Vascular dysfunction induced by AGE is mediated by VEGF via mechanisms involving reactive oxygen species, guanylate cyclase, and protein kinase C.

OBJECTIVE: These experiments were designed to elucidate mechanisms mediating vascular dysfunction induced by advanced glycation end products (AGEs). METHODS: Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 week later, granulation tissue that formed in the bottom of the chamber was exposed twice daily for 7 days to glycated rat serum albumin in the presence and absence of inhibitors of reactive oxygen intermediates, nitric oxide synthase and guanylate cyclase, protein kinase C (PKC), and a neutralizing vascular endothelial growth factor (VEGF) antibody. Vascular (125)I-albumin clearance and blood flow were quantified by use of a double isotope-dilution technique and radiolabeled microspheres, respectively. RESULTS: Albumin permeation and blood flow were increased dose-dependently to a maximum of 2 to 3 times controls by increasing the extent of glucose modification, the concentration, or the duration of exposure to glycated albumin. These increases were significantly attenuated by probucol and superoxide dismutase; N(G)-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase inhibitor; LY83583, a guanylate cyclase inhibitor; and LY333531, a beta-isoform-selective protein kinase C inhibitor. A neutralizing VEGF monoclonal antibody also markedly attenuated the permeability and blood flow increases induced by glycated albumin. CONCLUSIONS: These observations indicate potentially important roles for oxygen free-radicals and nitric oxide in mediating permeability and blood flow changes induced by glycated proteins via mechanisms involving increased protein kinase C activity and VEGF production. Striking similarities in the mechanism by which hyperglycemia and glycated proteins induce vascular dysfunction suggest that a common pathway mediates effects of these different metabolic imbalances on vascular dysfunction.

Vascular endothelial growth factor and basic fibroblast growth factor differentially modulate early postnatal coronary angiogenesis.

The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary growth that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar growth, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles (<15 microm) was markedly reduced, whereas the percentage of large arterioles (26 to 50 microm) more than doubled. Thus, inhibition of both growth factors negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary growth, (2) bFGF facilitates arteriolar growth, and (3) the two growth factors interact to establish the normal hierarchy of the arteriolar tree.

Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.

Inhibition of coronary thrombosis and local inflammation by a noncarbohydrate selectin inhibitor.

We tested the hypothesis that selectin inhibition with blocking antibodies or a small-molecular-weight inhibitor of L-, P-, and E-selectin, methoxybenzoylpropionic acid (MBPA), prevents thrombus formation in a canine coronary Folts' model. Cyclic flow variations (CFVs) were induced by crush injury and constriction of the left anterior descending coronary artery in dogs. Systemic infusion of antibodies to P- and L-selectin abolished CFVs, respectively, in 50% and 17% of treated dogs [P = not significant (NS)]. The combination of P- and L-selectin antibodies suppressed CFVs in 60% of treated dogs (P = NS). In contrast, systemic selectin blockade by intravenous infusion or local adventitial application of MBPA markedly reduced CFVs and, in addition, reduced myocardial myeloperoxidase (MPO) activity. We conclude that inhibition of L-, P-, and E-selectin binding by a small-molecular-weight, noncarbohydrate compound markedly reduces arterial thrombosis, whereas systemic administration of antibodies to L- and P-selectin fail to reproduce this antithrombotic effect. These results underscore the role of selectins in the pathogenesis of arterial thrombosis under high shear stress and suggest that inhibition of P- and L- selectin may not suffice to prevent thrombus formation in this model. The role of E-selectin in thrombus formation in this model awaits further testing.

A novel protein with homology to the junctional adhesion molecule. Characterization of leukocyte interactions.

