Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor

Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects.     

Proteinase-activated receptors: structural requirements for activity, receptor cross-reactivity, and receptor selectivity of receptor-activating peptides

We have used three distinct bioassay systems (rat aorta (RA) relaxation; rat gastric longitudinal muscle (LM) contraction; human embryonic kidney 293 (HEK293) cell calcium signal) to evaluate the activity and receptor selectivity of analogues of the receptor-activating peptides derived either from the thrombin receptor (TRAPs, based on the human receptor sequence, SFLLRNPNDK...) or the proteinase-activated receptor 2 (PAR2APs, based on the rat receptor sequence SLIGRL...). Our main focus was on the activation of PAR2 by PAR2APs and the cross-activation of PAR2 by the TRAPs. In the RA and LM assay systems, PAR2APs that were either N-acetylated (N-acetyl-SLIGRL-NH2) or had a reverse N-terminal sequence (LSIGRL-NH2) were inactive, either as agonists or antagonists. An alanine substitution at position 3 of the PAR2AP (SLAGRL-NH2) led to a dramatic reduction of biological activity, as did substitution of threonine for serine at position 1 (TLIGRL-NH2). However, alanine substitution at PAR2AP position 4 caused only a modest reduction in activity, resulting in a peptide (SLIARL-NH2) with a potency equivalent to that of the human PAR2AP, SLIGKV-NH2. The order of potency of the PAR2APs in the RA, LM, and HEK assay systems was SLIGRL-NH2 > SLIARL-NH2 > SLIGKV-NH2 > TLIGRL-NH2 > SLAGRL-NH2. In HEK cells, none of the PAR2APs activated the thrombin receptor (PAR1). However, in the HEK cell assay, the TRAP, SFLLR-NH2, activated or desensitized both PAR1 and PAR2 receptors, whereas the xenopus TRAP, TFRIFD-NH2, activated or desensitized selectively PAR1 but not PAR2. By constructing human-xenopus hybrid peptides, we found that the TRAPs, TFLLR-NH2, and SFLLFD-NH2 selectively activated the thrombin receptor in HEK cells without activating or desensitizing PAR2. In contrast, the TRAPs SFLLRD-NH2 and AFLLR-NH2 activated or desensitized both PAR1 and PAR2. The order of potency for the TRAPs in all bioassay systems was SFLLR-NH2 approximately equal to SFLLRD-NH2 approximately equal to TFLLR-NH2 > SFLLFD-NH2 > TFRIFD-NH2. We conclude that the N-terminal domain of the PAR2AP as well as positon 3 plays important roles for PAR2 activation. In contrast, the first and fifth amino acids in the TRAP motif, SFLLR-NH2, do not play a unique role in activating the thrombin receptor, but if appropriately modified can abrogate the ability of this peptide to cross-desensitize or activate PAR2, so as to be selective for PAR1. The PAR1- and PAR2-selective peptides that we have synthesized will be of use for the evaluation of the roles of the PAR1 and PAR2 receptor systems in vivo.     

Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist

A thrombin receptor has been described that is activated by thrombin cleavage generating a new N-terminus. The newly exposed SFLLR-containing "tethered-ligand" then activates the receptor. In these studies, we used 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn- Asp-Lys-amide (Mpapeptide) as a thrombin receptor antagonist. This compound was capable of preventing both thrombin- and SFLLR-peptide-induced platelet aggregation with little effect on collagen-induced platelet aggregation. It also prevented thrombin- and SFLLRNP-induced calcium mobilization with little effect on thromboxane receptor-activated platelet Ca2+ mobilization. Platelet membrane GTPase could be activated by peptides that activated the thrombin receptor, and the thrombin receptor antagonist also prevented receptor-stimulated GTPase activity. Platelet phospholipase A2 (PLA2) activity (measured as the release of radiolabeled arachidonic acid) and Na+/H+ exchange activation were stimulated by alpha-thrombin and by SFLLR-containing peptides. Activation of both processes with low concentrations of thrombin required thrombin's anion-binding exosite, as they were not activated by similar concentrations of gamma-thrombin, and the alpha- and zeta-thrombin activation was blocked by peptides mimicking the C-terminal region of hirudin. Stimulation of PLA2 and Na+/H+ exchange by both thrombin and SFLLR-containing peptides was inhibited by the thrombin receptor antagonist Mpa-peptide. These results support the hypothesis that thrombin stimulation of PLA2 activity and Na+/H+ exchange occurs via activation of the thrombin tethered-ligand receptor. Moreover, these data are consistent with the tethered-ligand receptor mediating most actions elicited by low concentrations of alpha-thrombin involved in human platelet activation.     

