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Structure−activity relationships were investigated on a novel series of tricyclic dihydropyridine-containing KATP openers. This diverse group of analogues, comprising a variety of heterocyclic rings fused to the dihydropyridine nucleus, was designed to determine the influence on activity of hydrogen-bond-donating and -accepting groups and their stereochemical disposition. Compounds were evaluated for KATP activity in guinea pig bladder cells using a fluorescence-based membrane potential assay and in a pig bladder strip assay. The inhibition of spontaneous bladder contractions in vitro was also examined for a subset of compounds. All compounds studied showed greater potency to inhibit spontaneous bladder contractions relative to their potencies to inhibit contractions elicited by electrical stimulation.

Structure−activity relationships were investigated on a novel series of sulfonyldihydropyridine-containing KATP openers. Ring sizes, absolute stereochemistry, and aromatic substitution were evaluated for KATP activity in guinea pig bladder cells using a fluorescence-based membrane potential assay and in a pig bladder strip assay. The inhibition of spontaneous bladder contractions in vitro was also examined for a select group of compounds. All compounds studied showed greater potency to inhibit spontaneous bladder contractions relative to their potencies to inhibit contractions elicited by electrical stimulation. In an anesthetized pig model of myogenic bladder overactivity, compound 14 and (−)-cromakalim 1 were found to inhibit spontaneous bladder contractions in vivo at plasma concentrations lower than those that affected hemodynamic parameters. Compound 14 showed approximately 5-fold greater selectivity than 1 in vivo and supports the concept that bladder-selective KATP channel openers may have utility in the treatment of overactive bladder.

The preparation and characterization of a series of C-10 substituted 5-allyl-2,5-dihydro-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines as a novel class of selective ligands for the glucocorticoid receptor is described. Substitution at the C-10 position of the tetracyclic core with linear, two-atom appendages (OCH3, OCF2H, NHMe, SMe, CH&dbd;CH2, C&tbd1;CH, CH2OH) provided molecules of high affinity (Ki = 2−8 nM) for the human glucocorticoid receptor (hGR) with limited cross-reactivity with other steroid receptors (PR, MR, AR, ER). Optimal analogues showed slightly less potent but highly efficacious E-selectin repression with reduced levels of GRE activation efficacy in reporter gene assays relative to prednisolone. Preliminary SAR of analogues containing substitution at the C-9 and C-10 positions identified the 9-OH, 10-OMe analogue 50 and the 9-OH, 10-Cl analogue 58 as compounds that demonstrated potent, GR-mediated inhibition in a conconavalin A stimulated T-cell proliferation assay in both rodent and human whole blood monocytes. When evaluated for their in vivo effects in carrageenan-induced paw edema in rats, 50, 58, and 10-OCF2H analogue 35 showed dose-dependent anti-inflammatory effects (50, ED50 = 16 mg/kg; 58, ED50 = 15 mg/kg; 35, ED50 = 21 mg/kg vs ED50 = 15 mg/kg for 18 and ED50 = 4 mg/kg for prednisolone).

The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure−activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED50 = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED50 = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (Ki = 2.4−9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED50 = 2.8 mpk for 13 vs ED50 = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of excitability in urinary bladder smooth muscle cells. Patch-clamp studies revealed that the current density was about 9-fold higher in the pig bladder smooth muscle cells, compared with guinea pig, although the rank order of potencies for suppression of electrical field-stimulated contraction of bladder strips by K(ATP) channel openers (KCOs) showed a nearly 1:1 correlation between pig and guinea pig. To investigate the existence of spare K(ATP) channels, P1075-evoked current and membrane potential responses were studied in bladder smooth muscle cells. During a 10-min exposure to P1075 (10 microM), K(ATP) currents ran down by approximately 30.5%, whereas membrane hyperpolarization remained constant. P1075 evoked membrane hyperpolarization with an EC(50) value of 0.20 +/- 0.02 microM, comparable to that required for smooth muscle relaxation (EC(50) = 0.11 +/- 0.01 microM). However, these potencies are 6-fold higher than those required for current activation (EC(50) = 0.73 +/- 0.4 microM). These findings demonstrate that the reduction in membrane excitability by KCOs is associated with membrane hyperpolarization, and that a low amount of K(ATP) channel opening is sufficient to suppress bladder smooth muscle contraction.

