Ernesto L. Schiffrin

Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats.

Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide produced in the endothelium of blood vessels that may play an important role in the control of local blood flow and could be involved in the pathogenesis of hypertension. We investigated immunoreactive ET-1 (ir-ET-1) levels in acid extracts from blood vessels of deoxycorticosterone acetate (DOCA)-salt and spontaneously hypertensive rats. We found that segments of thoracic aorta and the mesenteric vascular bed contain significantly more ir-ET-1 (11.84 +/- 0.84 and 17.30 +/- 1.89 fmol, respectively) than uninephrectomized control rats (1.78 +/- 0.20 and 9.19 +/- 0.63 fmol, respectively; p < 0.001). High performance liquid chromatography showed that ir-ET-1 of blood vessels of DOCA-salt hypertensive rats eluted in the same position as synthetic ET-1. Significantly increased ir-ET-1 was localized by immunohistochemistry in endothelial cells of aorta and large and small mesenteric arteries of DOCA-salt hypertensive rats. In contrast to the latter, in spontaneously hypertensive rats, vascular content of ir-ET-1 was similar to that of blood vessels of Wistar-Kyoto control rats, at both 6 and 16 weeks of age. High levels of vascular ET-1 may explain the downregulation of vascular endothelin receptors previously described in DOCA-salt hypertensive rats. Furthermore, this suggests that ET-1 may be involved in the maintenance of high blood pressure in mineralocorticoid hypertension.

Increased expression of endothelin-1 gene in blood vessels of deoxycorticosterone acetate-salt hypertensive rats.

We have recently shown that the content of immunoreactive endothelin-1 is increased in acid extracts from blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats compared with uninephrectomized control rats. We have also found by immunohistochemistry a significant increase in immunoreactive endothelin-1 in endothelial cells of aorta and mesenteric arteries of DOCA-salt hypertensive rats. In the present study, we investigated preproendothelin-1 gene expression in blood vessels of DOCA-salt hypertensive rats and uninephrectomized control rats. Northern blot analysis using a specific 32P-labeled complementary RNA probe for rat preproendothelin-1 of 319 base pairs revealed a fourfold to fivefold increase in abundance of preproendothelin-1 messenger RNA transcripts in both aorta and mesenteric arteries from DOCA-salt hypertensive rats. Thus, increased immunoreactive endothelin-1 content in blood vessels of DOCA-salt hypertensive rats is secondary to increased preproendothelin-1 gene expression. Exaggerated expression of the preproendothelin-1 gene in mineralocorticoid hypertension may contribute to the maintenance of elevated blood pressure.

Role of AT2 Receptors in Angiotensin II–Stimulated Contraction of Small Mesenteric Arteries in Young SHR

This study assesses the receptor subtype (AT1 and AT2) through which angiotensin II (Ang II) mediates contraction in small arteries of young and adult spontaneously hypertensive rats (SHR). Segments of third-order mesenteric arteries ( approximately 200 microm in lumen diameter) were mounted in a pressurized system. Systolic blood pressure and media:lumen ratio of small arteries were significantly greater (P<0.001) in young SHR and adult SHR than in age-matched Wistar-Kyoto rats (WKY). Ang II-induced contractile effects were significantly increased (P<0.05) in young SHR compared with age-matched WKY. AT1 blockade with losartan, and combined AT1 and AT2 blockade with losartan and PD123319, abolished Ang II-stimulated contraction in young and adult rats. AT2 blockade (PD123319) significantly reduced (P<0.01) Ang II-elicited contraction in young SHR but had no effect in WKY or adult SHR, indicating that AT2 receptors may contribute to Ang II-induced contraction in young SHR. To determine the Ang receptor status in rat mesenteric vessels, AT1 and AT2 receptor mRNA expression was determined by reverse transcription-polymerase chain reaction. AT1 and AT2 receptor protein expression were detected by Western blot analysis. AT1 receptor mRNA was equally expressed in age-matched rats, but expression was significantly lower in young rats compared with adult rats. AT2 receptor mRNA was weakly expressed in WKY and adult SHR. In vessels from young SHR, AT2 receptor mRNA expression was significantly increased compared with the other groups. AT1 receptor protein was equally expressed in adult rats of both strains but was undetectable in young rats. AT2 receptor protein was only detectable in young rats, with the magnitude of expression greater in SHR than WKY. In conclusion, Ang II-stimulated contractile responses are augmented in vessels from young SHR. These effects are reduced by selective AT2 blockade and abolished by AT1 blockade, indicating that both Ang receptor subtypes are involved in contraction in young SHR. In WKY and adult SHR, losartan, but not PD123319, inhibited Ang II-induced contraction, indicating the exclusive involvement of AT1 receptors. Thus, in SHR, in the phase of developing hypertension, enhanced Ang II-stimulated vascular contraction may be associated with changes in Ang II receptor status, as evidenced pharmacologically and by increased vascular AT2 receptor mRNA and protein expression.