Cyclooxygenase inhibition reveals synergistic action of vasoconstrictors on mesangial cell growth

Since endogenous vasoconstrictors promote mesangial cell growth and increase the biosynthesis of antiproliferative prostaglandins, the effects of cyclooxygenase inhibition on mesangial cell proliferation should be strongly dependent on the prevailing levels of neuroendocrine vasoconstrictors. We compared the effects of indomethacin (10(-6) M), a cyclooxygenase inhibitor, on [3H]thymidine incorporation by cultured rat mesangial cells in the presence of various combinations of angiotensin II (10(-10) M), [Arg8]vasopressin (10(-11) M), (-)-norepinephrine (10(-8) M) and endothelin-1 (10(-11) M). Indomethacin did not enhance [3H]thymidine incorporation in cells treated with each individual vasoconstrictor, or in cells treated with two-way combinations with the exception of modestly increased [3H]thymidine incorporation in cells treated with angiotensin II + (-)-norepinephrine or [Arg8]vasopressin + (-)-norepinephrine. In contrast, in cells treated with any three-way or the four-way combination, indomethacin markedly increased [3H]thymidine incorporation. Importantly, a highly significant interaction (P<0.0001) was observed for thymidine incorporation between the number of vasoconstrictors present and indomethacin treatment, thus demonstrating that cyclooxygenase inhibition reveals a synergistic action of vasoconstrictors on the DNA synthesis in mesangial cells.     

Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.     

Endothelin-1 mediates erythropoietin-stimulated glomerular endothelial cell-dependent proliferation of mesangial cells

These experiments were performed in an attempt to determine whether chronic stimulation of glomerular endothelial cells with recombinant human erythropoietin would alter mesangial cell proliferation. Glomerular endothelial cells in culture incubated with various concentrations of erythropoietin for up to 4 days exhibited dose-dependent endothelin-1 production. Moreover, the conditioned medium from erythropoietin-stimulated glomerular endothelial cells enhanced [3H]thymidine incorporation into mesangial cells. This enhancement was significantly attenuated in the presence of a endothelin A receptor antagonist, BQ-123. These results suggest that endothelin-1 mediates erythropoietin-stimulated glomerular endothelial cell-dependent mesangial cell proliferation, resulting in the progression of glomerulonephritis.     

HDL causes mesangial cell mitogenesis through a tyrosine kinase-dependent receptor mechanism

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.     

Regulation of smooth muscle alpha-actin expression and hypertrophy in cultured mesangial cells

BACKGROUND: Mesangial cells during embryonic development and glomerular disease express smooth muscle alpha-actin (alpha-SMA). We were therefore surprised when cultured mesangial cells deprived of serum markedly increased expression of alpha-SMA. Serum-deprived mesangial cells appeared larger than serum-fed mesangial cells. We hypothesized that alpha-SMA expression may be more reflective of mesangial cell hypertrophy than hyperplasia. METHODS: Human mesangial cells were cultured in medium alone or with fetal bovine serum, thrombin, platelet-derived growth factor-BB (PDGF-BB) and/or transforming growth factor-beta1 (TGF-beta1). Alpha-SMA expression was examined by immunofluorescence, Western blot, and Northern blot analysis. Cell size was analyzed by forward light scatter flow cytometry. RESULTS: Alpha-SMA mRNA was at least tenfold more abundant after three to five days in human mesangial cells plated without serum, but beta-actin mRNA was unchanged. Serum-deprived cells contained 5.3-fold more alpha-SMA after three days and 56-fold more after five days by Western blot. Serum deprivation also increased alpha-SMA in rat and mouse mesangial cells. The effects of serum deprivation on alpha-SMA expression were reversible. Mesangial cell mitogens, thrombin or PDGF-BB, decreased alpha-SMA, but TGF-beta1 increased alpha-SMA expression and slowed mesangial cell proliferation in serum-plus medium. Flow cytometry showed that serum deprivation or TGF-beta1 treatment caused mesangial cell hypertrophy. PDGF-BB, thrombin, or thrombin receptor-activating peptide blocked hypertrophy in response to serum deprivation. CONCLUSIONS: We conclude that increased alpha-SMA expression in mesangial cells reflects cellular hypertrophy rather than hyperplasia.     

