The kallikrein/kinin system in ocular function.

The kallikrein/kinin system uses two distinct serine proteases, plasma kallikrein and tissue kallikrein, to yield bradykinin and Lys-bradykinin (kallidin) from specific substrate kininogens. The kallikrein/kinin system is known to have a role in contact-activated coagulation mechanisms and in inflammatory responses, and recently has been shown to contribute to homeostatic and protective mechanisms in the cardiovascular and renal systems. This article reviews current knowledge of the ocular kallikrein/kinin system within the context of proposed roles for this system in other important organs and tissues. All components of the kallikrein/kinin system are present in the eye and are positioned to participate in key ocular functions. Plasma kallikrein binds to vascular endothelium and generates bradykinin, which may contribute to regulation of ocular blood flow, and, in excess, has been implicated in the pathogenesis of retinal edema in patients with proliferative diabetic retinopathy. Tissue kallikrein is expressed in retina, ciliary muscle, and trabecular meshwork cells and could be a significant factor in the protective mechanism of ischemic preconditioning, and in the modulation of aqueous dynamics. Improved understanding of the role of plasma and tissue kallikreins and kinins in such processes has the potential to identify significant new targets for the therapy of ocular dysfunction.

Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells.

Bradykinin stimulation of B(2) kinin receptors has been shown to promote matrix metallo-proteinase (MMP) secretion from trabecular meshwork cells and to increase conventional outflow facility. Because acute secretion of MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK1/2), experiments were performed to determine bradykinin effects on ERK1/2 in cultured human trabecular meshwork cells and the relationship of these effects to MMP-9 release. Treatment of cells with bradykinin produced a rapid 4-to 6-fold increase in ERK1/2 phosphorylation. Stimulation of ERK1/2 activity peaked within 2 min and then declined to control levels by 60 min. The response maximum occurred with 100nM bradykinin and the estimated EC50 was 0.7nM. Treatment of cells with the B2 kinin receptor agonist, Tyr8- bradykinin, also stimulated ERK1/2 phosphorylation while the B1 agonist, Lys- [Des-Arg9]- bradykinin had no significant effect. In addition, activation of ERK1/2 by bradykinin or Tyr8- bradykinin was blocked by the selective B2 receptor antagonist, Hoe-140. Inhibition of MAP kinase kinase (MEK) with U0126 also blocked bradykinin-induced ERK1/2 phosphorylation. Suppression of protein kinase C activity with the nonselective inhibitor, GF109203X, or by down-regulation with phorbol ester, diminished, but did not eliminate, bradykinin activation of ERK1/2. A similar decrease of ERK1/2 stimulation was observed when Src kinase was inhibited by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Finally, blockade of bradykinin-induced ERK1/2 activation substantially reduced the peptide's action to stimulate MMP-9 release into the extracellular environment. The data demonstrate that bradykinin promotes ERK1/2 activation in human trabecular meshwork cells. The effect is mediated by B2 kinin receptors, involves two different signaling pathways, and results in increased secretion of MMP-9.

Evidence for prostacyclin and cAMP upregulation by bradykinin and insulin-like growth factor 1 in vascular smooth muscle cells.

Although bradykinin (BK) and insulin like growth factor-1 (IGF-1) have been shown to modulate the functional and structural integrity of the arterial wall, the cellular mechanisms through which this regulation occurs is still undefined. The present study examined the role of second messenger molecules generated by BK and IGF-1 that could ultimately result in proliferative or antiproliferative signals in vascular smooth muscle cells (VSMC). Activation of BK or IGF-1 receptors stimulated the synthesis and release of prostacyclin (PGI(2)) leading to increased production of cAMP in VSMC. Inhibition of p42/p44(mapk) or src kinases prevented the increase in PGI(2) and cAMP observed in response to BK or IGF-1, indicating a role for these kinases in the regulation of cPLA(2) activity in the VSMC. Inhibition of PKC failed to alter production of PGI(2) in response to BK, but further increased both p42/p44(mapk) activation and the synthesis of PGI(2) produced in response to IGF-1. In addition, both BK and IGF-1 significantly induced the expression of c-fos mRNA levels in VSMC, and this effect of BK was accentuated in the presence a cPLA(2) inhibitor. Finally, inhibition of cPLA(2) activity and/or cyclooxygenase activity enhanced the expression of collagen I mRNA levels in response to BK and IGF-1 stimulation. These findings indicate that the effect of BK or IGF-1 to stimulate VSMC growth is an integrated response to the activation of multiple signaling pathways. Thus, the excessive cell growth that occurs in certain forms of vascular disease could reflect dysfunction in one or more of these pathways.

