Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT(1) and CysLT(2) receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT(2) receptor antagonist.Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT(1) and CysLT(2) receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts.In a CysLT(2) receptor reporter cell line, HAMI3379 antagonized leukotriene D(4)- (LTD(4)-) and leukotriene C(4)- (LTC(4)-) induced intracellular calcium mobilization with IC(50) values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT(1) receptor cell line (IC(50) > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT(2) and CysLT(1) receptor cell lines, HAMI3379 inhibited [(3)H]-LTD(4) binding with IC(50) values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC(4)-induced perfusion pressure increase and contractility decrease. The selective CysLT(1) receptor antagonist zafirlukast was found to be inactive in this experimental setting.HAMI3379 was identified as a potent and selective CysLT(2) receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT(2) receptor.
Background and purpose: Rho‐kinase (ROCK) has been implicated in the pathophysiology of altered vasoregulation leading to hypertension. Here we describe the pharmacological characterization of a potent, highly selective and orally active ROCK inhibitor, the derivative of a class of azaindoles, azaindole 1 (6‐chloro‐ N 4 ‐{3,5‐difluoro‐4‐[(3‐methyl‐1 H ‐pyrrolo[2,3‐ b ]pyridin‐4‐yl)oxy]‐phenyl}pyrimidine‐2,4‐diamine). Experimental approach: Pharmacological characterization of azaindole 1 was performed with human recombinant ROCK in vitro . Vasodilator activity was determined using isolated vessels in vitro and different animal models in vivo . Key results: This compound inhibited the ROCK‐1 and ROCK‐2 isoenzymes with IC 50 s of 0.6 and 1.1 nM in an ATP‐competitive manner. Although ATP‐competitive, azaindole 1 was inactive against 89 kinases (IC 50 >10 μM) and showed only weak activity against an additional 21 different kinases (IC 50 =1 ‐ 10 μM). Only the kinases TRK und FLT3 were inhibited by azaindole 1 in the sub‐micromolar range, albeit with IC 50 values of 252 and 303 nM, respectively. In vivo , azaindole 1 lowered blood pressure dose‐dependently after i.v. administration in anaesthetized normotensive rats. In conscious normotensive and spontaneously hypertensive rats azaindole 1 induced a dose‐dependent decrease in blood pressure after oral administration without inducing a significant reflex increase in heart rate. In anaesthetized dogs, azaindole 1 induced vasodilatation with a moderately elevated heart rate. Conclusions and implications: Azaindole 1 is representative of a new class of selective and potent ROCK inhibitors and is a valuable tool for the elucidation of the role of ROCK in the cardiovascular system. British Journal of Pharmacology (2007) 152 , 1070–1080; doi: 10.1038/sj.bjp.0707484 ; published online 15 October 2007
In the mouse keratinocyte line HEL-30 the epidermal mitogen transforming growth factor-a (TGF-a) stimulated the rapid release of arachidonic acid in a doseand time-dependent manner.The liberation of arachidonic acid was due to the activation of a Caz+-dependent cytosolic phospholipase Az (cPLAz).The activation mechanism critically depended on a functionally active epidermal growth factor receptor tyrosine kinase and occurred independently of phospholipase C-mediated increases in cellular diacylglycerol and inositol 1,4,5trisphosphate concentrations and protein kinase C activation.The activation included an increase in cytosolic PLAz (cPLAz) activity and an association of the enzyme with the membrane fraction.Both activation steps apparently occurred in the presence of basal cytoplasmic Caz+ concentrations.Moreover, cPLAz or a closely associated protein was found to be phosphorylated on tyrosine upon TGF-a challenge of the cells.The data suggest that tyrosine phosphorylation is involved in the TGF-a-induced activation of cPLAz.Phospholipases A2 (PLA2)' are a family of enzymes catalyzing the hydrolysis of the sn-2-acyl ester bond in membrane phospholipids.The mammalian enzymes are subdivided into two major families: the low molecular mass (10-14 kDa) enzymes which are either cell-associated or extracellular, and the structurally unrelated high molecular mass (85 kDa) enzymes localized in the cytosol (cPLA2).The former comprise type I or pancreatic PLA2s originally found in pancreatic secretions but are also present in other mammalian tissues, and type I1 or nonpancreatic PLA2s found at high levels in synovial fluids from patients with inflammatory diseases, but also in macrophages and thrombocytes (for reviews, see Refs. 1 and 2).cPLA2s have been purified to homogeneity from different cell types and tissues such as human platelets (3) and monocytes (4, 5), rat kidney (6), and mouse spleen (7).cDNAs of the corresponding human and mouse genes have
Inhibition of phosphodiesterase 5 (PDE5) decreases pulmonary pressure and improves symptoms in patients with pulmonary arterial hypertension. It is unclear however, whether inhibition of PDE5 can prevent myocardial remodelling during right-ventricular pressure overload.Right-ventricular pressure overload was produced in male rats in a pulmonary hypertension model (monocrotaline 60 mg/kg s.c.) or by surgical pulmonary artery banding. PDE5 inhibition using oral sildenafil (50 mg/kg/day in drinking water) or placebo was initiated 14 days after monocrotaline treatment and continued for 14 days until final examination. In the pulmonary artery banding groups, rats were treated with sildenafil (50 mg/kg/day) or placebo for 21 days following surgical pulmonary artery banding. At the final experiments, right-ventricular haemodynamics were measured and remodelling was analysed using histological, biochemical, and gene expression markers. Both monocrotaline and pulmonary artery banding increased right-ventricular systolic pressure to approximately 80 mmHg. In parallel, both interventions induced markers of hypertrophy (upregulation of natriuretic peptides, increase in myocyte diameter) and fibrosis (upregulation of collagen types 1A2 and 3A1) as well as mRNA expression of the tissue inhibitor of matrix metalloproteases 1 and osteopontin in the right ventricle. In monocrotaline model, sildenafil decreased pulmonary pressure, reduced right-ventricular hypertrophy, and prevented fibrosis marker gene upregulation. After pulmonary artery banding, in contrast, sildenafil increased markers of myocardial remodelling and right-ventricular myocyte diameter.Sildenafil prevents myocardial remodelling in pulmonary hypertension through an indirect action via right-ventricular unloading.
In response to epidermal growth factor (EGF), HeLa cells and A431 cells rapidly accumulate substantial amounts of phosphatidic acid (up to 0.16 and 0.2 micrograms/10(6) cells respectively), which represents approx. 0.17% of total phospholipid. Phosphatidic acid may be a potential product of diacylglycerol kinase and/or of phospholipase D. To evaluate the contribution of phospholipase D, the phosphatidyl-transfer reaction to a primary alcohol (mostly butan-1-ol; 0.2%) was measured; this reaction is known to be mediated exclusively by phospholipase D in intact cells. In HeLa and in A431 cells prelabelled with [1-14C]oleic acid, EGF (10 and 100 nM respectively) caused a 3-fold increase in radioactive phosphatidylbutanol within 5 min at the expense of labelled phosphatidic acid. Dose-response relationships showed 10 nM- and 100 nM-EGF to be maximally effective in HeLa cells and A431 cells respectively. Mass determinations showed that the phosphatidylbutanol formed within 5 min represented only part of the phosphatidic acid. Depletion of protein kinase C by pretreatment of A431 cells for 17 h with the phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) did not impair EGF-induced formation of phosphatidylbutanol, thus indicating that the reaction was independent of this enzyme. Since phosphatidic acid is suggested to exert second-messenger functions as well as to induce biophysical changes in cellular membranes, its formation, including that via the phospholipase D pathway, may represent an important link between extracellular signals and intracellular targets.
