SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.

Zolantidine (SK&F 95282) is a potent selective brain-penetrating histamine H2-receptor antagonist.

1. The novel benzthiazole derivative zolantidine (SK&F 95282) is a potent antagonist of histamine at H2-receptors in guinea-pig atrium and rat uterus. Only apparent pA2 values of 7.46 and 7.26 respectively could be calculated since the slopes of the Schild plots were significantly less than unity. 2. Zolantidine is equally potent as an antagonist at histamine H2-receptors in guinea-pig brain. The compound inhibited histamine stimulated adenylate cyclase (pKi 7.3) and dimaprit stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation (approx pA2 7.63), and competed with [3H]-tiotidine binding (pKi 7.17). 3. Zolantidine is at least 30 fold more potent at H2-receptors than at other peripheral and central receptors investigated. 4. Infusion of zolantidine into rats produces a brain concentration greater than the plateau blood concentration (brain/blood ratio 1.45). 5. Zolantidine is thus characterized as a potent selective brain-penetrating H2-receptor antagonist, and will be a valuable pharmacological tool for investigating possible physiological and pathological roles for histamine in the central nervous system.

Evidence for low brain penetration by the H1-receptor antagonist temelastine (SK&F 93944).

Competition by the potent selective H1-receptor antagonist temelastine (SK & F 93944) with [3H]mepyramine binding to mouse cortex H1-receptors has been measured in vitro and vivo. The data were compared with that obtained using the classical H1-receptor antagonists mepyramine and promethazine and indicated that temelastine has relatively low ability to penetrate the blood-brain barrier compared with the latter two compounds. These studies also revealed that temelastine has relatively low affinity for mouse cortex H1-receptors compared with its affinity for H1-receptors in guinea-pig ileum and cortex, suggesting that it may be a useful compound with which to investigate potential H1-receptor tissue and species-related differences.

The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.

Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.

3H-tiotidine binding to guinea-pig cortical and striatal membranes.

3H-Tiotidine has been identified as a suitable radioligand for the H2-receptor. We have confirmed and extended structure-binding affinity studies in the guinea-pig cortex, and established a structure-binding affinity relationship consistent with the H2-receptor in guinea-pig striatum. Cimetidine-displaceable 3H-tiotidine binding was observed also in the nucleus accumbens.

N-Methyldimaprit (SK&F92054). A chemical control for the histamine H2-receptor agonist dimaprit.

Dimaprit analogues having alkyl substituents on the isothiourea group have no detectable H2-agonist activity on the guinea-pig atrium. N-Methyldimaprit [S-[3-(N,N-dimethylamino)propyl]-N'-methylisothiourea] (SK&F92054) is identified as a compound which may serve as a suitable chemical control when studying the actions of dimaprit at putative H2-histamine receptors. Concentrations of N-methyl-dimaprit (up to 3 X 10(-3) M) had no agonist activity in vitro on the guinea-pig atrium (less than 0.005% of the activity of histamine), rat uterus (up to 2 X 10(-3) M,) or as a stimulant of guinea-pig ventricular adenylate cyclase (up to 10(-3) M). N-Methyldimaprit at 10(-3) M also did not significantly stimulate acid secretion in the rat isolated stomach preparation; it was a very weak stimulant of gastric acid secretion (less than 0.2% of the activity of histamine) in vivo in the lumen-perfused stomach of the anaesthetised rat but exhibited marked tachyphylaxis on repeated administration. N-Methyldimaprit has two basic centres, macroscopic pKa values 8.31 and 9.58 at 40 degrees C.

Inhibitors in urine of radioimmunoassay for the detection of gonococcal antigens.

Several substances in urine were found to inhibit the radioimmunoassay of added gonococcal antigens. The supernatants of two-thirds of urine samples from male patients with either gonorrhoea or non-specific urethritis (NSU) were inhibitory. The inhibition caused by many, but not all, samples was reduced or completely abolished by the addition of soybean trypsin inhibitor (STI); STI-sensitive inhibition is thought to be due to proteolytic enzymes, probably from pus cells. Their inhibitory effect was shown to be due to their action on gonoccocal antigens and not on antibodies in the assay system. Some supernatants contained other inhibitors unaffected by STI; some of these were dialysable and others were not. Sediments from the urine of patients with NSU or gonorrhoea were often strongly inhibitory, but treatment with STI annulled all but very slight inhibition. STI-treated sediments could, therefore, be used in an assay designed to detect gonococcal antigens.