Coronary Venular Endothelial Cells Research Articles

The type I TGFß receptor ALK‐1 and it’s ligands promote angiogenesis (544.2)

Control of angiogenesis is critical in diseases such as cancer, heart disease, or wound repair. Growth factors such as transforming growth factor beta (TGFß) and fibroblast growth factor 2 (FGF‐2) contribute to angiogenesis. The TGFß family signaling receptor Activin‐Like Kinase 1 Receptor (ALK‐1) is expressed in coronary venular endothelial cells (CVEC) at near confluent cell densities on gelatin, but not collagen but it is induced by TGFß + FGF‐2 during tube formation in collagen. Other genes induced during tube formation were Smad 6 and Fibroblast Growth Factor Receptor 1 (FGFR1). By contrast, ALK‐5, a related TGFß type 1 receptor, and TGFßRII were expressed in these cells in both gelatin and collagen. ALK‐1 is also expressed in other EC types, smooth muscle cells, cardiac fibroblasts, embryonic fibroblasts (3T3) but not epithelial cells. Three ligands: TGFß, activin, and bone morphogenetic protein 9 (BMP‐9), have been shown to bind ALK‐1. We show that endogenous activin binds ALK 1 and can be displaced by exogenous TGFß. It may function to regulate control of ALK 1 activation. The response of endothelial cells to various ALK‐1 ligands may depend on the cell matrix of endothelial cells and which other co‐receptors (type II receptors and endoglin) are expressed. We are: 1) testing expression of various ALK‐1 ligands and co‐receptors in endothelial cells under various conditions; and 2) we are generating mutants of ALK‐1, ALK‐5, and endoglin to further test their roles in angiogenesis.Grant Funding Source: Research support was from AHA‐TX and Dept Veteran’s Affairs grants (JH)

Regulation of protein expression by L-arginine in endothelial cells

L-Arginine is a conditionally essential amino acid for humans and plays an important role in the regulation of cardiovascular function and antioxidative defense. Previous studies have focused on the important role of L-arginine as a physiological precursor in the generation of nitric oxide and polyamines in endothelial cells (cells that line the interior surface of blood vessels). Because of the rapid development of high-throughput proteomics technology, there is now growing interest in studying roles for L-arginine in modulating endothelial-cell protein expression. Of particular interest, recent proteomics analysis has shown that treatment of coronary venular endothelial cells with a physiological level of L-arginine (e.g., 0.1 mM) increases expression of structural proteins (vimentin and tropomyosin) and cytochrome bc1 complex iii-chain A, while decreasing expression of stress-related proteins (PDZ domain containing-3), in these cells. These findings aid in elucidating the mechanisms responsible for the beneficial effect of physiological levels of L-arginine on the circulatory system.

Effect of mini-tyrosyl-tRNA synthetase/mini-tryptophanyl-tRNA synthetase on ischemic angiogenesis in rats: proliferation and migration of endothelial cells

The purpose of this study was to determine the mechanism of mini-tyrosyl-tRNA synthetase/mini-tryptophanyl-tRNA synthetase (mini-TyrRS/mini-TrpRS) on ischemic angiogenesis in rats with acute myocardial infarction and proliferation, migration, potential signaling pathways of rat coronary venular endothelial cells (RCVECs). The effects of mini-TyrRS/mini-TrpRS on RCVECs proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The potential involvement of Erk and PI3K signaling pathways was explored using selective chemical inhibitor or Western-blot analysis. Left coronary artery ligation was used to establish the model of acute myocardial infarction in rats (Sprague-Dawley male rats, 200-250 g, 2-3 months old), 20 μl of mini-TyrRS, mini-TrpRS, or PBS (vehicle) was injected subcutaneously every 12 h. The rats were randomly divided into four experimental groups: sham operated group; coronary artery ligation (CAL); CAL + mini-TyrRS (20 μl, twice daily, 600 μg kg(-1) day(-1)); and CAL + mini-TrpRS (20 μl, twice daily, 600 μg kg(-1) day(-1)). The experiment was carried out at four time points on the 3rd, 7th, 14th, and 28th day after ligation. To determine whether mini-TyrRS/mini-TrpRS affected the angiogenesis activity of rats with myocardial infarction, we measured the myocardial infarction size by TTC staining, and microvessel density (MVD) was determined by CD34 staining. The results show that proliferation and migration in RCVECs could be promoted by mini-TyrRS at concentrations of 1-100 μg/ml, and inhibited by mini-TrpRS. Phospho-PI3-kinase and Erk expression increased significantly when mini-TyrRS was added, but could be attenuated by mini-TrpRS. Compared to the CAL group, the myocardial infarction size of the mini-TyrRS group at the 3rd, 7th, 14th, and 28th day were decreased, while mini-TrpRS increased, but only in days 14 and 28 was there a significant difference. Except that, the microvessel density of RCVECs was promoted in mini-TyrRS group but inhibited in the mini-TrpRS group. These results indicated that angiogenesis could be either stimulated by mini-TyrRS or inhibited by mini-TrpRS.

Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability.

Neutrophil-induced microvascular leakage is an early event in ischemic and inflammatory heart diseases. The specific signaling paradigm by which neutrophils increase microvascular permeability is not yet established. We investigated whether the small GTPase RhoA and its downstream effector Rho kinase mediate neutrophil-stimulated endothelial hyperpermeability. We assessed the effect of neutrophils on Rho activity in bovine coronary venular endothelial cells (CVEC) with a Rho-GTP pull-down assay. Permeability to FITC-albumin was evaluated using CVEC monolayers. We then tested the role of Rho kinase in the permeability response to neutrophils using two structurally distinct pharmacological inhibitors: Y-27632 and HA-1077. Furthermore, neutrophil-stimulated changes in endothelial F-actin organization were examined with fluorescence microscopy. The results show that C5a-activated neutrophils induced an increase in permeability coupled with RhoA activation in CVEC. Inhibition of Rho kinase with either Y-27632 or HA-1077 attenuated the hyperpermeability response. Rho kinase inhibition also attenuated increases in permeability stimulated by the neutrophil supernatant. In addition, activated neutrophils caused actin stress fiber formation in CVEC, which was diminished by either Y-27632 or HA-1077. These findings suggest that RhoA and Rho kinase are involved in the mediation of neutrophil-induced endothelial actin reorganization and barrier dysfunction.

Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1.

Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.

Activated Neutrophils Induce Hyperpermeability and Phosphorylation of Adherens Junction Proteins in Coronary Venular Endothelial Cells

The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which beta-catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junction-cytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on beta-catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that beta-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for beta-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, beta-catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to beta-catenin and VE-cadherin.

Efficient protein transfection of cultured coronary venular endothelial cells.

Although it is well recognized that microvascular endothelial cells play an important role in the local regulation of tissue perfusion and exchange processes, the precise effect of specific endothelial proteins on microvascular function remains to be elucidated. The lack of information is partially due to methodological limitations, because pharmacological approaches that are routinely used in conventional microcirculatory studies produce nonspecific information. The purpose of this study was to develop an efficient method of transfecting endothelial cells with proteins for functional analysis. TransIT, a polyamine reagent, proved very successful for beta-galactosidase (beta-Gal) protein transfection of bovine coronary venular endothelial cells, because time-course and dose-dependent experiments showed that a transfection efficiency of 88 +/- 7% was possible. In control studies, beta-Gal was detected in transfected cells that were trypsinized and washed, indicating that the protein was not merely adhering to the cell surface. Furthermore, transfection of a cell-impermeable peptide inhibitor of protein kinase C (PKC) resulted in a decrease in PKC activity in comparison with control cells. This approach provides a technical basis for further transfection of endothelial cell monolayers with antibodies and constitutively active or dominant-negative proteins to study the molecular control of microvascular function.

Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function.

