Action of calcitonin gene-related peptide upon bovine vascular endothelial and smooth muscle cells grown in isolation and co-culture.

1. Bovine aortic endothelial cells (BAE) and smooth muscle cells (BASM) were grown separately and in co-culture. 2. Calcitonin gene-related peptide (CGRP) caused dose-dependent activation of adenylate cyclase in each cell type when grown in isolation. The concentration of CGRP causing half-maximal activation in BAE and BASM was 200 nM and 310 nM, respectively. 3. In cells grown in co-culture exposure to bradykinin produced dose-dependent elevations in cyclic GMP content which were maximal 1 min after application of the agonist. 4. CGRP (1 nM-1 microM) did not stimulate a rise in cyclic GMP in co-cultures. 5. Displaceable CGRP binding was identified throughout the wall of the bovine aorta. 6. We conclude that CGRP receptors coupled to adenylate cyclase are present on BAE and BASM, but there is no coupling of these receptors to the release of any agent (such as endothelium-derived relaxing factor) that activates guanylate cyclase.     

Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease.

In addition to their vasoactive action, endothelins are potent peptides in the regulation of both cell proliferation and the turnover of extracellular matrix. Using immunohistochemical, autoradiographic, and molecular analyses, we have studied the localization and expression of endothelin-1 and endothelin A (ETA) and B (ETB) receptors in scleroderma-associated fibrotic lung disease. Increased ET-1 immunoreactivity was found in sclerotic tissue compared with control and was associated with the vasculature, pulmonary interstitium, and bronchial and alveolar epithelium. Microautoradiographic analysis after 125I-labeled ET-1 binding showed a two- to threefold increase in the expression of total ET-1 receptors in scleroderma lung tissue localized to the alveolar epithelium and the pulmonary interstitium which was composed of mainly fibroblastic cells with macrophages and some microvessels. RNAse protection assay revealed significantly reduced ETA receptor and slightly raised ETB message levels in systemic sclerosis lung. Surface expression of functional ET receptors was examined by targeted receptor blocking using mixed and receptor-subtype-selective ligands. A consistent decrease in ETA receptor binding sites was noted primarily within the interstitium and vasculature, in contrast to a slight increase in ETB receptors. Elevated ET-1 and the cell-specific pattern of endothelin receptor expression suggest that the endothelins may represent important mediators that influence the pathology of scleroderma-associated lung disease and other fibrotic conditions.     

Chlorophyllin-enhanced excretion of urinary and fecal mutagens in rats given 2-amino-3-methylimidazo[4,5-f]quinoline.

Sodium/copper chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits antimutagenic activity in several short-term genotoxicity assays and inhibits carcinogen-DNA binding in vivo. The effect of CHL pretreatment on the excretion of mutagens in the urine and feces of male Sprague-Dawley rats has been studied using the Salmonella mutagenicity assay. Animals were given 1 percent CHL in the drinking water for 2 days before administering a single dose of 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) by oral gavage. Rats pretreated with CHL had higher levels of mutagens in the urine and feces compared with animals given IQ alone; 48 hr after IQ administration, the total mutagenic dose excreted was < 4% in controls vs. 18% in rats given CHL. Mutagenicity required the presence of an activation system, was unaffected by treatment with beta-glucuronidase or arylsulfatase, and in both the urine and feces was accounted for by increased elimination of unmetabolized parent compound. The results support the view that CHL may operate in vivo as a "desmutagen" or interceptor molecule, interacting with IQ in the gut and tissues, and reducing carcinogen bioavailability.     

Evidence for ras gene mutation in 2-amino-3-methylimidazo[4,5-f]quinoline–induced colonic aberrant crypts in the rat

Aberrant crypt foci (ACF) are putative peneoplastic lesions that develop after treatment of animals with colon carcinogens, including cooked-meat heterocyclic amines such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 rats given IQ by gavage on alternating days for 2 wk (130 mg/kg body weight) and killed 12 wk after the final carcinogen dose had an average of 4.4 ACF/colon and an average of 3.2 crypts/focus. The DNA from these ACF was amplified by the polymerase chain reaction and analyzed by 3′-primer mismatch and direct sequencing methods for mutations in the Ki-ras proto-oncogene. Of the 37 IQ-induced ACF screened, three contained a GGT→GAT mutation in codon 12 and one contained a GGC→GCC mutation in codon 13. The approximately 11% frequency of mutation in IQ-induced ACF is within the range of previous ACF studies of azoxymethane, which reported a 7–37% incidence of Ki-ras mutaion. These findings suggest that for both compounds, ras mutations occur during early stages of colorectal tumorigenesis. However, while ras mutations can be detected with increasing frequency in azoxymethane-induced adenomas and carcinomas, they are reportedly absent in IQ-induced colon tumors. Thus, for IQ and related compounds additional factors (possibly increased cell proliferation) may be important in the later stages of colorectal tumorigenesis. © 1995 Wiley-Liss Inc.     