Expression of the kallikrein/kinin system in human anterior segment.

Tissue kallikrein acts on the substrate, low molecular weight kininogen, to liberate bradykinin in a variety of tissues. Bradykinin stimulation of B(2) kinin receptors has been shown to initiate signaling in trabecular meshwork cells and increase conventional outflow facility. The objective of the present study was to determine if the components for kinin generation and response are expressed in tissues of the human anterior segment. Expression of mRNA encoding tissue kallikrein (KK), low molecular weight kininogen, and B(1) and B(2) kinin receptors was examined in human ciliary smooth muscle (CM), trabecular meshwork (TM) and non-pigmented epithelial (NPE) cells using RT-PCR. Expression of component proteins was also investigated by immunohistochemical analyses performed on parasagittal sections of human anterior segment and TM cells, and by immunoblot. KK mRNA was detected in NPE cells and in cultured CM and TM cells from multiple donors. Each cell type also expressed mRNAs encoding both B(1) and B(2) kinin receptors. Immunohistochemical analysis of KK protein in sectioned anterior segment supported the RT-PCR results. Intense KK immunofluorescence was observed in the epithelial lining of the ciliary body and KK protein was also detected in the ciliary muscle. KK protein expression within the TM was demonstrated by analyses of TM tissue and cultured TM cells. The presence of KK along with B(1) and B(2) receptor proteins was confirmed by immunoblots of cell lysates prepared from CM, NPE or TM cells. Finally, both CM and TM cells were found to possess enzymes for bradykinin inactivation. These data demonstrate that key components for kinin generation and regulation are localized within the human anterior segment. Further, multiple cell types express both B(1) and B(2) kinin receptors and are targets for kinin action. The results support the possibility that kinins produced within the eye may contribute to the regulation of aqueous outflow.

Kinin modulation of conventional outflow facility in the bovine eye.

The aim of this study was to investigate the effects of bradykinin on conventional outflow facility in relation to kinin effects on matrix metalloproteinase (MMP) secretion. Conventional outflow facility was measured in isolated bovine segments perfused at a constant pressure of 10 mmHg. Experiments were also performed in primary cultures of bovine trabecular meshwork cells to assess bradykinin effects on the secretion of MMP-9 assessed by western blot. Administration of bradykinin (10(-7) M) to perfused anterior segments produced a 50% increase in outflow facility above basal levels. The effect was slow to develop, requiring 100 min for a significant increase in facility and 4 h for the peak response to be observed. Pretreatment of anterior segments with the B(2) kinin receptor antagonist, HOE-140 (10(-6) M), or with the nonselective MMP inhibitor, GM6001 (10(-5) M) blocked the response to bradykinin (10(-7) M). Treatment of cultured trabecular meshwork cells with bradykinin (10(-7) M) for 120 min stimulated secretion of MMP-9 into the extracellular media, and this response was inhibited by HOE-140 (10(-6) M). These results demonstrate that bradykinin activates B(2) kinin receptors to increase conventional outflow in the perfused bovine eye and provide evidence that secretion and activation of MMPs within the conventional pathway may mediate the effect.

Regulation of human vascular smooth muscle cell migration by beta-adrenergic receptors.

