Ronald M. Burch

Recent developments in the understanding of bradykinin receptors

The dramatic activities of bradykinin and related peptides as mediators of pain, inflammation and hypotension have been intensely studied for several decades. More recently, the involvement of bradykinin in regulation of ion transport by epithelia, hormone release from endocrine organs, energy metabolism, tissue growth, and leukocyte activation have become topics of study. Kininogen precursors, synthetic kallikreins, and degradative kininases have been characterized in detail with regard to catalytic mechanisms, physical structure and gene regulation; however, the actual receptors for bradykinin are still only poorly understood. This situation is caused by the lack of availability of potent, specific receptor antagonists. However, specific bradykinin receptor antagonists became available in 1985, and several very potent classes of agents are now available; also, the first bradykinin receptor has been cloned.

Induction of vascular smooth muscle bradykinin B1 receptors in vivo during antigen arthritis

Antigen arthritis in rabbits was associated with induction of bradykinin B1 receptors in isolated aorta smooth muscle 24 h following intra-articular injection of antigen in sensitized animals. Control tissues developed responsiveness to desArg9-bradykinin or bradykinin during 3 h incubation, but failed to respond to either kinin at the beginning of experiments. Aorta from rabbits 24 h after induction of arthritis not only developed responsiveness to kinins more rapidly than controls, but also responded at the outset of experiments. Antigen arthritis was characterized by acute phase protein synthesis and joint swelling. This is the first demonstration of induction of smooth muscle responsiveness to desArg9-bradykinin during an immune complex disease.

The kallikrein-kininogen-kinin system in chronic inflammation

We examined bradykinins effects on macrophages and fibroblasts, two cell types important in the pathogenesis of chronic inflammation. Bradykinin stimulated release of proteins of 18 kDa from macrophages. These proteins caused increased thymocyte proliferation (interleukin 1, IL-1) and completely inhibited lipoprotein lipase (tumor necrosis factor, TNF). When fibroblasts were incubated with bradykinin, PGE2 synthesis was stimulated. Pretreatment with IL-1 or TNF dramatically amplified bradykinin-stimulated PGE2 synthesis. Thus, bradykinin is involved in a positive feedback loop in which bradykinin activates macrophages to release potent inflammatory cytokines; these in turn amplify responsiveness of bradykinin target tissues.

N-[9H-(2,7-dimethylfluorenyl-9-methoxy)carbonyl]-l-leucine, NPC 15669, prevents neutrophil adherence to endothelium and inhibits CD 11b/CD 18 upregulation

NPC 15669, a member of a new class of antiinflammatory agents termed leumedins, blocks inflammation in several animal models, including contact dermatitis and Arthus reaction, by inhibiting recruitment of neutrophils and lymphocytes into inflammatory lesions. These compounds do not block lipid metabolic enzymes, nor do they antagonize receptors for various chemoattractants, including LTB4, PAF, C5a, and fMLP. This report demonstrates that in vitro, pretreatment of stimulated neutrophils with NPC 15669 results in the dose-dependent inhibition of adherence to cultured human umbilical vein endothelial cells or to the protein substrate keyhole limpet hemocyanin. Adherence of HL-60 cells (a promyelocytic line) is unaffected when stimulated endothelial cells are pretreated with NPC 15669. Flow cytometric analysis of adhesion molecules expressed on neutrophils revealed that pretreatment of neutrophils with NPC 15669 prior to activation inhibits the increase in expression of the CD11b and CD18 adhesion molecule subunits. We conclude that (1) NPC 15669 acts on neutrophils to block activation-induced adherence, and (2) NPC 15669 inhibits the upregulation of the CD11b/CD18 (Mac-1) adhesion receptor.