We have cloned a novel cDNA belonging to the Ig superfamily that shows 44% similarity to the junctional adhesion molecule (JAM) and maps to chromosome 21q21.2. The open reading frame of JAM2 predicts a 34-kDa type I integral membrane protein that features two Ig-like folds and three N-linked glycosylation sites in the extracellular domain. A single protein kinase C phosphorylation consensus site and a PDZ-binding motif are present in the short intracellular tail. Heterologous expression of JAM2 in Chinese hamster ovary cells defined a 48-kDa protein that localizes predominantly to the intercellular borders. Northern blot analysis showed that JAM2 is preferentially expressed in the heart. JAM2 homotypic interactions were demonstrated by the ability of JAM2-Fc to capture JAM2-expressing Chinese hamster ovary cells. We further showed that JAM2, but not JAM1, is capable of adhering to the HSB and HPB-ALL lymphocyte cell lines. Neutralizing mouse anti-JAM2 polyclonal antibodies provided evidence against homotypic interactions in this assay. Biotinylation of HSB cell membranes revealed a 43-kDa counter-receptor that precipitates specifically with JAM2-Fc. These characteristics of JAM2 led us to hypothesize a role for this novel protein in adhesion events associated with cardiac inflammatory conditions.

Bradycardia-induced coronary angiogenesis is dependent on vascular endothelial growth factor.

A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily. Heart rate decreased by 32% for 20 to 40 minutes after injection and was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Arterial pressure and cardiac output were unchanged. Left ventricular capillary length density (mm/mm(3)) increased gradually with alinidine administration; a 15% increase after 2 weeks and a 40% increase after 3 weeks of alinidine treatment were documented. Left ventricular weight, body weight, and their ratio were not significantly altered by alinidine treatment. After 1 week of treatment, before an increase in capillary length density, VEGF mRNA increased >2-fold and then declined to control levels after 3 weeks of treatment. VEGF protein was higher in alinidine-treated rats than in controls after 2 weeks and increased further after 3 weeks of treatment. Injection of VEGF-neutralizing antibodies over a 2-week period completely blocked alinidine-stimulated angiogenesis. In contrast, bFGF mRNA was not altered by alinidine treatment. These data suggest that VEGF plays a key role in the angiogenic response that occurs with chronic bradycardia. The mechanism underlying this VEGF-associated angiogenesis may be an increase in stretch due to enhanced diastolic filling.

Development of novel monoclonal antibodies for the analysis of functional sites in FGF-2.

Fibroblast growth factor 2 (FGF-2) can function as a potent mitogen, as well as a survival factor for a variety of mammalian cell types. The biological effects of FGF-2 are mediated by its interaction with two types of cellular binding sites: (1) high affinity tyrosine kinase receptors; and (2) low affinity heparan sulfate proteoglycans (HSPGs) on the cell surface. Although numerous FGF-2 antibodies have been used previously to analyze its biological actions, few studies have utilized antibodies to analyze domains within FGF-2 involved in its interactions with the two binding sites. In this report, we describe the generation and use of two monoclonal antibodies against human recombinant FGF-2 (254F1 and 256A12) that inhibit FGF-2 function. However, these antibodies appear to target preferentially different domains within the FGF-2 molecule, and therefore differentially influence the interactions of FGF-2 with its low and high affinity receptors. 254F1 is a more effective inhibitor of the high affinity, receptor tyrosine kinase binding site, whereas 256A12 appears to be a better inhibitor of the low affinity, HSPG interactions. We also demonstrate that the two antibodies are potent inhibitors of FGF-2 stimulated vascular cell proliferation, and as such have potential use in the treatment of vascular hyperproliferative diseases.

Selectin blockade prevents antigen-induced late bronchial responses and airway hyperresponsiveness in allergic sheep.

