Peter Polgar

Mechanisms Regulating the Expression, Self-Maintenance, and Signaling-Function of the Bradykinin B2 and B1 Receptors

Bradykinin (BK) is a potent short‐lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G‐protein‐coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Gαi and Gαq G‐protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg10‐kallidin or desArg9‐BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin‐1 or tumor necrosis factor‐α. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and self‐maintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self‐maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews (Regoli and Barabé, 1980 , Pharmacol Rev 32:1–46; Regoli et al., 1990 , J Cardiovasc Pharmacol 15(Suppl 6):S30–S38; Regoli et al., 1993 , Can J Physiol Pharmacol 71:556–557; Marceau, 1995 , Immunopharmacology 30:1–26; Regoli et al., 1998 , Eur J Pharmacol 348:1–10). J. Cell. Physiol. 193: 275–286, 2002.

Regulation of lysyl oxidase and cyclooxygenase expression in human lung fibroblasts: interactions among TGF‐β, IL‐1β, and prostaglandin E

Prostaglandin E2, transforming growth factor‐β and interleukin‐1β variably regulate the expression of cyclooxygenase 1, cyclooxygenase 2, and lysyl oxidase in IMR90, human embryo lung fibroblasts. Prostaglandin E2 at 100 nM upregulates cyclooxygenase 1 mRNA by approximately three‐fold while it downregulates lysyl oxidase mRNA levels. Notably, prostaglandin E2 suppresses the enhancing effect of TGF‐β on basal levels of lysyl oxidase mRNA. These changes in steady state mRNA levels reflect transcriptional level control, at least in part. Corresponding changes are seen in the protein levels of lysyl oxidase, cyclooxygenase 1 and cyclooxygenase 2 and in catalytic activities of these enzymes, including net prostaglandin E2 synthesis. Cyclooxygenase 2 mRNA(t1 2, 30 min) is considerably less stable than that of cyclooxygenase 1 (t1 2, 4h) while lysyl oxidase mRNA is unusually stable (t1 2 > 14h). Taken together with the differing kinetics with which these genes respond to perturbation by these cytokines, the present results suggest a coordinated, autocrine‐like mechanism of regulation of cyclooxygenase 1 and cyclooxygenase 2 and further point to the potential of their metabolic product, prostaglandin E2, to suppress the expression of lysyl oxidase in the inflammatory response to injury.

Role of EP2 receptors and cAMP in prostaglandin E2 regulated expression of type I collagen α1, lysyl oxidase, and cyclooxygenase‐1 genes in human embryo lung fibroblasts

In a recent communication, we demonstrated that prostaglandin E2 (PGE2) lowers basal while it ablates interleukin‐1β( (IL‐1β) and transforming growth factor‐β (TGFβ) upregulated lysyl oxidase (LO) mRNA levels. Correspondingly, PGE2 increases cyclooxygenase‐1 (COX1) mRNA in diploid, human embryo lung fibroblasts (IMR90) [Roy et al., 1996]. We now report that these actions by PGE2 are routed through cAMP via the PGE2, EP2 receptor. Among the PGE2 receptor types, the IMR90 predominantly express the EP2 mRNA. These cells also express EP3 and EP4 mRNA at comparatively low levels. Northern blot analyses show that 11‐deoxy PGE1, an EP2/EP4 agonist, emulates the action of PGE2. In a similar manner to PGE2, 11‐deoxy PGE1 decreases basal and TGF‐β induced type I collagen α1 (COL) mRNA, basal and IL‐1β induced LO mRNA while it increases COX1 mRNA. Sulprostone, an EP3/EP1 agonist, has no effect on the expression of these three genes. Forskolin, an adenylate cyclase activator, acts in a very similar manner to PGE2or 11‐deoxy PGE1. It suppresses both basal and TGF‐β induced COL mRNA levels. Both PGE2 and 11‐deoxy PGE1 increase cAMP to a level comparable with forskolin. The role of the EP2 receptor in controlling collagen production is further underscored in the immortalized Rat‐1 fibroblasts, derived from Fischer rat embryos, which do not express detectable EP2 mRNA. In these cells, PGE2 has little effect on COL mRNA level, whereas forskolin increases it. Furthermore, forskolin increases cAMP level in Rat‐1 cells, whereas PGE2 does not. Overall, these results illustrate that much of the PGE2 action on the expression of COL, LO, and COX1 genes is mediated through the EP2 receptor and a subsequent increase in intracellular cAMP. J. Cell. Biochem. 71:254–263, 1998.

Stimulation of prostaglandin synthesis by ascorbic acid via hydrogen peroxide formation

Ascorbic acid causes an increase in prostaglandin (PG) synthesis in human lung fibroblasts in culture. This is accompanied by an increase in fatty acid release from cellular lipid stores. The effects of ascorbic acid on prostaglandin synthesis and fatty acid release are erased if the cultures are treated simultaneously with catalase, but not if treated with superoxide dismutase. The action of catalase points to a role of hydroperoxides in the synthesis of prostaglandins. The addition of hydrogen peroxide itself increases prostaglandin synthesis by these cells.

Prostaglandin production by type II alveolar epithelial cells

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.

