Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1.

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.

Immunocytochemical localization of plasminogen activator on porcine kidney cell strain: LLC-PK1 (LP100).

An antibody to plasminogen activator (PA) produced by the cultured cells of the pig kidney cell strain LLC-PK1 (LP100) was used to localize PA on the cell's free (unattached) surface. Localization was accomplished by the unlabeled antibody enzyme method (PAP) at the light microscopic level and at the electron microsopic level. Localization was commonly more intense at cell to cell junctions and was associated with blebs and vesiculation in this area. We are proposing that membrane shedding by blebs and vesiculation may be the mechanism of PA release in the LLC-PK1 (LP100) cell strain.

Porcine pancreatic phospholipase A2-induced contractions of guinea pig lung pleural strips.

The contractile nature of porcine pancreatic phospholipase A2 (PLA2) was characterized on paired pleural strips obtained from guinea pig lung. PLA2 (0.003-10 U/ml) produced concentration-related contractile responses which were sensitive to various drugs. The major component of the PLA2-induced contractions was derived from products of the cyclooxygenase pathway since a cyclooxygenase inhibitor or the combination of a thromboxane synthetase inhibitor and a thromboxane receptor antagonist produced a 54-65% reduction of the contractile responses. 5-Lipoxygenase products contributed to a smaller component of the PLA2-induced responses since 5-lipoxygenase inhibitors or the combination of a leukotriene (LT) B4 receptor antagonist and an LTD4/LTE4 receptor antagonist only suppressed the maximal responses 22-32%. PLA2-induced contractile responses were nearly abolished by altering both sides of the arachidonic acid cascade simultaneously. In contrast, a PAF receptor antagonist, a histamine (H1) receptor antagonist and an acetylcholine receptor antagonist, failed to significantly reduce PLA2-induced responses. These results demonstrate that exogenous administration of porcine pancreatic PLA2 produced concentration-dependent contractions of pleural strips mediated through the generation of eicosanoids.

Transgenic model for the discovery of novel human secretory non-pancreatic phospholipase A2 inhibitors.

Transgenic mice were created which overexpress human secretory non-pancreatic phospholipase A2 (sPLA2) pansomatically as a potential disease and drug-testing model. The mice were produced using a DNA construct in which the inducible mouse metallothionein gene promoter drives expression of a human sPLA2 minigene. High levels of sPLA2 were detected in several tissues by immunofluorescence localization. Expression in the testes caused hypospermia and male infertility. Circulating catalytically active sPLA2 could be induced to levels observed in patients undergoing a systemic inflammatory response but had no detectable effect on the mice. Therefore, these results suggest that sPLA2 hyperphospholipasemia alone may have only limited pathophysiological consequences. We further show that 3-[3-acetamide-1-benzyl-2-ethylindolyl-5-oxy]propane phosphonic acid LY311727), a potent new inhibitor of phospholipase A2 catalysis developed by our group, dramatically suppresses the circulating enzyme activity in these animals whereas 3-[3-acetamide-1-benzyl-2-propylindolyl-5-oxy]propane phosphonic acid (LY314024), a substantially less potent LY311727 analog, is without effect. These later results thus motivate the further development of this compound as a potential new therapeutic agent and valuable research tool.

Effect of carnitine analogs on carnitine acetyltransferase.

Carnitine analogs with various substituents on the nitrogen were tested for their effect on carnitine acetyltransferase from rat sperm and pigeon breast. A radiometric assay was used to measure the formation of acetylcarnitine in the presence of other enzymes that competed for acetyl coenzyme A in the sperm preparation. The apparent enzyme inhibition caused by the analogs was explained by the analogs serving as alternative substrates with higher Km and lower Vmax values. The analogs had no effect on whole sperm.

Evidence that phospholipase A2 (PLA2) inhibitors can selectively block PLA2-mediated contractions of guinea pig lung pleural strips.

We have demonstrated previously that porcine pancreatic phospholipase A2 (PLA2)-induced contractions of guinea pig lung pleural strips can be abated by inhibitors of cyclooxygenase and 5-lipoxygenase pathways, suggesting the liberation of arachidonic acid. To validate further the involvement of a PLA2-related mechanism, the effects of three known inhibitors of PLA2 were evaluated. Manoalogue, an irreversible inhibitor of PLA2, parabromophenacyl bromide, an irreversible, active site directed inhibitor and a transition-state analog, a competitive inhibitor of PLA2 were used. Transition-state analog (3-30 microM), added to the tissues for 30 min before PLA2, shifted the PLA2 curves to the right in a concentration-related manner. In contrast, the reported inactive enantiomer of transition-state analog failed to alter the PLA2 curves. Manoalogue and para-bromophenacyl bromide, at concentrations up to 40- and 50-fold higher than the enzyme concentration, respectively, were incubated in Krebs' buffer with the enzyme for 24 hr before challenging the tissues. Under these conditions, the PLA2-induced contractions were suppressed markedly. In contrast, neither reduced nor methylated manoalogue, incubated at a 20-fold molar excess with PLA2 for 24 hr, suppressed the maximal PLA2-induced responses. These results demonstrate that inhibitors of secretory PLA2, acting by different mechanisms, can alter the contractile responses induced by PLA2 on pleural strips from guinea pig lung.

