A solid-phase radioimmunoassay using digitonin-permeabilized cells to screen surface or intracellular membrane-bound antigens

Membrane-bound receptor or enzyme distribution between cell surface and cell interior can be determined using the non-ionic detergent digitonin. A solid-phase radioimmunoassay using myocardial cells from newborn rats was performed to screen hybridoma culture supernatants and the cells were rendered permeable with increasing concentrations of digitonin (0-0.1%). This was achieved in 5 min at 37 degrees C and did not require the continuous presence of detergent. A characteristic amount of cytoplasmic protein (approximately equal to 45%) was released with subunit molecular weights of up to 200,000. This allowed exogenous molecules such as immunoglobulin G to gain access to the intracellular targets. The binding to rat myocardial cells of 36 monoclonal antibodies was examined by this procedure. The binding assays were carried out for 30 min at 37 degrees C using cells previously treated for 10 min with 0.05% of digitonin. This appears to be a simple and rapid method of screening and selecting the hybridoma culture supernatants.     

SR 33557, a novel calcium-antagonist: interaction with [3H]-(±)-nitrendipine and [3H]-(−)-desmethoxy-verapamil binding sites in cerebral membranes

Summary We investigated the pharmacological properties of SR 33557, a novel compound with calcium-antagonist properties, in both functional tests in vitro and radioligand binding studies. SR 33557 potently antagonized calcium-induced contraction of potassium-depolarized rat aorta in vitro with an IC₅₀ value of 5.6 ± 0.9 nM, but was a much weaker inhibitor of noradrenaline-induced contraction of the same tissue (IC₅₀ = 96 ± 22 nM). SR 33557 totally inhibited [³H]-(±)-nitrendipine binding to rat brain membranes with a K _i value of 0.19 ± 0.03 nM. Diltiazem, which used alone increases [³H]-(±)-nitrendipine binding, reversed this inhibition indicating that SR 33557 allosterically regulates [³H]-(±)-nitrendipine binding. SR 33557 also fully inhibited [³H]-(–)-desmethoxyverapamil binding to cerebral membranes, but inhibition curves were biphasic. IC₅₀ value calculated for that part of the curve which reflects the high affinity binding site of SR 33557 (IC₅₀ = 0.20 ± 0.02 nM) was very similar to the K _i value determined for inhibition of [³H]-(±)-nitrendipine binding. Kinetic evidences indicate that SR 33557 binds to a site which is distinct from the 1,4-dihydropyridine or the phenylalkylamine binding sites associated with the calcium channel. To test the pharmacological specificity of these interactions, the ability of SR 33557 to interact with eight other receptors in cerebral or heart membranes was assessed by binding assays. No high-affinity interaction was observed between SR 33557 and any of the receptors investigated. We conclude that SR 33557 binds specifically and with a high affinity to a site closely associated with the voltage-operated calcium channel in cerebral membranes. Send offprint requests to: P. Chatelain at the above address     

Increase in plasma levels of active and inactive renin after inhibition of renin activity by SR 42128 in conscious Macaca monkeys

Plasma active renin (PAR), plasma inactive renin (PIR), and plasma renin activity (PRA) were determined after intravenous bolus injection of the renin inhibitor SR 42128, in sodium repleted and sodium depleted macacas. The kit renin of Pasteur Diagnostics allows determination of PAR after renin inhibition by SR 42128. PAR and total plasmatic renin (TPR) were determined before and after treatment of plasma using trypsin. IR = TPR-PAR. ARP was measured by RIA of angiotensin I. Sodium depletion induced a dramatic increase of PAR (1,678 + 11.5 pg/ml compared to 94.4 + 11.5, n = 6). PIR rose from 322.1 + 34.3 pg/ml to 1,137 + 206 (n = 6). In sodium repleted macacas, SR 42128 (3 mg/kg and 9 mg/kg) induced a PRA inhibition of 90 to 100 p. 100, for 4 h post-injection. PAR increased to reach maximal level after 90 min and remained constant up to 4 h post-injection (increase of 420 p. 100 at 3 mg/kg and 620 p. 100 at 9 mg/kg). PIR increased more slowly for 4 h (maximum increase of 250 p. 100). PRA was also inhibited in sodium depleted macacas by SR 42128 at the doses of 3 mg/kg and 9 mg/kg ARP was inhibited. PIR increased more slowly, but significantly at 9 mg/kg. We conclude that the activity of SR 42128 on PAR and PIR levels is the sole consequence of the inhibition of the Renin Angiotensin system.     