Both the Rho family of low-molecular-weight GTP-binding proteins and protein kinases C (PKCs) mediate responses to a variety of extracellular and intracellular signals. They share many downstream targets, including remodeling of the actin cytoskeleton, activation of p70(S6) kinase and c-jun N-terminal kinase (JNK), and regulation of transcription and cell proliferation. We therefore investigated whether Rho family GTP-binding proteins bind to PKCs. We found that Cdc42 associates with atypical PKCs (aPKCs) PKCzeta and -lambda in a GTP-dependent manner. The regulatory domain of the aPKCs mediates the interaction. Expression of activated Cdc42 results in the translocation of PKClambda from the nucleus into the cytosol, and Cdc42 and PKClambda colocalize at the plasma membrane and in the cytoplasm. Expression of activated Cdc42 leads to a loss of stress fibers, as does overexpression of either the wild type or an activated form of PKClambda. Kinase-dead PKClambda and -zeta constructs acted as dominant negatives and restored stress fibers in cells expressing the activated V12 Cdc42 mutant, indicating that Cdc42-dependent loss of stress fibers requires aPKCs. Kinase-dead PKClambda and -zeta and dominant-negative N17 Cdc42 also blocked Ras-induced loss of stress fibers, suggesting that this pathway may also be important for Ras-dependent cytoskeletal changes. N17 Rac did not block Ras-induced loss of stress fibers, nor did kinase-dead PKClambda block V12 Rac-stimulated loss of stress fibers. These results indicate that Cdc42 and Rac use different pathways to regulate stress fibers.

The ETS domain transcription factor PU.1 is necessary for the development of monocytes and regulates, in particular, the expression of the monocyte-specific macrophage colony-stimulating factor (M-CSF) receptor, which is critical for monocytic cell survival, proliferation, and differentiation. The bZIP transcription factor c-Jun, which is part of the AP-1 transcription factor complex, is also important for monocytic differentiation, but the monocyte-specific M-CSF receptor promoter has no AP-1 consensus binding sites. We asked the question of whether c-Jun could promote the induction of the M-CSF receptor by collaborating with PU.1. We demonstrate that c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter as well as a minimal thymidine kinase promoter containing only PU.1 DNA binding sites. c-Jun does not directly bind to the M-CSF receptor promoter but associates via its basic domain with the ETS domain of PU.1. Consistent with our observation that AP-1 binding does not contribute to c-Jun coactivation is the observation that the activation of PU.1 by c-Jun is blocked by overexpression of c-Fos. Phosphorylation of c-Jun by c-Jun NH2-terminal kinase on Ser-63 and -73 does not alter the ability of c-Jun to enhance PU.1 transactivation. Activated Ras enhances the transcriptional activity of PU.1 by up-regulating c-Jun expression without changing the phosphorylation pattern of PU.1. The activation of PU.1 by Ras is blocked by a mutant c-Jun protein lacking the basic domain. The expression of this mutant form of c-Jun also completely blocks 12-O-tetradecanoylphorbol-13-acetate-induced M-CSF receptor promoter activity during monocytic differentiation. We propose therefore that c-Jun acts as a c-Jun NH2-terminal kinase-independent coactivator of PU.1, resulting in M-CSF receptor expression and development of the monocytic lineage.

von Willebrand factor (vWF) was purified from pooled normal plasma, radiolabeled with iodine then cleaved by porcine pancreatic elastase. Cleavage was monitored by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-agarose electrophoresis, crossed immuno- electrophoresis, ristocetin and botrocetin cofactor activities, and ristocetin and botrocetin induced binding to fixed washed platelets. Cleavage of vWF by porcine elastase was dependent on both the concentration of porcine elastase and period of incubation. Incubation of vWF with concentrations of porcine elastase greater than 20 U/mL for 30 minutes resulted in loss of the 240 Kd vWF subunit and a corresponding loss of the high molecular weight multimers and formation of fragments with molecular weights on reduced SDS-PAGE of 150, 125, 115 Kd and minor bands at 44, 28, and 24 Kd and a band at 20 Kd, which increased in intensity as either the concentration of porcine elastase or the period of incubation increased. More than 50% of the ristocetin cofactor activity was lost during a five-minute incubation of vWF with 0.56 U/mL porcine elastase when no detectable structural changes in the vWF molecule had occurred. Botrocetin cofactor activity was more resistant. Similarly botrocetin induced vWF binding to platelets was retained by a fraction produced by digestion of vWF with porcine elastase, which contained predominantly a 20 Kd fragment of vWF. This fragment retained insignificant amounts of ristocetin related functions and therefore represents a useful piece of the vWF molecule for further exploration of the site involved in botrocetin induced binding to platelets.

Results of investigations of the factor VIII (FVIII) of a patient with an unusual variant form of von Willebrand's disease (vWD) are presented. A two-peak crossed-immunoelectrophoresis (CIE) pattern was seen when fresh plasma was electrophoresed, but the CIE pattern became normal by incubating the plasma at 37 degree C for more than 72 hr. The two peaks on CIE were separated by cryoprecipitation: the slow-moving peak precipitating and the fast-moving forms of FVIII remaining in the cryosupernate. An additional protein band was seen on multimeric analysis of FVIII. The platelet-rich plasma (PRP) from this patient did not respond to ristocetin, but agglutinated normally in response to botrocetin. Multimeric and CIE analysis of the FVIII post agglutination and 125I-FVIII binding studies to normal formalin-fixed platelets indicated that this patient's FVIII interacted normally with botrocetin but failed to interact with ristocetin. These data strongly suggest that the sites on the FVIII molecule or the multimeric forms involved for ristocetin and botrocetin are different and that the ristocetin reaction is more closely aligned to the physiologic function of FVIII.