Search PubMed⌕ Search

Biomedical subjects

R M Burch

Publications and source records attributed to R M Burch.

At least 37 records · Page 2Linked to original sources
PubMed1992

NPC 15669 inhibits the reversed passive Arthus reaction in rats by blocking neutrophil recruitment.

NPC 15669, N-carboxy-L-leucine,N-[(2,7-dimethylfluoren-9-yl)methyl]ester, has been shown to inhibit several inflammatory reactions that depend upon recruitment of neutrophils into the primary lesion. In the present study we examined the effects of NPC 15669 in the reversed passive Arthus reaction, an inflammatory reaction occurring in the skin of rats in response to intracutaneous injection of antigen followed by intravenous administration of antibody. In this model, immune complex formation activates complement, resulting in rapid recruitment of neutrophils to the site, which releases free radicals and proteases that damage capillaries, resulting in plasma leak. NPC 15669 inhibited the increased capillary permeability occurring in the reversed passive Arthus reaction in a dose-dependent manner, with an ED50 of 4 mg/kg. The agent similarly inhibited the recruitment of radiolabeled neutrophils as well as the accumulation of myeloperoxidase, a neutrophil marker. NPC 15669 in vitro inhibited the adherence of formyl-L-Met-L-Leu-L-Phe- or human recombinant C5a-activated neutrophils to endothelium, with IC50 values of 15 to 30 microM (ca. 4-9 micrograms/ml). Measurement of plasma NPC 15669 showed that at the ED50 dose, the average circulating concentration of drug was 5 micrograms/ml, consistent with the hypothesis that NPC 15669 exerts its anti-inflammatory effects by inhibiting neutrophil adherence to endothelium and recruitment into the inflammatory lesion.

Animals↗
PubMed1992

2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl] methyl)hexanamide (NPC 15437): a novel inhibitor of protein kinase C interacting at the regulatory domain.

NPC 15437 is a prototype member of a new class of synthetically derived protein kinase C (PKC) inhibitors. PKC activity and binding of phorbol ester to the enzyme were inhibited by NPC 15437, with IC50 values of 19 +/- 2 microM and 23 +/- 4 microM, respectively. No inhibition of cAMP-dependent or calcium/calmodulin-dependent protein kinases was observed at concentrations of NPC 15437 up to 300 microM. To investigate the mechanism by which NPC 15437 exerts its effects, a kinetic analysis of the inhibition with respect to three activators of the enzyme, phosphatidylserine, calcium, and phorbol ester, was performed. NPC 15437 was a competitive inhibitor of the activation of PKC by phorbol ester (Ki = 5 +/- 3 microM). Stimulation of PKC alpha by phosphatidylserine was competitively inhibited by NPC 15437 (Ki = 12 +/- 4 microM). The inhibition was mixed with respect to activation by calcium. These results suggest that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the enzyme. NPC 15437 inhibited PKC in intact cells, dose-dependently antagonizing the phorbol ester-induced phosphorylation of a 47-kDa protein in human platelets.

Animals↗
PubMed1991

Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope. Production, characterization, and interaction with the kinin receptor.

mAb against bradykinin, the prototypic member of the kinin family of vasodilator peptides, were generated by somatic cell fusion. The antibodies were isotyped as IgG1, kappa-type, and their target epitopes mapped with bradykinin, lysyl-bradykinin (kallidin), kinin receptor antagonists, and fragments thereof, revealing three distinct sets of mAb, i.e., mAb against bradykinin (MBK)1, MBK2, and MBK3. Comparison of the immunologic binding affinities and the known pharmacologic binding specificities of bradykinin derivatives disclosed a striking similarity in the binding profiles of mAb MBK3 and the B2 type of the kinin receptor. Anti-idiotypic antibodies against MBK1, MBK2, and MBK3 were raised in rabbit and sheep. Inhibition and competition experiments on the level of the Ag (ligand), the idiotype, and the anti-idiotype demonstrated the mutual specificity of the network system components. Anti-idiotypic antibodies against MBK3 recognized a particular idiotope that was conformation-dependent and associated with the Ag binding site of the antibody. Binding of anti-idiotypic antibodies to the B2 receptor expressed by human foreskin fibroblasts and guinea pig ileum demonstrated that the anti-idiotypes cross-react with the corresponding receptor across species. Specific stimulation of the inositol phosphate pathway in human fibroblasts and of the PG pathway in mouse fibroblasts, respectively, and inhibition of the latter effect by the B2 kinin receptor antagonist NPC 567 indicated that the anti-idiotypes bear the internal image of a bradykinin epitope. Furthermore, antibodies of the third generation (anti-anti-idiotypic antibodies) recognized the authentic Ag, i.e., bradykinin. Hence, the anti-idiotypic approach provides powerful tools to probe for the hitherto poorly characterized B2 kinin receptor.