VEGF levels in asthmatic airways do not correlate with plasma extravasation

BACKGROUND: Vascular permeability/vascular endothelial growth factor (VEGF) is a multifunctional cytokine which plays a role in chronic inflammation and angiogenesis. Its expression in bronchoalveolar lavage (BAL) has not been determined although VEGF may be relevant to the pathophysiology of asthma in which oedema is an important feature. METHODS: We studied VEGF, albumin and IgA immunoreactive levels in the BAL fluids obtained from 27 chronic stable asthmatics, nine untreated chronic bronchitis patients and 15 control subjects. RESULTS: BAL fluid levels of VEGF and VEGF normalized to IgA were not significantly different in any patient group. Both asthmatic steroid- and non-steroid-treated groups had significantly lower albumin levels in their BAL fluids explaining most of the 179% increased VEGF normalized to albumin ratios in non-steroid treated asthmatics. Moreover, VEGF and albumin markers correlated in control subjects (r = 0.73, P = 0.006) and in chronic bronchitics (r = 0.75, P = 0.03, Spearman test), but not in asthmatics. VEGF was inversely correlated with asthma severity (GINA/NHLBI scores) in non-steroid treated asthmatics (tau = - 0.52, P = 0.009, Kendall test). CONCLUSIONS: Thus, the potential role of VEGF in asthma requires further studies on bronchial biopsies and induced sputum.     

Inhibition of platelet phospholipase-A2 as a mechanism for the anti-aggregating effect of linoleic acid

Linoleic acid was incorporated into platelet phospholipids and then released after activation of phospholipase A₂ (PL-A₂) with thrombin or ionophore A23187. The rate of this release was tenfold lower for linoleic than for arachidonic acid. This observation strongly suggests that incorporation of linoleic acid in platelet phospholipids might inhibit platelet PL-A₂ and might explain the anti-aggregating effect of linoleic acid. In fact it has also been shown that linoleic acid inhibits PL-A₂ activity at concentrations which antagonize platelet aggregation. Moreover, in experimental conditions where platelet cyclo-oxygenase is totally inhibited, aggregation does occur and can still be blocked by linoleic acid. This latter observation led to the conclusion that the anti-aggregating effect of linoleic acid is independent from prostaglandin pathway and is probably related to the phospholipid metabolism.     

SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride], a new nonpeptide antagonist of the bradykinin B1 receptor: biochemical and pharmacological characterization

The biochemical and pharmacological properties of a novel non-peptide antagonist of the bradykinin (BK) B(1) receptor, SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride] were evaluated. SSR240612 inhibited the binding of [(3)H]Lys(0)-des-Arg(9)-BK to the B(1) receptor in human fibroblast MRC5 and to recombinant human B(1) receptor expressed in human embryonic kidney cells with inhibition constants (K(i)) of 0.48 and 0.73 nM, respectively. The compound selectivity for B(1) versus B(2) receptors was in the range of 500- to 1000-fold. SSR240612 inhibited Lys(0)-desAr(9)-BK (10 nM)-induced inositol monophosphate formation in human fibroblast MRC5, with an IC(50) of 1.9 nM. It also antagonized des-Arg(9)-BK-induced contractions of isolated rabbit aorta and mesenteric plexus of rat ileum with a pA(2) of 8.9 and 9.4, respectively. Antagonistic properties of SSR240612 were also demonstrated in vivo. SSR240612 inhibited des-Arg(9)-BK-induced paw edema in mice (3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p.). Moreover, SSR240612 reduced capsaicin-induced ear edema in mice (0.3, 3 and 30 mg/kg p.o.) and tissue destruction and neutrophil accumulation in the rat intestine following splanchnic artery occlusion/reperfusion (0.3 mg/kg i.v.). The compound also inhibited thermal hyperalgesia induced by UV irradiation (1 and 3 mg/kg p.o.) and the late phase of nociceptive response to formalin in rats (10 and 30 mg/kg p.o.). Finally, SSR240612 (20 and 30 mg/kg p.o.) prevented neuropathic thermal pain induced by sciatic nerve constriction in the rat. In conclusion, SSR240612 is a new, potent, and orally active specific non-peptide bradykinin B(1) receptor antagonist.