PAF binding to a single receptor in corneal epithelium plasma membrane.
(9/3334)
PURPOSE: To study the binding characteristics and the expression of platelet-activating factor receptors (PAF-R) in corneal epithelium to elucidate the site of action of PAF. METHODS: Binding of [3H]PAF was investigated in subcellular fractions of the epithelia of bovine corneas and in membranes from cultured rabbit corneal epithelial cells. Dose-response inhibition curves of [3H]PAF-specific binding were generated using increasing concentrations of several PAF-R antagonists. RNA from rabbit corneal epithelial cells was probed for PAF-R expression by reverse transcription-polymerase chain reaction (RT-PCR) with specifically designed degenerated primers. RESULTS: Scatchard analysis showed a high-affinity binding site in bovine and rabbit corneal epithelium. The dissociation constant (Kd) and the maximum binding sites (Bmax) in a bovine membrane preparation and similar rabbit fraction were 0.77+/-0.03 nM and 180+/-21 femtomoles/mg protein and 4.3 nM and 1.3 picomoles/mg protein, respectively. Specific PAF-binding sites were found in bovine preparations enriched in plasma membranes with a Kd = 69.6 pM and Bmax = 80 femtomoles/mg protein; no specific binding was found in nuclei or microsomal fractions. RT-PCR of rabbit corneal epithelium generated a single product of the predicted size (478 bp). The deduced amino acid sequence of the purified PCR product was 87% homologous to human PAF-R. The hetrazepines BN 50727 and BN 50730 and the PAF structural analogues CV 3988 and CV 6209 competitively inhibited [3H]PAF binding to corneal epithelium with similar potency. WEB 2086 BS was two orders of magnitude less active in antagonizing PAF binding. CONCLUSIONS: Corneal epithelium contains a single population of receptors localized in the plasma membrane. PAF antagonists exert their actions by blocking this PAF-R. The partial sequence deduced in rabbit corneal PAF-R show a higher homology to the human PAF-R. (+info)
Oestrogen receptor-alpha variant mRNA expression in primary human breast tumours and matched lymph node metastases.
(10/3334)
We have shown previously that the relative expression of a truncated oestrogen receptor-alpha variant mRNA (ER clone 4) is significantly increased in axillary node-positive primary breast tumours compared with node-negative tumours. In this study, we have examined the relative expression of clone 4-truncated, exon 5-deleted and exon 7-deleted oestrogen receptor-alpha variant mRNAs in 15 primary breast tumour samples and in synchronous axillary lymph node metastases. Overall, there were no significant differences between the primary tumours and the matched metastases in the relative expression of these three specific variant mRNAs. Furthermore, the pattern of all deleted oestrogen receptor-alpha variant mRNAs appeared conserved between any primary and its matched secondary tumour. (+info)
Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation.
(11/3334)
Bombesin-like peptides such as gastrin-releasing peptide (GRP) have been shown to play a role in cancer as autocrine growth factors that stimulate tumor growth through specific receptors. To search for potential clinical indications for GRP analogues, it is important to identify human tumor types expressing sufficient amounts of the respective receptors. In the present study, we have evaluated the expression of GRP receptors in human nonneoplastic and neoplastic prostate tissues using in vitro receptor autoradiography on tissue sections with 125I-Tyr4-bombesin as radio-ligand. GRP receptors were detected, often in high density, in 30 of 30 invasive prostatic carcinomas and also in 26 of 26 cases of prostatic intraepithelial proliferative lesions, corresponding mostly to prostatic intraepithelial neoplasias. Well-differentiated carcinomas had a higher receptor density than poorly differentiated ones. Bone metastases of androgen-independent prostate cancers were GRP receptor-positive in 4 of 7 cases. Conversely, GRP receptors were identified in only a few hyperplastic prostates and were localized in very low density in glandular tissue and, focally, in some stromal tissue. In all of the cases, the receptors corresponded to the GRP receptor subtype of bombesin receptors, having high affinity for GRP and bombesin and lower affinity for neuromedin B. These data demonstrate a massive GRP receptor overexpression in prostate tissues that are neoplastically transformed or, like prostatic intraepithelial neoplasias, are in the process of malignant transformation. GRP receptors may be markers for early molecular events in prostate carcinogenesis and useful in differentiating prostate hyperplasia from prostate neoplasia Such data may not only be of biological significance but may also provide a molecular basis for potential clinical applications such as GRP-receptor scintigraphy for early tumor diagnosis, radiotherapy with radiolabeled bombesin-like peptide analogues, and chemotherapy with cytotoxic bombesin analogues. (+info)
Binding properties of C-truncated delta opioid receptors.
