Thomas A. Morinelli

Mechanisms of MAPK activation by bradykinin in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is a prominent feature of the atherosclerotic process occurring after endothelial injury. A vascular wall kallikrein-kinin system has been described. The contribution of this system to vascular disease is undefined. In the present study we characterized the signal transduction pathway leading to mitogen-activated protein kinase (MAPK) activation in response to bradykinin (BK) in VSMC. Addition of 10(-10)-10(-7) M BK to VSMC resulted in a rapid and concentration-dependent increase in tyrosine phosphorylation of several 144- to 40-kDa proteins. This effect of BK was abolished by the B(2)-kinin receptor antagonist HOE-140, but not by the B(1)-kinin receptor antagonist des-Arg(9)-Leu(8)-BK. Immunoprecipitation with anti-phosphotyrosine antibodies followed by immunoblot revealed that 10(-9) M BK induced tyrosine phosphorylation of focal adhesion kinase (p125(FAK)). BK (10(-8) M) promoted the association of p60(src) with the adapter protein growth factor receptor binding protein-2 and also induced a significant increase in MAPK activity. Pertussis and cholera toxins did not inhibit BK-induced MAPK tyrosine phosphorylation. Protein kinase C downregulation by phorbol 12-myristate 13-acetate and/or inhibitors to protein kinase C, p60(src) kinase, and MAPK kinase inhibited BK-induced MAPK tyrosine phosphorylation. These findings provide evidence that activation of the B(2)-kinin receptor in VSMC leads to generation of multiple second messengers that converge to activate MAPK. The activation of this crucial kinase by BK provides a strong rationale to investigate the mitogenic actions of BK on VSMC proliferation in disease states of vascular injury.Vascular smooth muscle cell (VSMC) proliferation is a prominent feature of the atherosclerotic process occurring after endothelial injury. A vascular wall kallikrein-kinin system has been described. The contribution of this system to vascular disease is undefined. In the present study we characterized the signal transduction pathway leading to mitogen-activated protein kinase (MAPK) activation in response to bradykinin (BK) in VSMC. Addition of 10-10-10-7M BK to VSMC resulted in a rapid and concentration-dependent increase in tyrosine phosphorylation of several 144- to 40-kDa proteins. This effect of BK was abolished by the B2-kinin receptor antagonist HOE-140, but not by the B1-kinin receptor antagonist des-Arg9-Leu8-BK. Immunoprecipitation with anti-phosphotyrosine antibodies followed by immunoblot revealed that 10-9 M BK induced tyrosine phosphorylation of focal adhesion kinase (p125FAK). BK (10-8 M) promoted the association of p60 src with the adapter protein growth factor receptor binding protein-2 and also induced a significant increase in MAPK activity. Pertussis and cholera toxins did not inhibit BK-induced MAPK tyrosine phosphorylation. Protein kinase C downregulation by phorbol 12-myristate 13-acetate and/or inhibitors to protein kinase C, p60 src kinase, and MAPK kinase inhibited BK-induced MAPK tyrosine phosphorylation. These findings provide evidence that activation of the B2-kinin receptor in VSMC leads to generation of multiple second messengers that converge to activate MAPK. The activation of this crucial kinase by BK provides a strong rationale to investigate the mitogenic actions of BK on VSMC proliferation in disease states of vascular injury.

Oleic Acid–Induced Mitogenic Signaling in Vascular Smooth Muscle Cells A Role for Protein Kinase C