Self regulation of growth by human diploid fibroblasts via prostaglandin production

The prostaglandins comprise a family of related compounds which are capable of modulating a number of cellular functions including cell division [l-3] . These fatty acid-derived molecules are either extremely unstable in aqueous solution or are quickly metabolized, in vivo, to biologically less active or inactive substances [ 1 ] . The hormone-like action of these substances, therefore, it short range, generally on cells located in the vicinity of the site of their production. In this communication we report that prostaglandin (PG) production by human diploid fibroblasts in culture serves to enhance or inhibit their growth. We show that indomethacin and aspirin, when added to these cultures, initially inhibit and later stimulate cell growth while inhibiting prostaglandin production. We further note that PGEz added to these cells, at concentrations endogenous to these cultures, inhibits cell division while PGFm stimulates cell growth. Surprisingly, the effect on cell growth of hydrocortisone resembles that of indomethacin and aspirin. We find hydrocortisone also inhibits prostaglandin production in human diploid fibroblasts.

Strategic Plan for Lung Vascular Research: An NHLBI-ORDR Workshop Report

The Division of Lung Diseases of the National Heart, Lung, and Blood Institute, with the Office of Rare Diseases Research, held a workshop to identify priority areas and strategic goals to enhance and accelerate research that will result in improved understanding of the lung vasculature, translational research needs, and ultimately the care of patients with pulmonary vascular diseases. Multidisciplinary experts with diverse experience in laboratory, translational, and clinical studies identified seven priority areas and discussed limitations in our current knowledge, technologies, and approaches. The focus for future research efforts include the following: (1) better characterizing vascular genotype-phenotype relationships and incorporating systems biology approaches when appropriate; (2) advancing our understanding of pulmonary vascular metabolic regulatory signaling in health and disease; (3) expanding our knowledge of the biologic relationships between the lung circulation and circulating elements, systemic vascular function, and right heart function and disease; (4) improving translational research for identifying disease-modifying therapies for the pulmonary hypertensive diseases; (5) establishing an appropriate and effective platform for advancing translational findings into clinical studies testing; and (6) developing the specific technologies and tools that will be enabling for these goals, such as question-guided imaging techniques and lung vascular investigator training programs. Recommendations from this workshop will be used within the Lung Vascular Biology and Disease Extramural Research Program for planning and strategic implementation purposes.

Glycolysis as an energy source for stimulation of lymphocytes by phytohemagglutinin.

Abstract Various metabolic activities have been measured on cultures of lymphocytes from human blood which have been carefully freed of other types of cells. When these cultures were stimulated with phytohemagglutinin (PHA), a large increase in lactate production occurred which clearly correlated with the synthesis of DNA, RNA, and protein. In contrast, there was no demonstrable change in respiration. Deoxyglucose, which inhibits glycolysis, inhibited all synthetic activity back to the level of cultures not activated with PHA, and arrested the histological development of the cells. The effect of DOG was reversible; if the inhibitor was removed the culture could be activated with a new addition of PHA. Conversely, 2,4-dinitrophenol, which inhibits oxidative phosphorylation, had no effect on DNA synthesis, and only a partial inhibitory effect on RNA and protein synthesis. The lymphocytes continued to develop into blast forms. The results are interpreted as showing that the increased cellular activities following PHA activation are dependent on glycolysis rather than on respiration. This resembles in many respects what occurs in other rapidly dividing cells. The system may therefore be useful for studying metabolic controls associated with differentiation.

Effect of des arginine9-bradykinin and other bradykinin fragments on the synthesis of prostacyclin and the binding of bradykinin by vascular cells in culture

Bradykinin (BK) fragments, des arg1-BK, des arg1,pro2-BK, des phe8,arg9-BK and des pro7,phe8,arg9-BK were synthesized and along with des arginine9-BK (daBK), tested for their ability to induce prostacyclin synthesis in homogeneous cultures of cells from the calf pulmonary artery. Of the fragments daBK was the only peptide, in addition to bradykinin (BK), to activate the synthesis of prostacyclin (PGI2) and platelet activating factor (PAF) in endothelial cells and PGI2 in fibroblasts and smooth muscle cells. Half-maximal activation of PGI2 synthesis differed with the cell type. The other fragments tested did not directly affect PGI2 synthesis. These fragments also did not inhibit daBK or BK activation of PG synthesis.BK bound to endothelial cells with a dissociation constant (Kd) of 2.1 nM and a Bmax of 47.9 fmoles/106 cells. The Kd for the binding of BK to smooth muscle cells and fibroblasts was somewhat higher, 4.9 nM and 7.9 nM, respectively. None of the fragments tested, including daBK, altered the binding of BK. Des arg9[leu8]-BK, reported to be a competitive antagonist of the bradykinin B1 receptor, inhibited daBK induced PG of PAF synthesis in endothelial cells but had little effect of BK binding or BK induced PG synthesis. Finally, the BK antagonist [thi5,8, d-phe7]-BK blocked both BK binding and the ability of either BK or daBK to induce PG synthesis, thus substantiating that the binding of these kinins is a step in the activation of PG synthesis.

The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis

Cultured pulmonary artery endothelial cells produce PGI2 as their primary prostaglandin. Conditions which inhibit cell division have been shown to accelerate the synthesis of this compound. Exposure of endothelial cells to gamma radiation results in an irreversible cessation of growth and enhanced production of PGI2. The level of PGI2 measured after radiation exposure exceeds that observed in cultures rendered quiescent by serum reduction. This indicates a role for gamma radiation in the elevation of PGI2 levels which is distinct from its effect on cell division. Results presented indicate that exposure to gamma radiation does not, in and of itself, elevate PG levels but capacitates cells for enhanced production when presented with appropriate stimuli. Increased PGI2 synthesis appears to be a result of an observed increase in arachidonic acid release and an activation of cyclooxygenase.