(12/3334)
AIM: To study the role of C-terminal delta opioid receptor involved in ligand binding affinity and selectivity. METHODS: The 31 amino acid residues of C-terminal truncated delta opioid receptors and the wild-type were expressed stably in Chinese hamster ovary (CHO) cells, respectively. Then the ligand binding properties of the products were studied by receptor binding assay. RESULTS: A typical mutated receptor clone CHO-T and a wild-type receptor clone CHO-W were obtained. The Kd values of [3H] diprenorphine (Dip) and [3H]leucine-2-alanine enkephalin (DADLE) bound to CHO-T were similar to CHO-W. Both the specific [3H]Dip bindings of CHO-T and CHO-W were strongly inhibited by delta selective agonists with similar Ki, but neither by mu nor kappa selective agonists. CONCLUSION: The C-terminal of the delta opioid receptor is not involved in the ligands binding affinity and selectivity. (+info)
Endothelin receptor expression and pharmacology in human saphenous vein graft.
(13/3334)
1. We have investigated the expression and pharmacology of endothelin (ET) receptors in human aortocoronary saphenous vein grafts. 2. Subtype-selective ligands were used to autoradiographically identify ET(A) ([125I]-PD151242) and ET(B)([125I]-BQ3020) receptors. In graft saphenous vein ETA receptors predominated in the media, with few ET(B) receptors identified. Neither subtype was detected in the thickened neointima. 3. The ratio of medial ET(A):ET(B) receptors was 75%: 25% in both graft and control saphenous vein. 4. ET-1 contracted control (EC50 2.9 nM) and graft (EC50 4.5 nM) saphenous vein more potently than diseased coronary artery (EC50 25.5 nM). 5. In all three blood vessels ET-1 was 100 times more potent than ET-3 and three times more potent than sarafotoxin 6b (S6b). Little or no response was obtained in any vessel with the ET(B) agonist sarafotoxin 6c (S6c). 6. The ET(A) antagonist PD156707 (100 nM) blocked ET-1 responses in all three vessels with pKb values of approximately 8.0. 7. For individual graft veins the EC50 value for ET-1 and 'age' of graft in years showed a significant negative correlation. 8. In conclusion there is no alteration in ET receptor expression in the media of saphenous veins grafted into the coronary circulation compared to control veins. ETA receptors predominantly mediate the vasoconstrictor response to ET-1 in graft vein, with no apparent up-regulation of ET(B) receptors. The sensitivity of the graft vein to ET-1 increased with graft 'age', suggesting that these vessels may be particularly vulnerable to the increased plasma ET levels that are detected in patients with cardiovascular disease. (+info)
In vitro and in vivo characterization of intrinsic sympathomimetic activity in normal and heart failure rats.
(14/3334)
Clinical studies conducted with carvedilol suggest that beta-adrenoceptor antagonism is an effective therapeutic approach to the treatment of heart failure. However, many beta-adrenoceptor antagonists are weak partial agonists and possess significant intrinsic sympathomimetic activity (ISA), which may be problematic in the treatment of heart failure. In the present study, the ISAs of bucindolol, xamoterol, bisoprolol, and carvedilol were evaluated and compared in normal rats [Sprague-Dawley (SD)], in rats with confirmed heart failure [spontaneously hypertensive heart failure (SHHF)], and in isolated neonatal rat cardiomyocytes. At equieffective beta1-adrenolytic doses, the administration of xamoterol and bucindolol produced a prolonged, equieffective, and dose-related increase in heart rate in both pithed SD rats (ED50 = 5 and 40 microgram/kg, respectively) and SHHF rats (ED50 = 6 and 30 microgram/kg, respectively). The maximum effect of both compounds in SHHF rats was approximately 50% of that observed in SD rats. In contrast, carvedilol and bisoprolol had no significant effect on resting heart rate in the pithed SD or SHHF rat. The maximum increase in heart rate elicited by xamoterol and bucindolol was inhibited by treatment with propranolol, carvedilol, and betaxolol (beta1-adrenoceptor antagonist) but not by ICI 118551 (beta2-adrenoceptor antagonist) in neonatal rat. When the beta-adrenoceptor-mediated cAMP response was examined in cardiomyocytes, an identical partial agonist/antagonist response profile was observed for all compounds, demonstrating a strong correlation with the in vivo results. In contrast, GTP-sensitive ligand binding and tissue adenylate cyclase activity were not sensitive methods for detecting beta-adrenoceptor partial agonist activity in the heart. In summary, xamoterol and bucindolol, but not carvedilol and bisoprolol, exhibited direct beta1-adrenoceptor-mediated ISA in normal and heart failure rats. (+info)
Effects of BAY 10-6734 (Embusartan), a new angiotensin II type I receptor antagonist, on vascular smooth muscle cell growth.