As an initial step in testing the hypothesis that high oleic acid concentrations contribute to vascular remodeling in obese hypertensive patients by activating protein kinase C (PKC), the effects of oleic acid on primary cultures of rat aortic smooth muscle cells (RASMCs) were studied. Oleic acid, an 18-carbon cis-monounsaturated fatty acid (18:1 [cis]), from 25 to 200 mumol/L significantly increased [3H]thymidine uptake in RASMCs with an EC50 of 41.0 mumol/L and a maximal response of 196 +/- 15% of control (P < .01). Oleic acid from 25 to 200 mumol/L caused a concentration-dependent increase in the number of RASMCs in culture at 6 days, reaching a maximum of 210 +/- 13% of control at 100 mumol/L (P < .001). PKC inhibition with 4 mumol/L bisindolyImaleimide I and PKC depletion (alpha, mu, iota, and zeta) with 24-hour exposure to 200 nmol/L phorbol 12-myristate 13-acetate in RASMCs eliminated the mitogenic effects of oleic acid but did not reduce responses to 10% FBS. Stimulation of intact cells with oleic acid induced a peak increase of cytosolic PKC activity, reaching 328 +/- 8% of control (P < .001), but did not enhance PKC activity in the membrane fraction (105 +/- 4%, P = NS). The oleic acid-induced increase of PKC activity in cell lysates was similar in the presence and absence of Ca2+, phosphatidylserine, and diolein (maximum response, 360 +/- 4% versus 342 +/- 9% of control, P = NS). Unlike phorbol 12-myristate 13-acetate, oleic acid over 24 hours did not downregulate any of the four PKC isoforms detected in RASMCs. Oleic acid treatment activated mitogen-activated protein (MAP) kinase. PKC depletion in RASMCs eliminated the rise in thymidine uptake, activation of PKC, and activation of MAP kinase in response to oleic acid. In contrast to oleic acid, 50 to 200 mumol/L stearic (18:0) and elaidic (18:1 [trans]) acids, which are less effective activators of PKC than oleic acid, did not enhance thymidine uptake. These data suggest that oleic acid induces proliferation of RASMCs by activating PKC, particularly one or more of the Ca(2+)-independent isoforms, and raise the possibility that the higher oleic acid concentrations observed in obese hypertensive patients may contribute to vascular remodeling.

The conformational signature of β-arrestin2 predicts its trafficking and signalling functions

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based ‘signalsomes’. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor–arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand–receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

Potentiation of angiotensin II action by corticosteroids in vascular tissue

OBJECTIVES Studies were performed to determine if corticosteroids act directly on the vasculature to potentiate the vasoconstrictor action of angiotensin II and to determine whether corticosteroids upregulate angiotensin II receptors by receptor redistribution or by synthesis of new receptors. METHODS Aortic rings from normal Sprague-Dawley rats were incubated ex vivo with corticosteroids in aerated Krebs-Henseleit buffer to avoid secondary systemic effects prior to stimulated contraction. In cultured vascular smooth muscle cells, these experimental techniques were used: colchicine (blocker of microtubule assembly), chloroquine (inhibitor of endosomal pH gradients), measuring surface-bound 125I-Ang II internalization rate, immunoblotting of angiotensin AT1 receptor protein, and incorporation of [35S]methionine into AT1 receptor protein. RESULTS Contractions to 100 nM angiotensin II in rings incubated with 1 microM aldosterone or dexamethasone for 10 min ex vivo were not different from contractions in control rings. However, angiotensin II-stimulated (but not KCl-stimulated) contractions were enhanced by almost 100% if ex vivo incubation with aldosterone (or corticosterone) lasted for 24 h. Endothelium-dependent relaxation was not significantly reduced by aldosterone pre-incubation. Incubation of cultured vascular smooth muscle cells with a number of corticosteroids for > 8 h resulted in concentration-dependent upregulation of angiotensin II receptor binding and was reversible upon removal of the corticosteroid. Aldosterone did not affect the rate of internalization of surface-bound angiotensin II. In addition, concomitant incubation of colchicine or chloroquine with aldosterone did not hamper angiotensin II receptor upregulation. Incubation of cells with various concentrations of aldosterone for 24 h resulted in concentration-dependent increases in total cell angiotensin II receptor protein content and increases in [35S]methionine incorporation into immunoprecipitated AT1 receptor protein. CONCLUSIONS At least a portion of the enhancement of angiotensin II action by corticosteroids is via direct interaction of corticosteroids with the vasculature. Corticosteroids appear to upregulate angiotensin II receptors by synthesis of new receptor protein rather than by alterations in receptor trafficking.

Essential role of c-Cbl in amphiregulin-induced recycling and signaling of the endogenous epidermal growth factor receptor.