The transendothelial movement of solutes is a dynamic process controlled by a complex interaction between the cytoskeleton and adhesion proteins. The aim of this study was to examine whether protein tyrosine phosphorylation is involved in the regulation of endothelial barrier function. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused coronary venules. Tyrosine phosphatase inhibitors, including phenylarsine oxide and sodium orthovanadate, dose and time dependently increased basal Pa. Western blot analysis of cultured coronary venular endothelial cells revealed that inhibition of tyrosine phosphatase induced an increase in phosphotyrosine content in a number of proteins, including bands at 65-70 and 120-130 kDa, which were identified as paxillin and focal adhesion kinase (pp125FAK), respectively. The time course and dose responsiveness of protein tyrosine phosphorylation were tightly correlated with those of increases in Pa. Furthermore, stimulation of endothelial cells with histamine or phorbol myristate acetate (PMA) enhanced tyrosine phosphorylation of paxillin and pp125FAK, which was blocked by the tyrosine kinase inhibitor damnacanthal. Correspondingly, the increases in venular permeability elicited by histamine and PMA were abolished in damnacanthal-treated venules. Taken together, the data suggest a possible involvement of protein tyrosine phosphorylation in the control of endothelial barrier function. Paxillin and its associated focal adhesion proteins may play a specific role in agonist-induced hyperpermeability responses in the endothelium of exchange vessels.

Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF.

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase-polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N omega-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

Downregulation of Endothelial Constitutive Nitric Oxide Synthase Expression by Lipopolysaccharide

Lipopolysaccharide (LPS), a causal agent of sepsis, has been shown to induce systemic nitric oxide (NO) synthesis through complex mechanisms. However, the effect of LPS on endothelial cells is incompletely understood. To investigate the mechanism by which LPS influences the release of NO from endothelial cells, the effect of this compound on endothelial constitutive nitric oxide synthase (ecNOS) was studied in cultured bovine coronary venular endothelial cells. Western and Northern analyses showed that LPS decreased ecNOS expression at the protein and mRNA levels in a time-dependent and dose-responsive manner. Concurrent treatment of the endothelial cells with LPS and a transcription inhibitor, actinomycin D, resulted in decreased ecNOS mRNA within 8 hours. In contrast, treatment with actinomycin D had only a relatively insignificant effect on the ecNOS transcript level. This result suggests that the reduction of ecNOS by LPS resulted from an increased degradation rate of its transcript.

Multiple Ionic Mechanisms Activated by Bradykinin in Coronary Venular Endothelial Cells

In coronary endothelium, bradykinin (BK) modulates production of vasodilators by stimulating Ca2+ influx. To examine the ionic currents involved in this process, we applied BK (100 nM) to single bovine coronary venular endothelial cells (CVEC) while recording membrane potential (Em) or whole-cell current simultaneously with [Ca2+]i. The resting potential (Er) of unstimulated cells was bimodally distributed (-70 ± 9 mV, n = 26; -15 ± 8 mV, n = 30). Irrespective of Er, BK evoked a biphasic [Ca2+]i increase simultaneously with a change in Em. When Er was negative to -30 mV, depolarizations were typically observed. When Er was positive to -30 mV, transient hyperpolarizations were typically observed. Under voltage clamp, [Ca2+], increased as the membrane hyperpolarized and the ratio Δ[Ca2+]i/ΔEm was greater in the presence of BK than in unstimulated cells. Many, but not all, cells exhibited an outward K+ current that appeared to be Ca2+ dependent. When present, this current typically predominated over other cu...

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43.

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mM 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mM 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1-3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mM 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mM octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3-5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 microM monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mM 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.