Antigen challenge can elicit an allergic inflammatory response in the airways that involves eosinophils, basophils, and neutrophils and that is expressed physiologically as a late airway response (LAR) and airway hyperresponsiveness (AHR). Although previous studies have suggested that E-selectin participates in these allergic airway responses, there is little information concerning the role of L-selectin. To address this question, we examined the effects of administering an L-selectin-specific monoclonal antibody, DU1-29, as well as three small molecule selectin binding inhibitors, on the development of early airway responses (EAR), LAR and AHR in allergic sheep undergoing airway challenge with Ascaris suum antigen. Sheep treated with aerosol DU1-29 before antigen challenge had a significantly reduced LAR and did not develop postchallenge AHR. No protective effect was seen when sheep were treated with a nonspecific control monoclonal antibody. Treatment with DU1-29 also reduced the severity of the EAR to antigen. Similar results were obtained with each of the three small molecule selectin inhibitors at doses that depended on their L-, but not necessarily E-selectin inhibitory capacity. The inhibition of the EAR with one of the inhibitors, TBC-1269, was associated with a reduction in histamine release. Likewise, treatment with TBC-1269 reduced the number of neutrophils recovered in bronchoalveolar lavage (BAL) during the time of LAR and AHR. TBC-1269, given 90 min after antigen challenge also blocked the LAR and the AHR, but this protection was lost if the treatment was withheld until 4 h after challenge, a result consistent with the proposed time course of L-selectin involvement in leukocyte trafficking. These are the first data indicating that L-selectin may have a unique cellular function that modulates allergen-induced pulmonary responses.

Vascular remodeling in response to altered blood flow is mediated by fibroblast growth factor-2.

Vascular structures adapt to changes in blood flow by adjusting their diameter accordingly. The factors mediating this process are only beginning to be identified. We have recently established a mouse model of arterial remodeling in which flow in the common carotid artery is interrupted by ligation of the vessel near the carotid bifurcation, resulting in a dramatic reduction in vessel diameter as a consequence of inward remodeling and intimal lesion formation. In the present study, we used this model to determine the role of fibroblast growth factor-2 (FGF-2) in the remodeling response by maintaining neutralizing serum levels of a mouse monoclonal antibody against FGF-2 for 4 weeks. Morphometric analysis revealed that intimal lesion formation was not affected by the antibody. However, lumen narrowing was significantly inhibited, resulting in a greater than 3-fold increase in lumen area in anti-FGF-2-treated animals compared with controls. Treatment with anti-FGF-2 antibody significantly inhibited the reduction in vessel diameter (inward remodeling) and shortening of the internal elastic lamina in the ligated vessel. In addition, anti-FGF-2 treatment also caused outward remodeling of the contralateral carotid artery. These findings identify FGF-2 as an important factor in vascular remodeling, and its effects are likely to be mediated by increasing vascular tone. The results are consistent with the recent observation of reduced vascular tone in the FGF-2-deficient mouse.

Role for heparin-binding growth factors in glucose-induced vascular dysfunction.

Vascular hyperpermeability and excessive neovascularization are hallmarks of early and late vascular endothelial cell dysfunction induced by diabetes. Vascular endothelial growth factor (VEGF) appears to be an important mediator for these early and late vascular changes. We reported previously, using skin chambers mounted on backs of SD rats, that neutralizing antibodies directed against VEGF blocked vascular permeability and blood flow changes induced by elevated tissue glucose and sorbitol levels in a dosage-dependent manner. We report in this study, using the same skin chamber model and neutralizing antibodies directed against basic fibroblast growth factor (FGF-2), that another member of the heparin-binding growth factor family also mediates glucose- and sorbitol-induced vascular permeability and blood flow increases. In addition, we show that 1) TBC1635, a novel heparin-binding growth factor antagonist, blocks the vascular hyperpermeability and blood flow increases induced by elevated tissue levels of glucose and sorbitol and by topical application of VEGF and FGF-2 to granulation tissue in skin chambers, and 2) suramin, a commercially available growth factor antagonist, blocks glucose-induced vascular dysfunction. These results suggest an early role for heparin-binding growth factors in the vascular dysfunction caused by excessive glucose metabolism, possibly via the sorbitol pathway.

Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor.

