesthetized rats (ED50: 63 nmol/kg i.v.). Idrapril (0.04-23 mumol/kg i.v.) lowered the blood pressure dose dependently, up to 20-35%, in different models of hypertension (sodium-depleted spontaneously hypertensive rat, two-kidney-one-clip renal hypertensive rat, and aortic-coarctated rat), its profile being similar to that of captopril in terms of potency and efficacy. Idrapril and captopril reduced the blood pressure and potentiated substance P-induced bronchoconstriction in the guinea pig to the same extent, suggesting a similar degree of ACE inhibition in the circulation. However, idrapril potentiated capsaicin-induced bronchoconstriction (a model that has been related to the liability of ACE inhibitors to produce cough in patients) less effectively than captopril. We conclude that effective ACE inhibition in vitro and in vivo can be obtained with this novel class of compounds.

In VivoAdministration of Glucose Oxidase Conjugated with Monoclonal Antibodies to Angiotensin-converting Enzyme: The Tissue Distribution, Blood Clearance, and Targeting into Rat Lungs

A conjugate between glucose oxidase (GO) and monoclonal antibody to human angiotensin-converting enzyme (ACE) cross-reacting with rat ACE (MoAb9b9) has been prepared by oxidation of the cardohydrate moiety of the enzyme with sodium periodate. The conjugate (GO-MoAb9b9) thus obtained retained both antigen-binding capacity and enzymatic activity. The fate of the conjugate in vivo after intravenous injection was studied using conjugates containing radiolabeled enzyme. GO-MoAb9b9 was specifically accumulated in rat lungs upon in vivo administration, as compared with free enzyme and nonimmune IgG-conjugated glucose oxidase. The specificity of the conjugate accumulation expressed as the localization ratio (the ratio between radioactivity of gram tissue to that of blood) (Loc. Ratio) reached a value up to 50 on the second day after injection, in contrast to native enzyme and to IgG-conjugated enzyme (Loc. Ratio was less than 0.5 for both preparations). The Loc. Ratio of GO-MoAb9b9 was even higher than that of the original antibody MoAb9b9 and was equal to 20, which is probably explained by an extremely rapid blood clearance of the conjugate from the circulation. The administration of excess free MoAb9b9 dramatically inhibited the conjugate targeting in the lung without any effect on liver uptake. At doses ranging from 10 to 1,000 micrograms/rat, the conjugate was accumulated in the lung without saturation of the antigen determinants of the target. At minimal doses, the efficiency of targeting achieved 5 to 7% of the conjugate injected. With elevation of the dose, the efficiency of targeting decreased to 2.5% of the injected dose (1 mg of GO-MoAb9b9 per rat). A sixfold greater accumulation of unmodified radiolabeled MoAb9b9 compared with the GO-MoAb9b9 conjugate in rat lung has been observed, though the kinetics of desorption from the target organ was similar for both the antibody and the conjugate. In the bloodstream, the conjugate persisted for at least 5 days without binding to blood cells; all circulating radioactivity was associated with proteins. A considerable part of the conjugate (to 50%) circulated as a tight antibody-enzyme complex for several days. The conjugate retained its antigen-binding capacity for at least 24 h; during this period, its enzymatic activity decreased by less than 40%. The results obtained provide the experimental ground for further attempts to apply glucose oxidase conjugates for local modulation of inflammation and elimination of the target cells.

Tissue Angiotensin Converting Enzyme (ACE) During Changes in the Renin—Angiotensin System

To establish if physiological manipulations of the renin-angiotensin system modulates tissue angiotensin converting enzyme (ACE), rats were fed low, normal, or high salt diets, or high salt and DOCA, for 3 weeks, and then plasma and tissue ACE levels were determined by radioinhibitor binding studies. Urinary sodium excretion (mmol/24 h) was 0.03 +/- 0.01 (low salt), 0.58 +/- 0.09 (normal salt), and 10.4 +/- 1.3 (high salt), and plasma angiotensin II (pg/ml) was 73.5 +/- 8.6 (low salt), 49.3 +/- 8.5 (normal salt), 32.2 +/- 8.5 (high salt), and 6.9 +/- 0.6 (high salt plus DOCA) in the third week of dietary treatment. ACE was studied by Scatchard analysis of radioinhibitor binding in plasma and tissue homogenates. [125I]MK351A bound to ACE was measured under standardized conditions in the presence of MK351A (10(-13) to 10(-5) M) and MK351A binding sites and equilibrium dissociation constant calculated. There were no significant changes in binding site concentration in plasma, lung, aorta, epididymus, brain, or kidney preparations across the range of salt states studied. The equilibrium dissociation constant appeared uniform within organs, but varied between organs. Further studies were undertaken with captopril, enalapril, and cilazoprilic acid in kidney and lung preparations. Concentration of inhibitor required for equal displacement of bound [125I]MK351A was consistently greater for kidney than for lung. Binding studies of rat ACE using [125I]MK351A showed that manipulation of salt status did not influence rat tissue ACE. Binding data suggest ACE from different tissues may have variations at the active site.

Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.

A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, alpha-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a greater than 1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.