Application of control chart statistics to blood pressure measurement variability in the primary care setting

The thrombin receptor has been shown to be a novel member of the family of G-protein coupled receptors (Vu, T.-K. H., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. (1991) Cell 64, 1057-1068). This receptor appears to be activated through a thrombin-mediated proteolytic mechanism which exposes a "tethered ligand" responsible for receptor activation. In order to investigate the initial interactions of thrombin with this receptor, we have constructed cell lines which express high levels of the human thrombin receptor and studied the binding of various forms of thrombin to the cell surface. Analysis of transfected cells with thrombin receptor monoclonal antibodies identified a particular cell line (clone #5-18) which displayed > 150,000 thrombin receptors per cell. Clone #5-18 appeared to express functional receptors since treatment with thrombin resulted in both a 15-20 fold increase of cytoplasmic phosphoinositide levels and a comparable shift in the EC50 of thrombin-mediated calcium mobilization when compared to non-transfected CHO cells. Binding of 125I-alpha-thrombin to clone #5-18 did not reach equilibrium at 37 degrees C. However, direct binding studies of 125I-alpha-, 125I-diisopropylphospho (DIP)-alpha-, and 125I-beta-thrombin to clone #5-18 demonstrated that binding at 4 degrees C was saturable and reversible for each ligand. Analysis of the binding data revealed Kd's of 0.8 nM, 0.7 nM and 9.7 nM for 125I-alpha-, 125I-DIP-alpha- and 125I-beta-thrombin respectively. Association of 125I-alpha-, DIP-alpha, and beta-thrombin could be competed by unlabelled alpha- and DIP-alpha-thrombin. Unlabelled beta-thrombin, which has a modified anion-binding exosite, was a poor competitor for 125I-alpha- and 125I-DIP-alpha-thrombin, but did compete for 125I-beta-thrombin. In addition, the hirudin54-65 peptide competed at submicromolar concentrations for the binding of alpha- and DIP-alpha-thrombin, but not for beta-thrombin. This peptide binds specifically at the anion-binding exosite of alpha-thrombin and has been shown to have a lower affinity for beta-thrombin. These results demonstrate directly a high affinity interaction between thrombin and its receptor, and suggest that an important component is the high affinity association of the thrombin receptor with the anion-binding exosite of thrombin.     

Plasma apolipoprotein VLDL-II and egg production in laying hens: establishment of an ELISA method

The thrombin receptor on platelets is an integral membrane protein and is cleaved by thrombin to expose a "tethered ligand" that binds to and triggers the receptor. Here we have explored the power of phage selection technology to make a peptide antagonist of this receptor using platelets directly for the selection. To focus the selection to the thrombin receptor, we eluted the phage with a peptide agonist of the thrombin receptor. A repertoire (1 x 10(7) phage clones) displaying peptide sequences based on the sequence of the tethered ligand, was constructed and selected by binding to the platelets. After several rounds of selection, we identified phage clones that were able to immunoprecipitate the thrombin receptor from platelets and the encoded peptides were sequenced. This revealed some features in common with the tethered ligand, in particular an arginine residue followed by a proline. Several of the peptides were synthesized chemically and one of the peptides was shown to antagonise platelet aggregation triggered by the agonist peptide, and to inhibit serotonin release and tyrosine phosphorylation triggered by either thrombin or the agonist peptide. Anti-aggregatory activity was about ten-fold higher than that of previously reported peptide antagonists of the thrombin receptor.     

The thrombin receptor elevates intracellular calcium in adult rat ventricular myocytes

While there is evidence that thrombin receptor activation leads to contractile dysfunction and induces arrhythmias in ischemic/reperfused cardiac tissue, thrombin is variably reported to modulate intracellular calcium in cardiomyocytes. The present study demonstrates that thrombin receptor activation leads to a rise in intracellular calcium in adult ventricular myocytes and serves to reconcile previous discrepant findings. The thrombin receptor-derived agonist peptide (SFLLRN, a portion of the tethered ligand created by thrombin's proteolytic actions) increases cytosolic calcium and twitch amplitude in cardiomyocytes isolated from adult ventricles. The truncated control peptide FLLRN has no effect, establishing that the response to SFLLRN results from a specific agonist peptide-receptor interaction. However, the response to SFLLRN occurs only at high agonist peptide concentrations and thrombin itself is inactive. This result is not compatible with an action of SFLLRN at a distinct protease-activated receptor (PAR-2; which is activated by SFLLRN, but not by thrombin), since SLIGRL (a ligand which is selective for PAR-2, but not the thrombin receptor) has no effect. Rather, the enzyme-based cell isolation procedure may partially cleave the thrombin receptor and influence cell responses, since concentrations of SFLLRN which are sub-threshold in enzymatically disaggregated myocytes significantly increase the force of isometric contraction of intact rat papillary muscles. These studies provide the first evidence that thrombin receptor activation leads to a change in intracellular calcium and a positive inotropic response in adult ventricular myocardium.     