Characterization of the contractile effects of human recombinant nonpancreatic secretory phospholipase A2 (PLA2) and other PLA2s on guinea pig lung pleural strips.

Contractile activities of nPLA2, pPLA2 and hPLA2 were characterized on pleural strips of guinea pig lung. The rank order of potency for these PLA2s was nPLA2 > pPLA2 > hPLA2. The concentration-related contractions induced by nPLA2 (0.0002-0.67 micrograms/ml), pPLA2 (0.006-20 micrograms/ml) and hPLA2 (0.1-30 micrograms/ml) appear to be mediated primarily by the formation of cyclooxygenase products and to a lesser extent by 5-lipoxygenase products, as revealed by experiments using indomethacin and BW A4C. To further support a PLA2-related mechanism, the selectivity and inhibitory effects of two irreversible PLA2 inhibitors, parabromophenacyl bromide (pBPB) and manoalogue, were evaluated against the contractile responses induced by each PLA2. Various concentrations of manoalogue and pBPB were incubated with individual PLA2s for 24 hr before initiating experiments. Both agents suppressed each PLA2-induced contractile activity in a concentration-related manner. The inhibitory effects of pBPB were similar at the highest concentration, whereas manoalogue was more effective in blocking contractions induced by pPLA2 and hPLA2. Conversely, methylated manoalogue, an inactive analog, failed to reduce the PLA2-induced contractions. These results demonstrate that hPLA2 has the ability to catalytically induce the release of arachidonic acid and the formation of proinflammatory eicosanoids that contract the pleural strips. This tissue bath preparation may be a useful model for the evaluation of novel PLA2 inhibitors as potentially useful therapeutic agents.

Characterization and specificity of a high titre polyclonal goat anti-murine IL-1 antibody.

The immunization of goats with an HPLC-purified IL-1 preparation derived from the supernatants of LPS-induced P388D1 cultures has resulted in the derivation of a high titre antiserum specific for murine IL-1. This antiserum exhibits a 50% inhibition of an IL-1-dependent thymocyte costimulation assay at a reciprocal dilution of 30,000 and antibody activity is still detected at 100,000-fold dilution. The goat anti-murine IL-1 serum is specific for murine IL-1 synthesized by several murine macrophage cell lines and does not neutralize human, rabbit or porcine (catabolin) IL-1. The antiserum also inhibits antigen-induced proliferation of the D10.G4 helper T-cell line. In addition to the reaction against IL-1, the antiserum also detects three additional peptides with molecular weights between 20,000 and 30,000.

Interleukin 1-induced pathophysiology: induction of cytokines, development of histopathologic changes, and immunopharmacologic intervention.

In this report, we describe a murine system in which treatment with recombinant human interleukin 1 (IL-1) induced an acute lethal state with pathologic changes similar to septic shock at high doses and development of arthritic and other tissue changes following more prolonged treatment with lower doses. We have demonstrated that both recombinant human interleukin 1 alpha and recombinant human interleukin 1 beta could be administered to an endotoxin hyporesponsive strain, C3H/HeJ, and produce these pathologic changes. Induction of tumor necrosis factor (TNF) and colony-stimulating factor activity was noted. The ability to induce these changes was dose and time dependent. Histopathologic changes included lesions in the lung, heart, liver, adrenal glands, intestines, and joints. Neutrophil infiltration was a prominent feature in many organs. Drugs, immunotherapy, or other treatments which have been effective in delaying or preventing a lethal syndrome induced following high dose interleukin 2 therapy were not effective in preventing the interleukin 1-induced lethal syndrome. Interestingly, pretreatment with low nonlethal doses of IL-1 (but not lipopolysaccharides or TNF) could prevent deaths from an LD100 challenge dose of IL-1.

Recombinant human secretory phospholipase A2 released thromboxane from guinea pig bronchoalveolar lavage cells: in vitro and ex vivo evaluation of a novel secretory phospholipase A2 inhibitor.