Migration and proliferation of vascular smooth muscle cells (VSMCs) are two events involved in atherosclerosis, restenosis after balloon angioplasty, and stenosis of grafted vessels. Platelet-derived growth factor (PDGF) found in stenotic vessels is known to induce migration of VSMCs. VSMCs express both alpha- and beta-adrenergic receptors on their surface, and blood vessels are innervated by the adrenergic nervous system and exposed to circulating epinephrine. We examined the role of these receptors on PDGF-induced migration of VSMCs. VSMCs were cultured from saphenous vein segments. Migration was stimulated by PDGF. Effect of pretreatment of VSMCs with the beta-agonist isoproterenol, the alpha-agonist phenylephrine, or forskolin on PDGF-induced migration was examined with a modified Boyden chamber. Cell migration was quantitated by spectrophotometry. Intracellular cyclic AMP was determined by radioimmunoassay. PDGF significantly induced VSMC migration. Isoproterenol (0.1 and 1.0 microM) inhibited PDGF-induced migration by 30 per cent and 50 per cent, respectively. Forskolin (10 microM) completely blocked PDGF-induced migration. The migration inhibition by isoproterenol or forskolin was associated with a significant elevation of intracellular cyclic AMP. In contrast, phenylephrine had no effect on PDGF-induced migration or on cyclic AMP. Activation of beta-adrenergic receptors and the consequent rise in intracellular cyclic AMP inhibits migration of VSMCs induced by PDGF. These results are consistent with the notion that adrenergic agonists with substantial beta-receptor affinity, such as isoproterenol, can inhibit smooth muscle cell migration.

Mechanisms of vascular angiotensin II surface receptor regulation by epidermal growth factor.

We investigated mechanisms by which epidermal growth factor (EGF) reduces angiotensin II (AngII) surface receptor density and stimulated actions in vascular smooth muscle cells (VSMC). EGF downregulated specific AngII radioligand binding in intact cultured rat aortic smooth muscle cells but not in cell membranes and also inhibited AngII-stimulated contractions of aortic segments. Inhibitors of cAMP-dependent kinases, PI-3 kinase, MAP kinase, cyclooxygenase, and calmodulin did not prevent EGF-mediated downregulation of AngII receptor binding, whereas the EGF receptor kinase inhibitor AG1478 did. Total cell AngII AT1a receptor protein content of EGF-treated and untreated cells, measured by immunoblotting, did not differ. Actinomycin D or cytochalasin D, which interacts with the cytoskeleton, but not the protein synthesis inhibitor cycloheximide, prevented EGF from downregulating AngII receptor binding. Consistently, EGF inhibited AngII-stimulated formation of inositol phosphates in the presence of cycloheximide but not in the presence of actinomycin D or cytochalasin D. In conclusion, EGF needs an intact signal transduction pathway to downregulate AngII surface receptor binding, possibly by altering cellular location of the receptors.

Bacterial lipopolysaccharide-stimulated release of tumor necrosis factor-alpha from the isolated rat heart: the effect of aprotinin and forskolin.

Aprotinin has been reported to reduce plasma levels of inflammatory cytokines associated with cardiopulmonary bypass (CPB). Because CPB is also associated with elevated levels of bacterial lipopolysaccharide (LPS) and LPS stimulates release of inflammatory cytokines from the heart we tested the hypothesis that aprotinin would inhibit cardiac release of tumor necrosis factor-alpha (TNF) provoked by LPS. Isolated rat hearts were perfused Langendorf style. After 30 minutes of equilibration LPS (100 ng/mL) was infused for 60 minutes. Timed samples of coronary effluent were collected at 0, 30, 60, 90, 120, and 150 minutes after the initiation of LPS for the measurement of coronary flow and the determination of TNF and cyclic AMP. Other hearts were perfused with buffer containing aprotinin [137 kallikrein-inhibiting units (KIU)/mL or 250 KIU/mL] and then infused with LPS. An additional group received forskolin (10 microM) and LPS. In hearts perfused as controls with buffer alone no TNF was detected in the coronary effluent. In hearts perfused with LPS TNF was reliably detected in the coronary effluent at 60 minutes (606 +/- 450 pg/min) and increased with time to a level of 1792 +/- 650 pg/min at 150 minutes. The addition of aprotinin had no significant effect on LPS-stimulated TNF release. For instance in hearts perfused with 137 KIU/mL aprotinin LPS-stimulated release at 150 minutes was 2141 +/- 732 pg/min and in hearts perfused with 250 KIU/mL LPS-stimulated TNF release was 2049 +/- 789 pg/min. Forskolin administration was associated with release of cyclic AMP from the heart and completely inhibited LPS-stimulated TNF release. We conclude that LPS stimulated release of TNF from the heart. Adding aprotinin to the perfusion buffer in either high or low concentrations did not attenuate LPS-stimulated cytokine release. Elevating myocardial cyclic AMP with forskolin completely attenuated LPS-stimulated TNF release.