Amino Acid Sequence↗
PubMed1991

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-[( 1-(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 PKC alpha 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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Hyperalgesia induced in the rat by the amino-terminal octapeptide of nerve growth factor.

Nerve growth factor (NGF) in the mouse submandibular gland undergoes cleavage of its amino-terminal octapeptide when salivation is induced by epinephrine. The significance of this event is uncertain; cleaved NGF demonstrates bioactivity and no function has been attributed to the octapeptide produced (NGF-OP; Ser-Ser-Thr-His-Pro-Val-Phe-His). Enzyme inhibition studies indicating structural relatedness of NGF-OP and bradykinin (BK) prompted us to determine whether NGF-OP would elicit BK-like actions. We found that like BK, NGF-OP induced a decrease in mechanical nociceptive threshold (i.e., produced hyperalgesia) in the hairy skin of the rat. This effect was dose-dependent and sequence-specific; like BK it was attenuated by sympathectomy and indomethacin pretreatment. However, NGF-OP actions appeared to be distinct from those for BK in that tissue injury was required for NGF-OP to induce hyperalgesia. Furthermore, we found no evidence that NGF-OP bound to or activated BK receptors. Our data indicate that NGF-OP is a distinct mediator of hyperalgesia. We suggest that NGF-OP alters pain threshold in the injured target regions of NGF-responsive neurons.

Animals↗
PubMed1991

Cyclic AMP mediates the direct antiproliferative action of mismatched double-stranded RNA.

Previous experiments have demonstrated that double-stranded RNAs (dsRNAs) can exert an antiproliferative effect on human tumor cells, independent of interferon (IFN) induction. However, the mechanism by which dsRNAs inhibit tumor growth has not been elucidated. As a first step in determining the molecular events responsible for growth arrest, we have explored the role of signal transduction through the cAMP system in the antiproliferative effect of the mismatched dsRNA, r(I)n.r(C12,U)n (Ampligen). These studies utilized the human glioma cell line A1235, which does not produce detectable levels of IFN-alpha, -beta, or -gamma in response to mismatched dsRNA treatment. Treatment of A1235 cells with mismatched dsRNA in combination with either 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), which inhibits cAMP-dependent protein kinase and protein kinase C, or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), which preferentially inhibits the cAMP-dependent protein kinase, yielded an antagonism of the mismatched dsRNA-induced antiproliferative effect. Measurement of adenylate cyclase activation showed a dose-dependent increase in activity at antiproliferative mismatched dsRNA concentrations, but not at lower, nonantiproliferative doses. This increase in activity was rapid, seen as early as 30 sec after initiation of treatment, and it was sustained at peak levels for 1-2 hr. Analysis of the intracellular cAMP concentration gave similar kinetics of induction. Exposure of cells to the stable cAMP analogue dibutyryl cAMP yielded dose-dependent inhibition of cell growth. The cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also inhibited proliferation. In contrast, neither H-7 nor HA1004 had an effect on growth inhibition induced by human natural IFN-alpha treatment. In addition, antiproliferative doses of IFN-alpha did not increase cAMP concentrations. These results indicate that the cAMP system is utilized by mismatched dsRNA as an early signal transduction mechanism for growth control. Furthermore, the antiproliferative effects induced by mismatched dsRNA and IFN can occur by different mechanisms of action.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

N-(fluorenyl-9-methoxycarbonyl) amino acids, a class of antiinflammatory agents with a different mechanism of action.

Several members of a series of N-(fluorenyl-9-methoxycarbonyl) amino acids were found to possess a broad spectrum of antiinflammatory activity. The compounds were active against oxazolone dermatitis in mice and adjuvant arthritis in rats, models in which activated T lymphocytes are implicated. The compounds also inhibited T-lymphocyte activation in vitro, assessed by using the mixed lymphocyte reaction. The compounds inhibited the reversed passive Arthus reaction in rats and arachidonic acid-induced dermatitis in mice, models in which leukocyte infiltration is responsible for the inflammatory reaction. More complete evaluation was made of one compound, N-(fluorenyl-9-methoxycarbonyl)leucine (NPC 15199). On histologic examination after arachidonic acid administration, NPC 15199 was found to block recruitment of neutrophils into the inflammatory site. The compound was not a general myelotoxin. Prolonged treatment of animals did not alter bone-marrow progenitor number or the numbers of circulating white blood cells. Further, several white cell functions were not inhibited in vitro, including neutrophil respiratory burst and macrophage phagocytosis. NPC 15199 was effective in blocking antigen arthritis in rabbits and was effective in a therapeutic protocol, reversing oxazolone edema. These studies suggest that N-(fluorenyl-9-methoxycarbonyl) amino acids may be valuable therapeutic agents for inflammatory diseases.