D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P acts as a biased agonist toward neuropeptide and chemokine receptors

Substance P derivatives are potential therapeutic compounds for the treatment of small cell lung cancer and can cause apoptosis in small cell lung cancer cells in culture. These peptides act as broad spectrum neuropeptide antagonists, blocking calcium mobilization induced by gastrin-releasing peptide, bradykinin, cholecystokinin, and other neuropeptides. We show that [D-Arg1,D-Phe5,D-Trp7,9, Leu11]substance P has unique agonist activities in addition to this described antagonist function. At doses that block calcium mobilization by neuropeptides, this peptide causes activation of c-Jun N-terminal kinase and cytoskeletal changes in Swiss 3T3 fibroblasts and stimulates migration and calcium flux in human neutrophils. Activation of c-Jun N-terminal kinase is dependent on the expression of the gastrin-releasing peptide receptor in rat 1A fibroblasts, demonstrating that the responses to the peptide are receptor-mediated. We hypothesize that [D-Arg1,D-Phe5,D-Trp7,9, Leu11]substance P acts as a biased agonist on neuropeptide and related receptors, activating certain guanine nucleotide-binding proteins through the receptor, but not others.     

Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells

OBJECTIVE: While natriuretic peptides can inhibit growth of vascular muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth. METHODS: Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein. RESULTS: In rat VSMC, passages 4-12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR. CONCLUSION: Our findings indicate that the guanyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanyl-cyclase-linked receptors.     

Low density lipoprotein enhances the thrombin-induced growth of vascular smooth muscle cells

OBJECTIVE: In the present study we investigated whether low density lipoprotein is able to enhance the growth promoting effects of thrombin in vascular smooth muscle cells. METHODS: DNA synthesis was examined by measurement of the [3H]thymidine incorporation into the cell DNA. Cell count was measured with a Neubauer cell box. Thrombin receptor mRNA was determined by Northern blotting. Ca2+ was measured by the fura 2-method. RESULTS: Thrombin (5 nmol/l), thrombin receptor activating protein (3 mumol/l) and low density lipoprotein (33 nmol/l) induce a 652 +/- 80%, 593 +/- 80% and a 316 +/- 60% increase in [3H]thymidine incorporation into DNA (mean +/- SD, n = 3), respectively. A coincubation of thrombin or thrombin receptor activating protein with low density lipoprotein led to a 1245 +/- 160% or 1200 +/- 40% increase of DNA synthesis (mean +/- SD, n = 3). Thus, coincubation of low density lipoprotein and thrombin causes a synergistic rather than an additive mitogenic effect on smooth muscle cells. Thrombin and low density lipoprotein induced a 22 +/- 8.4% and a 29% +/- 6% increase in cell number, respectively. Simultaneous treatment of vascular smooth muscle cells with thrombin and low density lipoprotein caused a 63 +/- 14% increase in cell number (mean +/- SD, n = 3). To further elucidate the underlying mechanism, we studied the effect of low density lipoprotein on the expression of thrombin receptor mRNA. Low density lipoprotein caused a 2.5-fold increase of thrombin receptor mRNA within 24 h, as assessed by Northern analysis. Preincubation of cells for 24 h with 33 nmol/l low density lipoprotein resulted in an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration from 538 +/- 54 to 923 +/- 75 nmol/l (mean +/- SD, n = 4). CONCLUSION: In summary, low density lipoprotein may enhance the mitogenic effect of thrombin probably by an up-regulation of thrombin receptor gene expression in vascular smooth muscle cells or by an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration.     

Functional thrombin receptor PAR1 in primary cultures of human glioblastoma cells

In this study we investigated primary cultures obtained from two glioblastomas surgically removed from a 64-year-old man and a 50-year-old woman, respectively. The presence of the tethered ligand thrombin receptor PAR1 (protease-activated receptor 1) in these cells was demonstrated at the level of receptor binding by using immunofluorescence studies with the monoclonal anti-PAR1 antibody Mab 31-2. Stimulation of human glioblastoma cells both with alpha-thrombin and the thrombin receptor activating peptide TRAP-6 resulted in a series of [Ca+]i spikes as shown by confocal laser fluorescence microscopy with fluo-3 as calcium sensitive fluorescence indicator. This effect was completely blocked with the thrombin receptor antagonist peptide T1. Our results demonstrate functional thrombin receptors (PAR1) in primary cultures of human glioblastomas for the first time.     