Bradykinin enhancement of PGE2 signalling in bovine trabecular meshwork cells.

Kinins and prostaglandins activate signalling pathways in cells of the trabecular meshwork and have opposing effects on outflow resistance to aqueous humor. Consequently, interactions between these pathways may be important in the regulation of intraocular pressure. In the present study, the influence of bradykinin on PGE(2) signalling was examined in primary cultures of bovine trabecular meshwork cells. Incubation of cells with bradykinin produced a concentration-dependent (EC(50)=3.6+/-0.7 nM) elevation of intracellular free Ca(2+). At a maximal concentration of 100 nM, the increase in Ca(2+) was rapid, peaking in 30 sec, and then slowly returned to baseline. This effect was completely inhibited in cells pretreated with the selective B(2) kinin receptor antagonist, Hoe-140. Treatment of trabecular meshwork cells with PGE(2), in comparison, had no effect on cellular Ca(2+) but produced a concentration-dependent increase in adenosine 3', 5'-cyclic monophosphate (cAMP) formation. Bradykinin had no effect on basal cAMP. However, incubation of cells with PGE(2) in combination with bradykinin resulted in a 3- to 5-fold enhancement of PGE(2)-stimulated cAMP production. Bradykinin enhancement of cAMP stimulation was concentration-dependent with an EC(50) of 3.6+/-1.8 nM. Treatment of cells with bradykinin increased the response maximum for PGE(2) signalling, while the EC(50) for PGE(2) was not changed. This action of bradykinin was again blocked in cells pretreated with Hoe-140. Bradykinin also produced a 2- to 3-fold increase in isoproterenol and cholera toxin-stimulated cAMP accumulation. However, when adenylyl cyclase was stimulated directly with forskolin, bradykinin failed to alter cAMP production. These results indicate that bradykinin activates B(2) kinin receptors in trabecular meshwork cells to amplify PGE(2)-stimulated cAMP formation by facilitating the interaction between activated G(s) and the catalytic unit of adenylyl cyclase.

Cyclic AMP inhibits production of interleukin-6 and migration in human vascular smooth muscle cells.

BACKGROUND: Gene expression induced by tumor necrosis factor-alpha (TNF-alpha) is involved in the regulation of vascular smooth muscle cell (VSMC) proliferation and migration, two events critical to formation of stenotic vascular lesions. In some systems, elevating adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits TNF-alpha induced gene transcription. We recently demonstrated that interleukin-6 (IL-6) was chemotactic to VSMC. Therefore, we tested the hypothesis that elevating cyclic AMP would inhibit TNF-alpha-mediated IL-6 expression and VSMC migration. MATERIALS AND METHODS: VSMC were cultured from saphenous vein remaining after coronary artery bypass grafting. Migration of VSMC through a porous membrane was determined. Intracellular cyclic AMP was elevated by exposing the cells to forskolin or 8-Br-cyclic AMP and was measured by radioimmunoassay. IL-6 was measured by enzyme-linked immunosorbent assay. RESULTS: TNF-alpha induced migration of VSMC in a concentration-dependent manner. Incubation of cells with forskolin significantly increased cyclic AMP. Co-incubation of cells with TNF-alpha in combination with 8-Br-cyclic AMP or forskolin inhibited migration by approximately 25 and 70%, respectively. Incubation with TNF-alpha increased release of IL-6 from VSMC 18-fold over basal. This stimulated release was inhibited by either 8-Br-cyclic AMP or forskolin. In cells stimulated with TNF-alpha, addition of an antibody to IL-6 reduced migration by 25%. CONCLUSIONS: These data show that IL-6 produced by VSMC contributes to cell migration induced by TNF-alpha. Further, elevating cyclic AMP inhibited TNF-alpha-induced release of IL-6, and migration of VSMC. These results are consistent with the notion that mechanisms that increase intracellular cyclic AMP, such as activation of beta-adrenergic receptors on VSMC, act as a brake on cell migration.