The primary objective of this study was to develop a functional assay that could provide rapid and reliable information on some pharmacologic characteristics of a novel inhibitor of human secretory phospholipase A2 (sPLA2). Guinea pig bronchoalveolar lavage (BAL) fluid, containing predominantly macrophages, eosinophils and epithelial cells, released thromboxane A2, as measured by thromboxane B2, in a concentration-dependent manner on exposure to recombinant human sPLA2 (rh-sPLA2). Similarly, n-formyl-L-methionyl-L-leucyl-L-phenylalanine (n-F-Met-Leu-Phe) or arachidonic acid also released this lipid mediator. Indomethacin, a cyclooxygenase inhibitor, blocked synthesis of thromboxane in response to these agents. p-Bromophenacylbromide-inactivated rh-sPLA2 was substantially less effective than the untreated enzyme in causing release of thromboxane. LY311727 is a potent indole-derived inhibitor of the isolated enzyme (IC50 = 23 nM). Incubation of this agent with BAL cells, just before addition of rh-sPLA2, reduced release of thromboxane with an IC50 = 1.8 x 10(-6) M. Specificity for sPLA2 was demonstrated in that LY311727, unlike indomethacin, did not reduce synthesis and subsequent release of thromboxane A2 in response to arachidonic acid. Using this technique as a basis, we determined whether LY311727 could sufficiently accumulate in lung after i.v. administration to inhibit rh-sPLA2-induced thromboxane A2 release from BAL cells. The compound, given i.v. to guinea pigs 5 min before collecting BAL fluid, produced a dose-dependent inhibition of rh-sPLA2 with an ED50 = 50 mg/kg. Thus, new in vitro and ex vivo assays were developed that permit functional evaluation of novel sPLA2 inhibitors. These techniques should serve as secondary assays for evaluation of human sPLA2 inhibitory activity from a chemical series and in addition provide initial data related to metabolic stability and distribution to the lung.

Structure of recombinant human rheumatoid arthritic synovial fluid phospholipase A2 at 2.2 A resolution.

Phospholipases A2 (PLA2s) may be grouped into distinct families of proteins that catalyse the hydrolysis of the 2-acyl bond of phospholipids and perform a variety of biological functions. The best characterized are the small (relative molecular mass approximately 14,000) calcium-dependent, secretory enzymes of diverse origin, such as pancreatic and venom PLA2s. The structures and functions of several PLA2s are known. Recently, high-resolution crystal structures of complexes of secretory PLA2s with phosphonate phospholipid analogues have provided information about the detailed stereochemistry of transition-state binding, confirming the proposed catalytic mechanism of esterolysis. By contrast, studies on mammalian nonpancreatic secretory PLA2s (s-PLA2s) have only recently begun; s-PLA2s are scarce in normal cells and tissues but large amounts are found in association with local and systemic inflammatory processes and tissue injury in animals and man. Such s-PLAs have been purified from rabbit and rat inflammatory exudate, from synovial fluid from patients with rheumatoid arthritis and from human platelets. Cloning and sequencing shows that the primary structure of the human s-PLA2 has about 37% homology with that of bovine pancreatic PLA2 and 44% homology with that of Crotalus atrox PLA2. The human s-PLA2 is an unusually basic protein, yet contains most of the highly conserved amino-acid residues and sequences characteristic of the PLA2s sequenced so far. Here we report the refined, three-dimensional crystal structure at 2.2 A resolution of recombinant human rheumatoid arthritic synovial fluid PLA2. This may aid the development of potent and specific inhibitors of this enzyme using structure-based design.

Thrombolytic activity of a novel plasminogen activator, LY210825, compared with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis.

LY210825, a recombinant tissue-type plasminogen activator (rt-PA), which contains the kringle-2 and serine protease functional domains of native tissue-type plasminogen activator, was previously produced by site-directed mutagenesis in a Syrian hamster cell line. We studied the thrombolytic potential of this molecule in a canine thrombosis model. Male hounds (16-22 kg) were anesthetized; a 2.0-cm segment of the left circumflex coronary artery (LCX) was isolated proximal to the first main branch, and the dogs were instrumented with an electromagnetic flow probe to measure coronary blood flow. An occlusive thrombus was formed after injury of the intimal surface of the LCX with an electrical current applied by a needle-tipped anode placed distal to the electromagnetic flow probe. After 1 hour of occlusion, either LY210825 or rt-PA was administered intravenously according to the following protocols: 1) a 1-hour infusion of either 0.25 mg/kg LY210825 or 0.4 mg/kg rt-PA, 2) single injections of 0.15-0.6 mg/kg LY210825, and 3) a single injection of 0.45 mg/kg LY210825 and a 3-hour infusion of 1.0 or 1.7 mg/kg rt-PA. Plasma half-lives of LY210825 and rt-PA were 58 +/- 7 and 3.3 +/- 0.3 minutes, respectively. LY210825 produced more rapid reperfusion of the LCX than did rt-PA. In the third study, 90% of the rt-PA-treated vessels reoccluded within 1 hour after cessation of drug, whereas only 25% of the LY210825-treated vessels reoccluded during a 4-hour washout period. There were significant, but relatively small, reductions produced by both plasminogen activators on plasma fibrinogen and plasminogen (25-35% decreases). Because of its longer plasma half-life, LY210825 could be administered intravenously as a single injection. In a canine model of coronary artery thrombosis, LY210825 was a more effective thrombolytic agent than was rt-PA.