The purpose of these experiments was to investigate a potential role for vascular endothelial growth factor (VEGF) in mediating vascular dysfunction induced by increased glucose flux via the sorbitol pathway. Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 wk later, granulation tissue in the chamber was exposed twice daily for 7 d to 5 mM glucose, 30 mM glucose, or 1 mM sorbitol in the presence and absence of neutralizing VEGF antibodies. Albumin permeation and blood flow were increased two- to three-fold by 30 mM glucose and 1 mM sorbitol; these increases were prevented by coadministration of neutralizing VEGF antibodies. Blood flow and albumin permeation were increased approximately 2.5-fold 1 h after topical application of recombinant human VEGF and these effects were prevented by nitric oxide synthase (NOS) inhibitors (aminoguanidine and N(G)-monomethyl L-arginine). Topical application of a superoxide generating system increased albumin permeation and blood flow and these changes were markedly attenuated by VEGF antibody and NOS inhibitors. Application of sodium nitroprusside for 7 d or the single application of a calcium ionophore, A23187, mimicked effects of glucose, sorbitol, and VEGF on vascular dysfunction and the ionophore effect was prevented by coadministration of aminoguanidine. These observations suggest a potentially important role for VEGF in mediating vascular dysfunction induced by "hypoxia-like" cytosolic metabolic imbalances (reductive stress, increased superoxide, and nitric oxide production) linked to increased flux of glucose via the sorbitol pathway.

A cyclic hexapeptide is a potent antagonist of alpha 4 integrins.

The alpha 4 integrins mediate leukocyte adhesion to specific counter-receptors, including vascular cell adhesion molecule-1 (VCAM-1), the fibronectin splice variant containing connecting segment 1 (CS1), and mucosal addressin cell adhesion molecule-1. A series of cyclized peptides based on the LDV sequence of CS1 were synthesized and assayed for inhibition of alpha 4 integrin binding. The most potent peptide, C*WLDVC* (where * indicates disulfide-linked residues), inhibited alpha 4 beta 1-dependent binding of lymphocytes to VCAM-1 and CS1 with half-maximal inhibition achieved at 1 to 3 microM of peptide. The peptide proved more potent when the lymphocytes were activated with 1 mM MnCl2; half-maximal inhibition was reached at 0.4 and 0.05 microM for VCAM-1 and CS1, respectively. This represents a 100- to 800-fold increase in potency over a linear CS1 peptide in these same assays. C*WLDVC* also inhibited alpha 4 beta 7-dependent lymphocyte binding to the ligands mucosal addressin cell adhesion molecule-1, VCAM-1 and CS1. Immunoprecipitation of radiolabeled integrin indicated that the peptide could bind alpha 4 beta 1 and alpha 4 beta 7 directly and elute alpha 4 beta 1 from a CS1-conjugated agarose resin. The peptide showed selectivity for alpha 4 integrins in that it effectively inhibited alpha 4 beta 1-dependent, but not alpha 5 beta 1-dependent, binding of cells to intact fibronectin. Due to its small size and potency, C*WLDVC* may serve as a useful tool for the study of alpha 4 integrin biology and the development of small molecule therapeutics.

Brain nicotinic receptors isolated by a monospecific antibody against a synthetic alpha-3 subunit receptor peptide compared to a monoclonal anti-idiotypic (to nicotine) antibody.

Nicotinic cholinergic receptor proteins purified from rat brain by immunoaffinity chromatography were characterized using the anti-S3 polyclonal antibody vs. the anti-idiotypic monoclonal antibody 422F11 (generated against an antibody to nicotine). Anti-S3 IgG was purified to homogeneity; anti-S3-Sepharose 4B and 422F11-Sepharose 4B each depleted 3H-nicotine binding sites from brain. Nicotinic receptors isolated from both immunoaffinity columns showed major bands (silver-stained) at 55K and 70K. Using anti-S3 serum as probe, Western blots of nicotinic receptors isolated by the two immunoaffinity gels also showed major bands at 55 and 70K. However, Western blots of fresh brain extracts revealed a major band at 80K and minor bands at 55K and 70K. These results show similar nicotinic cholinergic receptor proteins isolated by the anti-S3 and 422F11 anti-idiotypic antibodies; 80K was dominant only when fresh brain extract was subjected to Western blotting without prior immunoaffinity purification.

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.

Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM. A constitutive form of P-450 is also implicated in nicotine metabolism, while purified P-450IA1 and P-450IIC6 show no detectable activity. The lack of P-450IA1 activity substantiates work by others who also failed to observe an increase in the efficiency of nicotine metabolism to cotinine by microsomes from rats that had been pretreated with benzanthracene. This result is in contrast to work with purified rabbit liver enzymes, in which P-450IA1 exhibited low but measurable activity. Our results support the notion that nicotine metabolism to cotinine by P-450 enzymes is highly species dependent. Thus, it is unwise in some cases to extrapolate results obtained by animal model study to the possible role of specific forms of the P-450 enzymes in nicotine metabolism in humans.