Amino Acids↗
PubMed1991

2,6-Diamino-N-([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 microM and 23 +/- 4 microM, 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 microM) and phosphatidylserine (PS) (Ki = 12 +/- 4 microM). 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 micrograms/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Sucralfate induces proliferation of dermal fibroblasts and keratinocytes in culture and granulation tissue formation in full-thickness skin wounds.

Sucralfate is used to induce healing of gastrointestinal tract ulcers. We evaluated its potential utility in the healing of skin wounds. Initial experiments examined the effects of the sucralfate on proliferation of cultured dermal fibroblasts and keratinocytes. Sucralfate induced proliferation in quiescent cultures of both cell types. Additionally, sucralfate enhanced prostaglandin E2 synthesis in basal keratinocytes and in interleukin-1-stimulated keratinocytes and dermal fibroblasts. Basal interleukin-1 and 6 release were not affected by sucralfate, but the agent enhanced interleukin-1-stimulated interleukin-6 release from fibroblasts. When applied daily to full-thickness wounds in rats, sucralfate increased the thickness of granulation tissue when assessed at day 12.

Animals↗
PubMed1991

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.

Computers↗
PubMed1991

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin, a potent antagonist of smooth muscle BK2 receptors and BK3 receptors.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin (NPC 16731) inhibited bradykinin (BK) binding and BK-induced contraction in guinea-pig ileum, being markedly more potent than D-Phe7-BK analogues as a BK2 receptor antagonist. In isolated trachea NPC 16731, unlike other BK2 antagonists, inhibited BK binding and BK-induced contraction, and 45Ca2+ efflux in tracheal smooth muscle cells. That NPC 16731 potently inhibits BK effects in trachea provides further evidence for the existence of the airway BK3 receptor.

Amino Acid Sequence↗
PubMed1991

A bradykinin antagonist modifies allergen-induced mediator release and late bronchial responses in sheep.

We assessed the role of bradykinin (BK) in allergen-induced early and late bronchial responses, airway inflammation, mediator release, and antigen-induced airway hyperresponsiveness in allergic sheep by studying the effects of the BK B2 receptor antagonist, NPC-567 (D-Arg-[Hyp3, D-Phe7]-BK), on these parameters. Antigen challenge was performed on two occasions greater than 3 wk apart, once with placebo (control) and once after high-dose (10 mg/ml) and low-dose (5 mg/ml) treatments with aerosol NPC-567. In the control trials (n = 14) antigen challenge resulted in an early and late increase in specific lung resistance (SRL). The early response was associated with increases (p less than 0.05) in prostaglandin (PG) D2, immunoreactive kinin, tosyl-L-arginine methyl ester (TAME)-esterase, and PGE2 in bronchoalveolar lavage (BAL) fluid. The late response was associated with increases (p less than 0.05) in leukotrienes (LT) B4 and C4, thromboxane (TX) B2, 6-keto-PGF10, and PGE2. There was a significant influx of neutrophils in the BAL fluid during the late response, and airway hyperresponsiveness to carbachol aerosol was apparent 4 h after challenge. In six sheep the high-dose NPC-567 treatment (given before, during, and 4 h after antigen challenge) did not attenuate the early bronchoconstrictor response or the early release of mediators but caused a significant reduction in the late response (p less than 0.05). This protective effect was accompanied by reductions (p less than 0.05) in both the concentrations of all the mediators associated with the late response and the severity of the BAL neutrophilia. High-dose NPC-567 did not attenuate the airway hyperresponsiveness or the cellular inflammatory response seen 24 h after challenge. In eight sheep treated with the low dose of NPC-567 (given before, during, and 4, 8, and 24 h after challenge) the early response was not blocked but the late response was again inhibited, as were the mediators associated with the late response. At the low dose the drug did not prevent the airway inflammation at 8 or 24 h. The additional treatments did, however, prevent the 24 h hyperresponsiveness. These data suggest that kinin generation during antigen-induced airway anaphylaxis may be important for controlling the release of arachidonic acid metabolites from airway inflammatory cells that contribute to the development of the late response in the allergic sheep model.

Airway Resistance↗