Characterization of a functional angiotensin IV receptor on coronary microvascular endothelial cells

A new class of angiotensin receptors has recently been identified that exhibits both high specificity and affinity for the hexapeptide (3–8) fragment of angiotensin II, angiotensin IV (AngIV). Here, utilizing radioligand binding, we fully characterize AngIV binding at the AT4 receptor on cultured bovine coronary venular endothelial cells (CVEC), and report that when AngIV and bFGF are presented simultaneously an enhancement of DNA synthesis results that is significantly greater than that produced by bFGF alone. The level of DNA synthesis was determined by the incorporation of [ 3H]thymidine into quiescent CVEC monolayers following exposure to 10 nM AngIV and 10 ng/ml bFGF for 1, 3, 5, 7, 9, or 11 days. A significant enhancement of DNA synthesis ( P < 0.01) was seen following 3, 5, 7, 9 and 11 days exposure. In addition, AngIV does not bind to bFGF or heparin, and conversely, bFGF is unable to compete for AngIV binding which suggests that this synergistic response is mediated by independent receptors for these ligands. Results of this study indicate that microvascular endothelial cells are significantly more responsive to bFGF in the presence of nanomolar concentrations of AngIV.

Oxidative injury of coronary venular endothelial cells depletes intracellular glutathione and induces HSP 70 mRNA

Vascular endothelium is one of the first tissues exposed to reactive oxygen species produced during myocardial ischemia-reperfusion. Bovine coronary venular endothelial cells (CVEC) were evaluated for intracellular glutathione (GSH) levels and heat shock protein 70 (HSP 70) mRNA and protein during in vitro oxidative stress. CVEC were incubated with 0.01875 U/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (HX) for 30 min and then allowed to recover for 0, 1, 2, or 3 h. Relative GSH levels were determined by evaluation of monochlorobimane fluorescence. GSH fluorescence was significantly lower in CVEC treated with XO+HX for 30 min than in controls. GSH fluorescence was also decreased in heat-shocked CVEC. After oxidative stress, GSH levels were higher than in controls at 1 h, but by 2 or 3 h after treatment, GSH fluorescence fell below control values. HSP 70 mRNA was induced in CVEC by a 30-min treatment with XO+HX exposure. These data suggest that CVEC respond to oxidative stress by reducing intracellular GSH levels and inducing HSP 70 mRNA, although significant increases in HSP 70 protein were not detected at the time points tested.

Chloride and cation currents activated by bradykinin in coronary venular endothelial cells.

Bradykinin (BK) is known to activate several types of ion channels in endothelial cells, including a K+ channel and a nonselective cation channel. The predominant BK-activated current in most endothelial cells appears to be an outward, Ca(2+)-activated K+ current. We consistently recorded a rapidly activated, spontaneously inactivated inward current stimulated by BK in bovine coronary venular endothelial cells (CVECs). With the use of a whole cell, perforated patch recording mode, the average magnitude of the current was -293 +/- 38 pA. Simultaneous measurements of current and intracellular Ca2+ concentration ([Ca2+]i) showed that the inward current correlated closely with transient increases in [Ca2+]i due to Ca2+ release from intracellular stores. The current could be blocked by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) but not by La3+, and it persisted in Ca(2+)-free/Na(+)-free solution. When intra- and/or extracellular Cl- concentrations were altered, the reversal potential of the current shifted according to the calculated Cl- -equilibrium potential, indicating that the current was carried primarily by Cl-. Another inward current was also activated by BK. This current was slower to activate, could be blocked by La3+, but was not blocked by DIDS. The time course of the slowly activated current correlated with the plateau phase of the BK-stimulated [Ca2+]i increase, which was similar to the behavior of a nonselective cation current reported previously. We propose that these two currents may contribute to the depolarizations and net inward currents induced by BK in this cell line.

Nuclear accumulation of exogenous basic fibroblast growth factor in endothelial, fibroblast, and myoblast cell lines results in diverse biological responses

