Pharmacology of SB-273779, a nonpeptide calcitonin gene-related peptide 1 receptor antagonist.

Calcitonin gene-related peptide (CGRP), a potent vasodilatory and cardiotonic peptide, has a potential role for CGRP in diverse physiologic and pathophysiologic situations such as congestive heart failure, diabetes, migraine, and neurogenic inflammation. Although a peptide CGRP receptor antagonist, CGRP(8-37,) is available, its utility presents significant limitations for these indications. Here, we describe the properties of SB-(+)-273779 [N-methyl-N-(2-methylphenyl)-3-nitro-4-(2-thiazolylsulfinyl)nitrobenzanilide], a selective nonpeptide antagonist of CGRP(1) receptor. SB-(+)-273779 inhibited (125)I-labeled CGRP binding to SK-N-MC (human neuroblastoma cells) and human cloned CGRP(1) receptor with K(i) values of 310 +/- 40 and 250 +/-15 nM, respectively. SB-(+)-273779 also inhibited CGRP (3 nM)-activated adenylyl cyclase in these systems with IC(50) values of 390 +/-10 nM (in SK-N-MC) and 210 +/-16 nM (recombinant human CGRP receptors). Prolonged treatment (>30 min) of SK-N-MC cells with SB-(+)-273779 followed by extensive washing resulted in reduction in maximum CGRP-mediated adenylyl cyclase activity, suggesting that this compound has irreversible binding characteristics. In addition, SB-(+)-273779 antagonized CGRP-mediated 1) stimulation of intracellular Ca(2+) in recombinant CGRP receptors in HEK-293 cells, 2) inhibition of insulin-stimulated [(14)C]deoxyglucose uptake in L6 cells, 3) vasodilation in rat pulmonary artery, and 4) decrease in blood pressure in anesthetized rats. SB-(+)-273779 tested at 3 microM had no significant affinity for calcitonin, endothelin, angiotensin II, and alpha-adrenergic receptors under standard ligand binding assays. SB-(+)-273779 also did not inhibit forskolin and pituitary adenylate cyclase-activating polypeptide. These results suggest that SB-(+)-273779 is a valuable tool for studying CGRP-mediated functional responses in complex biological systems.

Impaired endothelium-dependent relaxation by adrenomedullin in monocrotaline-treated rat arteries.

The effects of adrenomedullin were evaluated in isolated vascular rings from rats treated with monocrotaline (60 mg/kg, s.c.) causing pulmonary hypertension and right ventricular hypertrophy within 3 to 4 weeks. Sham animals (NaCl-treated rats) were used for comparison. The relaxing effects of adrenomedullin (10(-8) M) and acetylcholine (10(-6) M) were determined in thoracic aorta and pulmonary artery rings precontracted with phenylephrine (10(-7) M). In sham animals, adrenomedullin caused significant vasorelaxation of aorta and pulmonary artery although of different amplitude (24 +/- 3% and 40 +/- 2%, respectively). A greater relaxation was observed in response to acetylcholine. Monocrotaline-treated rats exhibited a reduction in adrenomedullin relaxation in pulmonary artery (54 and 68% loss of effect, at 3 and 4 weeks, respectively, P < 0.01 vs. sham) and comparable reductions in acetylcholine responses. The decrease in adrenomedullin relaxing effect was less pronounced in aorta than in pulmonary artery, suggesting a distinct tissue sensitivity to monocrotaline. In contrast, the relaxing effect of acetylcholine on aorta was decreased at 4 weeks (36% reduction, P < 0.01 vs. sham). In this model, the adrenomedullin-induced relaxation of the pulmonary artery was impaired due to a severe endothelial dysfunction which may contribute partly to the evolving pathophysiological process.

Relationship between biochemical and functional effects of protein phosphatase 1 inhibitors in rabbit cardiac skinned fibers.

Tautomycin (TT) and calyculin A (CyA) are inhibitors of protein phosphatases type 1 and 2 (PP1, PP2). Inhibitors 1 and 2 are specific for PP1, which is the major phosphatase functionally relevant in heart and able to dephosphorylate phospholamban (PLB). TT and CyA maintain PLB in its phosphorylated state, thereby increasing calcium uptake. Rabbit saponin skinned fibers (SF) are used to assess calcium load of the sarcoplasmic reticulum (SR). The present investigation aimed to examine the effects of PP1 inhibitors on SR calcium load assessed by caffeine-induced tension transient (CITT), and to correlate this activity with the PLB phosphorylation state. TT and CyA (100 nm) applied during the uptake phase increased the amplitude of CITT by 10 and 20%, respectively,P<0.05 without effect on the release phase. Both CyA and TT were devoid of calcium sensitizing effect when studied on Triton X-100 SF. After skinning procedure, SF were grinded for biochemical studies. SDS-PAGE electrophoresis and immunoblots using a monoclonal PLB antibody showed that cAMP or Ca2+/calmodulin-dependent protein kinases phosphorylated PLB in an additive fashion. Inhibition of PP1 by inhibitor 1, CyA and TT maintained PLB in its phosphorylated state in a dose-dependent manner. The results of this study in which functional and biochemical experiments in cardiac SF were combined demonstrate that strong correlation exists between the phosphorylation-dephosphorylation cycle of PLB and calcium uptake.