NPC 15669, an inhibitor of neutrophil recruitment, is efficacious in acetic acid-induced colitis in rats

BACKGROUND The efficacy of the leukocyte recruitment inhibitor, N-[9H-2,7-dimethylfluoren-9-ylmethoxy)carbonyl]-L-leucine (NPC 15669) was compared with drugs used to treat inflammatory bowel diseases in a rat model, acetic acid-induced colitis. METHODS Colonic damage assessed by visual inspection, histological quantitation of tissue injury, vascular permeability, myeloperoxidase (MPO) accumulation, and synthesis of inflammatory mediators were measured. RESULTS Intrarectal pretreatment with NPC 15669 results in a significant reduction of all measured indices of inflammation. The median effective dose (ED50) of NPC 15669 for inhibition of MPO accumulation and vascular permeability is 13.2 mg/kg and 31 mg/kg, respectively. The active moiety of sulfasalazine, 5-aminosalicylic acid (5-ASA), the antioxidant/5-lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA) and the corticosteroids dexamethasone and hydrocortisone, yielded ED50 values (MPO accumulation) of 68 mg/kg, 95 mg/kg, 0.7 mg/kg, and 13 mg/kg, respectively. When formulated suspensions of NPC 15669, 5-ASA, or dexamethasone were used, potency was increased 10-40-fold. Furthermore, NPC 15669 (10 mg/kg) administered 7 hours after acetic acid and evaluated 24 hours after acetic acid administration significantly attenuated neutrophil influx (70% inhibition of MPO accumulation), whereas 5-ASA (100 mg/kg) displayed no therapeutic effects. CONCLUSIONS NPC 15669 may be useful in the treatment of inflammatory disorders.

NPC 15669 enhances survival and reverses leukopenia in endotoxin-treated mice

We now report on the effects of NPC 15669 to reverse endotoxin-mediated leukopenia and to reduce mortality from endotoxic shock in the conscious mouse

NPC 15437 interacts with the C1 domain of protein kinase C : an analysis using mutant PKC constructs

We recently demonstrated that 2,6,diamino‐N‐([I‐(oxotridecyl)‐2‐piperidinyl]methyl)‐hexanamide (NPC 15437) is a selective inhibitor of PKC interacting at the regulatory domain of the enzyme. To further investigate the interaction of NPC 15437 with PKC we expressed a series of cDNAs encoding mutant PKC molecules in COS7 cells. NPC 15437 had no effect on the protein kinase activity of mutants lacking the N‐terminal region of the C1 domain. Further, NPC 15437 was a competitive inhibitor of the activation of PKCα by phorbol ester and attenuated the binding of phorbol ester to the enzyme in intact cells. The present study demonstrates that mutant enzyme constructs can be used to localize the site of interaction of NPC 15437 with PKC to residues 12–42, which encodes the pseudosubstrate binding domain and part of the first cysteine‐rich repeat sequence.

Thromboxane and Stable Prostaglandin Endoperoxide Analogs Stimulate Water Permeability in the Toad Urinary Bladder

The effects of thromboxane B(2) and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9alpha - 11alpha - (epoxymethano)prosta - 5Z,13E - dienoic acid (U44069) and (15Z)-hydroxy -11alpha,9alpha-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A(2) synthesis, thromboxane B(2) stimulated water flow in a dose-dependent manner. U44069 (1 muM) stimulated water flow from 3.6+/-0.8 to 12.4+/-1.2 mg/min per 10 cm(2) hemibladder surface area, while U46619 (1 muM) stimulated water flow from 2.8+/-1.0 to 21.8+/-2.0 mg/min per 10 cm(2). The prostaglandin endoperoxide/thromboxane A(2) antagonist trans- 13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B(2)- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 muM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P < 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A(2)-like compounds stimulate water flow in the toad urinary bladder.

2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): A selective inhibitor of protein kinase C

NPC 15437 inhibited protein kinase C (PKC) activity and [3H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC50 values, 19±2 μM and 23±4 μM, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (Ki=5±3 μM) and phosphatidylserine (PS) (Ki=12±4 μM). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC50=175 μg/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.

Mass receptor screening for new drugs.

Mass receptor screening is capable of identifying drug candidates in large compound libraries. Our laboratory has developed a mass screening technology by standardizing assay protocols that can be transferred from receptor to receptor. The entire operation, from disbursement of compounds to data analysis, is computerized to handle vast numbers of experimental results. The success of this method depends upon strict definitions of compound activity, with rapid elimination of compounds that do not fulfill all criteria. Finally, we approach automation with caution. While certain items, such as automatic harvesters, are essential for high-throughput screening, much time can be spent optimizing gadgets instead of gathering data.