Characterization of nicotinic receptors on cultured cortical neurons using anti-idiotypic antibodies and ligand binding.

Monoclonal anti-idiotypic antibodies that represent the internal image of nicotine's natural isomer, L-nicotine, were used in conjunction with L-[3H]nicotine binding to characterize nicotinic receptors on neurons cultured from fetal rat cortex. Of the antibodies tested, two (422F11 and 420G11) were found that recognized a class of high affinity [3H]nicotine binding sites present on neuronal cells, but not on glia. The binding properties and pharmacological specificity of these sites compared well with those determined previously for putative nicotinic cholinergic receptors in adult rat brain. The binding of [3H]nicotine to neuronal receptors was effectively inhibited by both antibodies. Receptor-bearing cells were identified using indirect immunofluorescence. Approximately 20 to 30% of the cells were labeled specifically by the anti-idiotypes. Labeling was blocked by L-nicotine and other nicotinic agonists, but not by antagonists or by alpha and neuronal bungarotoxins. The majority of cells which were labeled had either bipolar or pyramidal morphology. Fluorescent labeling was associated with cell bodies as well as with axonal and dendritic processes, consistent with the proposed roles of neuronal nicotinic receptors in neuromodulation and synaptic transmission. The results suggest that anti-idiotypic antibodies may provide a new tool suitable for studying the locations, structure and functional significance of high affinity neuronal nicotinic receptors at the cellular level.

Interaction of cotinine with rat hepatic microsomal P-450. Comparison with metyrapone and immunomodulation of cotinine and metyrapone binding by monoclonal anti-cotinine antibodies.

The ability of the major nicotine metabolite, cotinine, to interact with rat liver microsomal cytochrome P-450 and the immunomodulatory effects of anti-cotinine antibodies were studied. Cotinine induced type II spectral changes with both microsomes from phenobarbital (PB)-induced rats and purified P-450 with apparent Ks values of 97 and 750 microM, respectively. In contrast, the Ks value was 0.3 microM for metyrapone and 5 microM for nicotine with both the microsomes and purified enzyme. The apparent Ki value for cotinine inhibition of 7-pentoxyresorufin O-dealkylase activity with the microsomes (87 microM) was approximately 87- and 870-fold higher than for nicotine and metyrapone, respectively. Monoclonal antibodies produced against cotinine cross-reacted equally well with metyrapone. They specifically blocked enzyme binding of both drugs based on dose-dependent inhibition of spectral changes, and reversed the metyrapone-induced inhibition of microsomal O-dealkylase activity. In contrast, antibodies to nicotine did not cross-react with cotinine or metyrapone and had no effect on their activity, although they did block the action of nicotine. These results demonstrate that cotinine binding to P-450 from PB-induced rats and inhibition of functional activity in vitro are qualitatively like the effects of metyrapone and nicotine, and that monoclonal anti-cotinine antibodies are useful molecular probes of the interactions between cotinine and metyrapone with the enzyme.

Idiotype-anti-idiotype hapten immunoassays: assay for cotinine.