Boson analyses in the Ge isotopes

The renin-angiotensin system is activated during vascular development and injury. Furthermore, angiotensin II (Ang II) is a comitogen for fetal mesangial cells in vitro and it may be important in vascular smooth cell growth in disease states. Since fibronectin is an important extracellular matrix protein for vascular development and it too is overexpressed in the mesangium of diseased glomeruli, we explored the interrelationship of fibronectin and Ang II in fetal mesangial cell growth. In human fetal kidney, Ang II type 2 receptors (AT2) were detected in abundance by ex vivo autoradiography. When mesangial cells were isolated from fetal kidney and grown in culture, Ang II type 1 receptors (AT1) were also detected. To explore the mitogenic properties Ang II and fibronectin as well as the effects of Ang II on fibronectin metabolism, studies were performed in vitro, isolated from the potentially confounding variables of hemodynamic influence and circulating growth factors and cytokines. In vitro, mesangial cells expressed a single class of AT1 receptors that were not altered by growth on various substrates. Ang II (10(-7) M) significantly increased thymidine incorporation by confluent human fetal mesangial cells (twofold). When subconfluent, Ang II-stimulated proliferation was greater (fourfold). Ang II significantly increased cell-associated and secreted fibronectin as determined by immunoprecipitation at concentrations that also stimulate mitogenesis. Both of these Ang II-mediated responses were inhibited by the AT1 receptor antagonist DuP-753 (10(-5) M) but not by AT2 receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

The emerging role of paclitaxel in breast cancer therapy

Bradykinin and thrombin caused a time- and dose-dependent stimulation of prostanoid biosynthesis in human dental-pulp fibroblasts, as assessed by the release of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of prostacyclin). The stimulatory effect of bradykinin and thrombin on PGE2 biosynthesis was maximal within 5-10 min. The concentration of bradykinin producing half-maximal stimulation (EC50) of PGE2 and prostacyclin formation was 10 nM. EC50 for thrombin-induced formation of PGE2 and prostacyclin were 0.05 and 0.2 U/ml, respectively. Bradykinin analogues with affinity to the bradykinin B2 receptor, but not those with affinity to the B1 receptor, caused a burst of PGE2 formation. The stimulatory action of bradykinin and thrombin on PGE2 biosynthesis was abolished by two structurally different cyclo-oxygenase inhibitors and significantly reduced by two corticosteroids. Thrombin dose-dependently enhanced the incorporation of [3H]-thymidine into DNA in pulpal fibroblasts by a mechanism that was unrelated to the effect on prostanoid biosynthesis. Bradykinin did not affect thymidine incorporation. Thrombin, but not bradykinin, stimulated the biosynthesis of type 1 collagen in the pulpal fibroblasts. The stimulatory effect of thrombin on collagen biosynthesis was not affected by cyclo-oxygenase inhibitors. These data show that human dental-pulp fibroblasts are equipped with receptors for bradykinin and thrombin linked to enhanced prostanoid biosynthesis. Occupancy of the thrombin receptors also leads to a prostaglandin-independent stimulation of cell proliferation and collagen biosynthesis.     

Glomerular epithelial cells produce endothelin-1

Endothelin-1, a vasoactive peptide originally isolated from vascular endothelial cell culture supernatants, has constricting or mitogenic effects on smooth muscle and glomerular mesangial cells. Whether or not cultured rat glomerular epithelial cells synthesize endothelin-1 was assessed. Under basal culture conditions, the synthesis and release of endothelin-1 peptide by glomerular epithelial cells was time dependent, reaching 0.231 +/- 0.017 pg/1,000 cells at 24 h. For comparison, unstimulated bovine pulmonary artery endothelial cells and rat mesangial cells produced 0.982 +/- 0.237 and 0.004 +/- 0.002 pg of endothelin-1 peptide/1,000 cells per 24 h, respectively. In addition to endothelin-1 peptide, unstimulated glomerular epithelial cells expressed preproendothelin-1 mRNA. Transforming growth factor-beta, complement C5b-9, thrombin, and phorbol myristate acetate significantly enhanced endothelin-1 peptide synthesis in glomerular epithelial cells (45, 15, 55, and 25% above basal levels at 24 h, respectively), whereas epidermal growth factor had no effect. Thrombin and phorbol myristate acetate appeared to stimulate endothelin-1 peptide by activating protein kinase C, because the protein kinase inhibitor 1-(5-isoquinolinyl-sulfonyl)-3-methyl-piperazine abolished the thrombin- and phorbol myristate acetate-induced rise in endothelin-1 but had no effect on basal production. The stimulatory effect of thrombin was also markedly diminished in glomerular epithelial cells that had been depleted of protein kinase C by prolonged preincubation with a high dose of phorbol myristate acetate. Thus, glomerular epithelial cells may be an important source of endothelin-1, which might influence glomerular vasoconstriction or proliferation of target cells, particularly in the presence of proinflammatory molecules in the glomerulus.