(15/3334)
Angiotensin II (AII), an important hypertrophic factor in the cardiovascular system, exerts most of its known effects in vivo through the AII receptor type 1 (AT1) subclass of AII receptors. These receptors are also responsible for the growth-related effects of AII in cultured vascular smooth muscle cells (VSMCs). We presently investigated the effects of BAY 10-6734 (Embusartan), a new orally active AT1 antagonist, on VSMC growth and proliferation of cultured VSMCs isolated from the aortae of Wistar Kyoto rats and spontaneously hypertensive rats. BAY 10-6734 and losartan (considered as AT1 receptor antagonist of reference), as well as their respective active metabolites, were studied for their inhibition of: 1) [125I]AII binding to its receptors, 2) AII-induced DNA and protein synthesis (by measuring the incorporation of 5-bromo-2'-deoxyuridine and [3H]L-leucine, respectively), and 3) AII-induced variations in intracellular Ca2+ concentration, using cells labeled with Fura-2. All of the tested compounds inhibited the aforementioned parameters in a concentration-dependent manner. Half-maximal inhibitory concentration values indicated that BAY 10-6734 was significantly more potent than losartan and that spontaneously hypertensive rat-derived VSMCs were more sensitive than Wistar Kyoto rat-derived ones. Neither BAY 10-6734 nor losartan affected the intracellular Ca2+ concentration of unstimulated VSMCs but both compounds inhibited both AII-induced Ca2+ mobilization from internal stores and Ca2+ influx. Neither compound affected arginine-vasopressin-, basic fibroblast growth factor-, or serum-induced DNA and protein synthesis. BAY 10-6734 appears therefore as a potent and specific new inhibitor of AII-induced growth-related events in VSMCs. (+info)
Characterization of the ATP-sensitive potassium channels (KATP) expressed in guinea pig bladder smooth muscle cells.
(16/3334)
ATP-sensitive K+ (KATP) channels play an important role in the regulation of smooth muscle membrane potential. To investigate the properties of KATP channels in guinea pig urinary bladder smooth muscle cells, fluorescence-based assays were carried out with the membrane potential-sensitive probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)]. The prototypical channel openers, including pinacidil, (-)-cromakalim, and diazoxide, elicited concentration-dependent decreases in membrane potential that were attenuated by glyburide. Similar responses were evoked by a reduction in intracellular ATP levels by metabolic inhibition. The observed rank order potency (EC50) for evoking membrane potential changes by potassium channel openers, P1075 (53 nM) approximately Bay X 9228 > (-)-cromakalim approximately ZD6169 approximately pinacidil > Bay X 9227 approximately ZM244085 > diazoxide (59 microM), showed a good correlation with that of bladder smooth muscle relaxation, as assessed by isolated tissue bath studies. The maximal efficacies of (-)-cromakalim, pinacidil, Bay X 9228, and ZD6169 were comparable with the response achieved by the reference activator P1075. Whole cell currents in bladder smooth muscle cells were increased in both inward and outward directions by P1075 and were reversed by glyburide to control levels. The molecular composition assessed by reverse transcriptase-polymerase chain reaction analysis using subunit-specific primers revealed the presence of mRNA for inward rectifying potassium channel (KIR6.2) and sulfonylurea receptors (SUR)2B and SUR1. The subunit profile together with pharmacological properties suggests that the KATP channel in bladder smooth muscle cells could be composed of SUR2B associated with a single inward rectifier, KIR6.2. In summary, these studies have characterized the pharmacological profile using fluorescent imaging plate reader-based membrane potential techniques and provide evidence for the molecular identity of KATP channels expressed in guinea pig bladder smooth muscle cells. (+info)