Practical application of the idiotype-anti-idiotype reaction to hapten immunoassays has been demonstrated with cotinine as an example. The assay relies on the ability of cotinine, a major nicotine metabolite, to inhibit binding between a monoclonal anti-cotinine antibody (the idiotype) and a second monoclonal antibody (the anti-idiotype) specific for the antigen combining region on the idiotype. A solid phase enzyme-linked immunoadsorbent assay (ELISA) format was adopted in which fluid phase anti-cotinine and cotinine present either as a standard or in a test sample were incubated in microtiter plate wells coated with F(ab')2 fragments of the anti-idiotype. Horseradish peroxidase-labeled protein A and o-phenylenediamine were used to detect idiotype-anti-idiotype binding. Under optimal assay conditions, 0.9 ng cotinine inhibited immune binding by 50% and as little as 0.04 ng could be detected. In contrast, nearly 70 times more trans-3'-hydroxycotinine, a major urinary metabolite, and over 1000-fold more nicotine were required for 50% inhibition. Several other metabolites and structurally related compounds also were poor competitors. Assay reliability was good over a range of cotinine concentrations from 5 to 500 ng/ml saliva with intraassay coefficients of variation between 6 and 10% and interassay values between 6 and 13%. Also, there was a strong correlation (R2 = 0.994) between the cotinine levels found in saliva from 35 cigarette smokers with the idiotype-anti-idiotype assay and a cotinine-anti-cotinine ELISA. Because only monoclonal antibodies and antigen are required, the idiotype-anti-idiotype immunoassay offers a high degree of standardization without the need to prepare labeled hapten derivatives or macromolecular conjugates for solid phase assays.

Anti-idiotypic antibody probes of neuronal nicotinic receptors.

Monoclonal anti-idiotypic antibodies specific for the combining site on a monoclonal antinicotine were used in immunocytochemistry to localize nicotine binding sites on rat brain cortical sections and in immunoaffinity chromatography to isolate receptor from solubilized brain tissue. The receptor, which consists of two subunits with Mr values of 43 and 50 kDa, was eluted from the antiidiotype column with either pH3 citrate buffer or 25 mM (-)-nicotine, but was not present in eluates from immobilized anti-Electrophorus acetylcholine receptor or anti-methotrexate. The anti-idiotypes specifically inhibited [3H]nicotine binding to rat brain homogenate and (-)-nicotine inhibited anti-idiotype binding to brain sections based on abrogation of immunofluorescence staining. These results are consistent with the operational definition of the anti-idiotypes as the internal image of nicotine, and demonstrate their value as immunochemical probes of nicotinic receptors.

Monoclonal antibody ELISA for cotinine in saliva and urine of active and passive smokers.

The value of a monoclonal antibody-based ELISA for measuring cotinine in saliva and urine of active and passive smokers was assessed. Cotinine (mean +/- SEM) was detected in all 26 saliva (392 +/- 74 ng/ml) and 27 urine (4264 +/- 508 ng/mg creatinine; 2566 +/- 364 ng/ml) samples from smoking parents, but in only two of 36 salivas and one of 37 urines from nonsmokers (P less than 0.001). Similarly, mean cotinine levels in 30 salivas (4.67 +/- 1.10 ng/ml) and 33 urines (35.5 +/- 8.8 ng/mg creatinine; 25.3 +/- 8.1 ng/ml) from passively exposed children were significantly higher (P less than 0.001) than in fluids of 36 unexposed children. Children's levels showed a strong correlation (P less than 0.001) with the number of cigarettes smoked in the home, but only when data from nonsmoking households were included in the analysis. In adult smokers there was a positive correlation between salivary and urinary cotinine (P = 0.002) and a close relationship between urinary cotinine and cigarettes smoked per day (P = 0.066). The ELISA gives a reliable quantitative measure of cotinine as an indicator of active and passive exposure to tobacco smoke. However, correlations with cotinine can be overestimated if large numbers of nonsmokers are included in the comparison.

Monoclonal anti-idiotypic antibodies that recognize the binding site for nicotine on rat brain receptor.

Anti-idiotypic monoclonal antibodies have been prepared that represent the internal image of nicotine and are specific for the nicotine binding site on rat brain receptor. Specificity of these antibodies for the combining site on anti-nicotine was demonstrated by their ability to inhibit binding of monoclonal anti-nicotine to immobilized nicotine-polylysine. Furthermore, purified rat brain nicotine receptor but not acetylcholine receptor from fish electric organ effectively competed with anti-nicotine for immobilized nicotine and for immobilized anti-idiotype. Only 9 pmoles of naturally occurring (-)-nicotine inhibited idiotype-anti-idiotype binding by 50% whereas 11 times more (+)-nicotine was required. Acetylcholine, several cholinergic agonists and antagonists, nicotine metabolites, and other structurally related compounds were poor inhibitors.