The intracellular processing of the epidermal growth factor receptor (EGFR) induced by epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) has been studied meticulously, with the former resulting in EGFR degradation and the latter in EGFR recycling to the plasma membrane. However, little is known about how other EGF family growth factors affect the trafficking of the EGFR. Additionally, although both EGF and TGF-alpha have been shown to effectively induce initial c-Cbl (ubiquitin ligase)-mediated ubiquitination of the EGFR, limited information is available regarding the role of c-Cblin the trafficking and signaling of recycling EGFR. Thus, in this study, we investigated the roles of c-Cblin endogenous EGFR trafficking and signaling after stimulation with amphiregulin (AR). We demonstrated that a physiological concentration of AR induced recycling of the endogenous EGFR to the plasma membrane, which correlated closely with transient association of the EGFR with c-Cbl and transient EGFR ubiquitination. Most importantly, we used c-Cbl small interfering RNA (siRNA) duplexes and ac-Cbl dominant negative mutant to show that c-Cbl is critical for the efficient transition of the EGFR from early endosomes to a recycling pathway and that c-Cbl regulates the duration of extracellular signal regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) phosphorylation. These data support novel functions of c-Cbl in mediating recycling of EGF receptors to the plasma membrane, as well as in mediating the duration of activation (transient vs sustained) of ERK1/2 MAPK phosphorylation.

Enzymatic processing of angiotensin peptides by human glomerular endothelial cells

The intraglomerular renin-angiotensin system (RAS) is linked to the pathogenesis of progressive glomerular diseases. Glomerular podocytes and mesangial cells play distinct roles in the metabolism of angiotensin (ANG) peptides. However, our understanding of the RAS enzymatic capacity of glomerular endothelial cells (GEnCs) remains incomplete. We explored the mechanisms of endogenous cleavage of ANG substrates in cultured human GEnCs (hGEnCs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and isotope-labeled peptide quantification. Overall, hGEnCs metabolized ANG II at a significantly slower rate compared with podocytes, whereas the ANG I processing rate was comparable between glomerular cell types. ANG II was the most abundant fragment of ANG I, with lesser amount of ANG-(1-7) detected. Formation of ANG II from ANG I was largely abolished by an ANG-converting enzyme (ACE) inhibitor, whereas ANG-(1-7) formation was decreased by a prolylendopeptidase (PEP) inhibitor, but not by a neprilysin inhibitor. Cleavage of ANG II resulted in partial conversion to ANG-(1-7), a process that was attenuated by an ACE2 inhibitor, as well as by an inhibitor of PEP and prolylcarboxypeptidase. Further fragmentation of ANG-(1-7) to ANG-(1-5) was mediated by ACE. In addition, evidence of aminopeptidase N activity (APN) was demonstrated by detecting amelioration of conversion of ANG III to ANG IV by an APN inhibitor. While we failed to find expression or activity of aminopeptidase A, a modest activity attributable to aspartyl aminopeptidase was detected. Messenger RNA and gene expression of the implicated enzymes were confirmed. These results indicate that hGEnCs possess prominent ACE activity, but modest ANG II-metabolizing activity compared with that of podocytes. PEP, ACE2, prolylcarboxypeptidase, APN, and aspartyl aminopeptidase are also enzymes contained in hGEnCs that participate in membrane-bound ANG peptide cleavage. Injury to specific cell types within the glomeruli may alter the intrarenal RAS balance.

N-Acetylcysteine Decreases Angiotensin II Receptor Binding in Vascular Smooth Muscle Cells

Antioxidants seem to inhibit angiotensin II (Ang II) actions by consuming stimulated reactive oxygen species. An alternative hypothesis was investigated: Antioxidants that are also strong reducers of disulfide bonds inhibit the binding of Ang II to its surface receptors with consequent attenuation of signal transduction and cell action. Incubation of cultured vascular smooth muscle cells, which possess Ang II type 1a receptors, with the reducing agent n-acetylcysteine (NAC) for 1 h at 37 degrees C resulted in decreased Ang II radioligand binding in a concentration-dependent pattern. NAC removal restored Ang II binding within 30 min. Incubation with n-acetylserine, a nonreducing analogue of NAC, did not lower Ang II binding, and oxidized NAC was less effective than reduced NAC in lowering Ang II binding. NAC did not decrease Ang II type 1a receptor protein content. Other antioxidants regulated Ang II receptors differently: alpha-Lipoic acid lowered Ang II binding after 24 h, and vitamin E did not lower Ang II binding at all. NAC inhibited Ang II binding in cell membranes at 21 or 37 but not 4 degrees C. Dihydrolipoic acid (the reduced form of alpha-lipoic acid), which contains free sulfhydryl groups as NAC does, decreased Ang II receptor binding in cell membranes, whereas alpha-lipoic acid, which does not contain free sulfhydryl groups, did not. Ang II-stimulated inositol phosphate formation was decreased by preincubation with NAC (1 h) or alpha-lipoic acid (24 h) but not vitamin E. In conclusion, certain antioxidants that are reducing agents lower Ang II receptor binding, and Ang II-stimulated signal transduction is decreased in proportion to decreased receptor binding.