During studies comparing 125I-bFGF internalization between endothelial cells and other cell types, we found, unexpectedly, internalization and nuclear translocation of exogenously added 125I-bFGF in two cell lines: Chinese hamster ovary cells (CHO) and rat L6 myoblasts. These cell lines were previously reported to be devoid of FGF receptors. Furthermore, CHO cells showed a weak mitogenic response to added bFGF, while L6 cells were mitogenically unresponsive. By comparison, coronary venular endothelial cells (CVEC), BALB/c 3T3 fibroblasts, and BHK-21 cells, demonstrated internalization and nuclear translocation of added 125I-bFGF, and mitogenic responsiveness to the growth factor. Insulin alone stimulated DNA synthesis in all cell types, yet augmented bFGF-dependent DNA synthesis only in CVEC, 3T3, and BHK. All five cell types expressed FGF receptors as assessed by covalent crosslinking with 125I-bFGF and immunoblotting with anti-FGF receptor antibodies. Differing rates of cytoplasmic and nuclear accumulation of 125I-bFGF and partial inhibition of internalization by pretreatment of CVEC with chlorate support a recent model that bFGF can internalize by two mechanisms. Insulin did not significantly affect 125I-bFGF internalization or metabolism in any cell type. bFGF treatment resulted in weak inhibition of RNA synthesis in L6 cells. bFGF appears firmly bound to the nuclear matrix as little nuclear-bound 125I-bFGF in CVEC is released by DNAse I or RNAse A digestion, while washes with 0.5 M NaCl result in partial release. Nuclear bFGF may thus be involved in regulation of nuclear events (e.g., gene transcription and/or DNA replication).

Tyrosine kinase inhibitors impair fibroblast growth factor signaling in coronary endothelial cells

We examined the effect of various tyrosine kinase inhibitors on basic fibroblast growth factor (bFGF)-induced cell signaling and DNA synthesis in coronary venular endothelial cells (CVEC). Two tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, showed reversible, dose-dependent inhibition of bFGF-stimulated DNA synthesis in CVEC with half-maximal inhibitory concentrations of 12 and 3 microM, respectively. Both compounds exhibited preferential inhibition of bFGF vs. serum-induced DNA synthesis. bFGF stimulated increased tyrosine phosphorylation of CVEC cellular proteins, including the FGF receptor, which were visible within 1 min of treatment. Concomitant with their effect on DNA synthesis, both compounds exhibited dose-dependent inhibition of tyrosine phosphorylation of intracellular substrates induced by bFGF. A 2-h pretreatment of quiescent CVEC with genistein blocked nuclear translocation but not cytoplasmic internalization of bFGF, whereas the same treatment with methyl 2,5-dihydroxycinnamate inhibited both processes. These results suggest that activation of bFGF receptor tyrosine kinase activity plays a role in nuclear translocation of bFGF and initiation of DNA synthesis in endothelial cells.

Calcium entry, mobilization, and extrusion in postcapillary venular endothelium exposed to bradykinin.

The effect of bradykinin (BK) on cytosolic calcium in coronary venular endothelial cells (CVEC) was studied using the intracellular calcium indicator indo 1. At normal extracellular calcium levels, CVEC responded to BK at concentrations as low as 0.1 pM; maximum cytosolic calcium spikes occurred at 10 nM. In calcium-free medium, poststimulation cytosolic calcium concentration returned to levels below prestimulation values, implying that BK modulates calcium extrusion mechanisms that are normally masked by calcium influx into the cell. To test this hypothesis, we depleted internal stores of calcium using two approaches: preconditioning or blockade of the endoplasmic reticulum calcium pump with the sesquiterpene lactone, thapsigargin. Depletion by preconditioning consisted of two prior doses of BK followed by a third stimulus of the agonist. Under these conditions, the final dose of BK caused a fall, rather than rise, in cytosolic calcium. Thapsigargin blocked the endoplasmic reticulum calcium pump, leading to a steady-state rise in intracellular calcium concentration. Subsequent exposure of these cells to BK also led to a fall in cytosolic calcium. The preconditioning and thapsigargin studies are consistent with a modulation of calcium extrusion processes by BK in CVEC. The signals responsible for this modulation are unknown.

Angiotensin(s) induce the release of prostacyclin and the growth of coronary venular endothelial cells

Angiotensin(s) induce the release of prostacyclin and the growth of coronary venular endothelial cells

Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial cells

The vasodilating effect of substance P (SP) at the microvascular level is endothelium-dependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.