Utilization of a radioimmunoassay to detect the generation of Arg-Pro-Pro-Gly-Phe, a stable endproduct of bradykinin metabolism (from cultured rat aortic smooth muscle cells exposed to bradykinin).

The peptide hormone bradykinin is a mediator in many physiological and pathological processes. The generation and, to a limited extent, metabolism occur at the sites of action. The short half-life of bradykinin (approximately 15 s) renders measurements of its concentration in bodily fluids difficult. As a result, recent methods utilizing either ELISA or HPLC/mass spectrometry, have been developed for the measurement of the stable metabolic endproduct of bradykinin, i.e., the pentapeptide Arg-Pro-Pro-Gly-Phe (RPPGF; BK[1-5]). Both have been successfully used to quantitate levels of RPPGF in plasma, pulmonary and nasal exudates. In this study, we demonstrate the characterization and utility of a radioimmunoassay for the measurement of RPPGF, using a newly synthesized radioiodinated analogue of RPPGF, i.e., RPPG(125I)F. This radioimmunoassay shows an IC50 of 80.5 +/- 7.4 pg/tube (n = 23) with a linear range of detection between 10 and 500 pg/tube. The assay was used to demonstrate the time-dependent generation of RPPGF by cultured rat aortic vascular smooth muscle cells exposed to bradykinin (100 ng/ml). Peak generation of RPPGF was 74.5 +/- 9.7 pmol/well (n = 5) after 24 h of incubation. Captopril, an inhibitor of angiotensin-converting enzyme (kininase II), inhibited generation in a concentration-dependent manner. The results characterize a new radioimmunoassay for the stable metabolic endproduct of bradykinin, RPPGF.

Isoproterenol inhibits transcription of cardiac cytokine genes induced by reactive oxygen intermediates.

BACKGROUND: Cytokines such as tumor necrosis factor alpha (TNF-alpha) are produced by the myocardium in heart disease and might be stimulated by reactive oxygen. In some cell types, cyclic adenosine monophosphate (AMP) inhibits TNF-alpha production. The authors tested the hypothesis that stimulation of cardiac beta-adrenergic receptors would inhibit cytokine gene transcription induced by reactive oxygen. METHODS: Rat hearts were perfused with buffer containing hypoxanthine. Reactive oxygen intermediates were generated by infusion of xanthine oxidase. Myocardial mRNA encoding 11 cytokines was determined. TNF-alpha, interleukin-6, and cyclic AMP were measured in the coronary effluent. RESULTS: In control hearts, of the screened RNA, only mRNA encoding interleukin-1beta, -4, and -6 was detected. Stimulation with hypoxanthine-xanthine oxidase (HX-XO) induced detectable mRNA for TNF-alpha and interleukin-5 and increased mRNA band density for interleukin-1beta, -4, and -6. Simultaneous infusion of isoproterenol inhibited HX-XO-stimulated cytokine gene expression and caused release of cyclic AMP into the coronary effluent. In control hearts, TNF-alpha was not detected in the coronary effluent. After HX-XO administration, TNF-alpha was reliably detected at 60 min and interleukin-6 at 90 min. Simultaneous infusion of isoproterenol inhibited TNF-alpha and interleukin-6 release. Inclusion of propranolol in the perfusion buffer blocked the isoproterenol-induced inhibition of HX-XO-stimulated TNF-alpha release and release of cyclic AMP into the coronary effluent. In addition, elevating myocardial cyclic AMP with forskolin also blocked release of TNF-alpha stimulated by HX-XO. Finally, delaying infusion of isoproterenol until 30 min after HX-XO administration still suppressed release of TNF-alpha. CONCLUSIONS: Reactive oxygen species activate cytokine gene transcription in the myocardium. The sympathetic nervous system, acting through beta-receptors to elevate myocardial cyclic AMP, regulates cardiac cytokine production by inhibition of transcription.