Adjunct effect of loratadine in the treatment of acute sinusitis in patients with allergic rhinitis.

H1-blockers are often added to the standard treatment of acute sinusitis, but this is not supported by a controlled study. A multicentric, randomized, double-blind, placebo-controlled, parallel-group study was done in 139 allergic patients (15-65 years) to assess the adjunct efficacy of loratadine in acute exacerbation of rhinosinusitis. Sinusitis was diagnosed by symptoms and confirmed by rhinoscopy and sinus radiograph. Allergy was characterized by skin tests, RAST, and history. Patients were treated with antibiotics (14 days), oral corticosteroids (10 days), and loratadine (10 mg OD) or placebo (28 days). Treatment efficacy was assessed over 28 days by symptom scores quoted daily by patients. Physicians also rated total symptom scores at entry and at day 28. At entry, both groups had similar symptoms. Placebo-treated patients improved significantly, but patients who received loratadine had a significantly greater improvement in sneezing (P = 0.003) after 14 days, and in nasal obstruction (P = 0.002) after 28 days. Physicians found that patients receiving loratadine were significantly improved compared to placebo patients (P = 0.0125). Loratadine in addition to standard therapy was found to improve the control of some symptoms of sinusitis.

Effects of cyclopiazonic acid, an inhibitor of the Ca++ ATPase of sarcoplasmic reticulum, on Ca++ transport, contraction and relaxation in cardiac muscle.

Cyclopiazonic acid is a potent inhibitor of cardiac sarcoplasmic reticulum Ca++ ATPase. It scarely affects inotropism but significantly impairs lusitropism suggesting a greater role for cardiac sarcoplasmic reticulum in the control of cardiac relaxation than in the control of cardiac contraction.

A novel class of cardiotonic agents: synthesis and biological evaluation of 5-substituted 3,6-dihydrothiadiazin-2-ones with cyclic AMP phosphodiesterase inhibiting and myofibrillar calcium sensitizing properties.

As part of a search for new cardiotonic agents significantly sensitising the myocardial contractile proteins to calcium, together with cardiac cyclic AMP-PDE inhibitory activity, we have discovered that novel 5-substituted 3,6-dihydrothiadiazin-2-ones may fulfill both properties. The sensitising effect of the contractile proteins to calcium, assessed by the shift in the calcium sensitivity of canine cardiac myofibrillar magnesium-dependent ATPase, is determined by steric and electronic requirements. The requirements for phosphodiesterase inhibition, especially that of a near-planar arrangement for the phenyl and thiadiazin-2-one ring are consistent with those already described for analogous pyridazinones. The synthesis and structure-activity relationships are discussed.

Anti-islet immunity and thymic dysfunction in the mutant diabetic C57BL/KsJ db/db mouse.

Anti-islet immune reactions were studied in vitro in genetically diabetic homozygote C57BL/KsJ db/db mice, using murine islet cells as a target. Spleen lymphocytes inhibited insulin secretion by the islet cells. This inhibition was abolished when T-cells were eliminated by treatment with anti-Thy 1.2 monoclonal antibody in the presence of complement. Anti-islet complement-dependent antibody (CDA) and antibody-dependent cell cytotoxicity (ADCC) were also found in the sera of these mice. This anti-islet immunity was detectable as early as the tenth day of life and lasted throughout the entire life span of the animals. A significant lymphopenia was detected in thymus and spleen cell populations. None of these anomalies was found in control heterozygote mice. Thymic function was explored in the same mice by evaluating their serum thymic factor (FTS) levels using a rosette assay. The age-dependent decline of FTS levels was significantly accelerated in diabetic mice as compared with heterozygous littermates. Furthermore, FTS inhibitory immunoglobulins were detected in db/db mouse sera, which inactivated in vitro the biologic potency of synthetic FTS. Histologically, the thymus displayed an accelerated involution. It was shown by indirect immunofluorescence using anti-FTS monoclonal antibodies that the number of FTS+ cells was reduced in db/db mouse thymuses. Histologic study of the islets of Langerhans showed early signs of beta-cell hyperactivity and hypertrophy, followed by beta-cell rarefaction and profound dislocation of islet architecture. Insulitis was not detected.