The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers

Background: Hemodynamic regulation involves extensive cross-talk between the renin-angiotensin and kallikrein-kinin systems. Results: In vascular smooth muscle, “biased” AT1 agonists inhibit both AT1 and B2 signaling by internalizing AT1-B2 heterodimers. Conclusion: AT1 antagonists and arrestin-selective biased AT1 agonists have opposing effects on B2 signaling. Significance: Negative allosteric modulation of B2 signaling by biased AT1 agonists may impact their clinical utility. The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar1,Ile4,Ile8]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar1,Ile4, Ile8]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar1,Ile4,Ile8]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar1,Ile4,Ile8]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar1,Ile4,Ile8]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar1,Ile4,Ile8]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions.

Characterization of thromboxane A2/prostaglandin H2 receptors in human vascular smooth muscle cells

The present study utilizes a newly synthesized TXA2/PGH2 mimetic, I-BOP, to characterize the TXA2/PGH2 receptor in suspensions of cultured human vascular smooth muscle cells. [125I]-BOP bound in a saturable and specific manner (Kd = 2.6 +/- 0.6 nM; Bmax = 33,540 +/- 6,200 sites/cell; 69 fmoles/mg protein, n = 12). Competition binding assays were performed with [125I]-BOP and the TXA2/PGH2 receptor antagonists SQ29548, L657925 and L657926 and the receptor agonist U46619. I-BOP induced concentration-dependent increases in intracellular free calcium which were inhibited by SQ29548. The results provide radioligand binding evidence for the presence of a TXA2/PGH2 receptor in human vascular smooth muscle cells.

Identification of functional bradykinin B2 receptors endogenously expressed in HEK293 cells

The human embryonic kidney (HEK) 293 cell line is widely used in cell biology research. Although HEK293 cells have been meticulously studied, our knowledge about endogenous G protein-coupled receptors (GPCR) in these cells is incomplete. While studying the effects of bradykinin (BK), a potent growth factor for renal cells, we unexpectedly discovered that BK activates extracellular signal-regulated protein kinase 1 and 2 (ERK) in HEK293 cells. Thus, we hypothesized that HEK293 cells possess endogenous BK receptors. RT-PCR demonstrated the presence of mRNAs for BK B(1) and BK B(2) receptors in HEK293 cells. Western blotting with BK B(1) and BK B(2) receptor antibodies confirmed this result at the protein level. To establish that BK receptors are functional, we employed fluorescent measurements of intracellular Ca(2+), measured changes in extracellular acidification rate (ECAR) as a reflection of the Na(+)/H(+) exchange (NHE) with a Cytosensortrade microphysiometer, and assessed ERK activation by Western blotting with a phospho-specific ERK antibody. Exposure of HEK293 cells to BK produced a concentration-dependent rise in intracellular Ca(2+) (EC(50)=36.5+/-8.0 x 10(-9)M), a rapid increase in tyrosine phosphorylation of ERK (EC(50)=9.8+/-0.4 x 10(-9)M), and elevation in ECAR by approximately 20%. All of these signals were blocked by HOE-140 (B(2) receptor antagonist) but not by des-Arg(10)-HOE-140 (B(1) receptor antagonist). We conclude that HEK293 cells express endogenous functional BK B(2) receptors, which couple to the mobilization of intracellular Ca(2+), increases in ECAR and increases in ERK phosphorylation.