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION: Protection by green tea, black tea, and indole-3-carbinol against 2-amino-3-methylimidazo[4,5-f]quinoline-induced DNA adducts and colonic aberrant crypts in the F344 rat

Male F344 rats were exposed for 8 weeks to extracts of greentea (2% w/v) or black tea (1% w/v), or to 0.1% dietary indole-3-carbinol(I3C). In weeks 3 and 4 of the study, rats were given 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) every other day by oral gavage (50 mg/kgbody wt) in order to induce aberrant crypt foci (ACF) in thecolon. Compared with controls given IQ alone, all three inhibitorsreduced the number of total aberrant crypts per colon, and greentea and I3C inhibited significantly the mean number of ACF (P< 0.05). Rats pre-treated with green tea, black tea, or I3Cand given a single p.o. injection of 50 mg IQ/kg body wt 24–48h before sacrifice had reduced levels of IQ-DNA adducts in theliver, and excreted lower amounts of IQ and other promutagensin the urine and feces. Inhibitors also reduced the excretionof IQ-sulfamate in the urine, but increased the relative amountsof IQ-5-O-sulfate and IQ-5-O-glucuronide. Western blotting togetherwith assays for 7-ethoxyresorufin O-deethylase and methoxyresorufinO-demethylase established that I3C preferentially induced cytochromeP4501A1 over 1A2, consistent with the altered profile of urinarymetabolites. However, both teas caused slight induction of cytochromeP4501A2 versus 1A1, which would be predicted to enhance theactivation of IQ. Thus, green tea and black tea are likely toprotect against IQ-DNA adducts and ACF by mechanisms other thaninduction of cytochromes P450, such as inhibition of enzymeswhich activate IQ or the scavenging of reactive intermediates.     

Enhanced relaxation of diabetic rabbit cavernosal smooth muscle in response to nitric oxide: potential relevance to erectile dysfunction

New Zealand white rabbit cavernosal smooth muscle strips (n=6) were mounted in organ baths. Relaxations to nitric oxide (10(-7)-10(-4) mol/l) were measured and the same procedure was repeated on strips from rabbits 6 months after alloxan-induced diabetes (n=6). Transverse cavernosal sections were obtained from the same penises. Low and high resolution autoradiographs were prepared using [(3)H]-L-N(G)-nitroarginine (an index of nitric oxide binding sites) and analysed densitometrically. Histochemical analysis was performed on adjacent sections using NADPH diaphorase (an index of nitric oxide synthase activity).Nitric oxide relaxed control rabbit cavernosal smooth muscle strips in a concentration-dependent manner. Diabetic rabbit cavernosal smooth muscle strips were significantly (P<0.03) more sensitive to nitric oxide (mean IC(50)=3.9 x 10(-6) mol/l). Nitric oxide synthase binding sites were localised to the cavernosal endothelium and smooth muscle. Nitric oxide synthase activity was increased in 6 month diabetic cavernosal smooth muscle. These findings suggest impairments in the L-arginine-nitric oxide pathway may play a role in the pathophysiology of diabetic erectile dysfunction.     

Inhibitory Activity of Green and Black Tea in a Free Radical-generating System Using 2-Amino-3-methylimidazo[4,5-f]quinoline as Substrate

Green tea and black tea inhibit colon carcinogenesis in rats exposed to the cooked meat mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In the present investigation, green tea, black tea and (-)-epigallocatechin gallate (EGCG) were shown to block the production of oxygen free radicals derived from IQ in the presence of NADPH-cytochrome P450 reductase. In kinetic studies using IQ as tbe substrate and DMPO as a free radical spin trap, EGCG increased the Km of the reaction without altering Vmax, suggesting competitive enzyme inhibition (Ki=9.% mUM). This was confirmed in spectrophotometric studies using cytochrome c as the substrate, in which EGCG acted as a competitive inhibitor of NADPH-cytochrome P450 reductase (Ki=9.7 μM). These results suggest that the inhibitory activities of green tea and black tea in electron spin resonance assays using IQ as the substrate for the rednctase are related to an indirect effect on the enzyme rather than via direct scavenging of the free radicals. The possible implications of these findings are discussed in the context of pathways involved in the activation and detoxification of IQ in the colon.