Mitogen-Activated Protein/Extracellular Signal–Regulated Kinase Inhibition Attenuates Angiotensin II–Mediated Signaling and Contraction in Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells

This study investigates the role of extracellular signal-regulated kinases (ERKs) in angiotensin II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+ concentration, ie, [Ca2+]i, and pHi) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+]i, pHi, and contractile responses to Ang II (10(-12) to 10(-6) mol/L) in the absence and presence of PD98059 (10(-5) mol/L). [Ca2+]i and pHi were measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II-stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca2+]i and pHi and contracted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (41.9+/-5.1% reduction in cell length relative to basal length) than in WKY (28.1+/-3.0% reduction in cell length relative to basal length). Basal [Ca2+]i, but not basal pHi, was higher in SHR compared with WKY. [Ca2+]i and pHi effects of Ang II were enhanced (P<0.05) in SHR compared with WKY (maximum Ang II-induced response [Emax] of [Ca2+]i, 576+/-24 versus 413+/-43 nmol/L; Emax of pHi, 7.33+/-0.01 versus 7.27+/-0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II-elicited contractile responses in SHR (Emax,19. 3+/-3% reduction in cell length relative to basal length). Ang II-stimulated [Ca2+]i (Emax, 294+/-55 nmol/L) and pHi (Emax, 7. 27+/-0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In conclusion, Ang II-stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR.

ETA receptor antagonist prevents blood pressure elevation and vascular remodeling in aldosterone-infused rats

Increased endothelin-1 may be associated with elevation of blood pressure (BP) and promotion of vascular hypertrophy, especially in salt-sensitive hypertension. Mineralocorticoid hypertension has been associated with activation of the endothelin system. We evaluated whether in aldosterone-infused rats the selective endothelin type A receptor-antagonist BMS 182874 prevents BP elevation and vascular hypertrophy. Rats were infused with aldosterone (0.75 &mgr;g/h) subcutaneously via a mini-osmotic pump and were offered 1% NaCl in the drinking water±BMS 182874 (40 mg/kg in food) for 6 weeks. Systolic BP was monitored by the tail-cuff method, and vascular changes of mesenteric arteries were evaluated using a pressurized myograph. Aldosterone-infusion significantly increased BP to 151±7 mm Hg compared with controls (108±4 mm Hg, P <0.01). BMS 182874 normalized BP (117±4 mm Hg). Media cross-sectional area of aorta was significantly increased by aldosterone infusion (P <0.05), and BMS treatment normalized it (P <0.001). Aldosterone infusion increased media width and media-to-lumen ratio of mesenteric resistance arteries (17.6±0.4 &mgr;m and 7.5±0.4%) compared with controls (14.2±0.5 &mgr;m, P <0.01, and 5.9±0.1%, P <0.05). BMS 182874 normalized media and media-to-lumen ratio (15.1±0.6 &mgr;m and 5.7±0.1%, both P <0.01). In conclusion, the endothelin type A receptor antagonist attenuated BP elevation and prevented vascular remodeling or hypertrophy of aorta and mesenteric resistance arteries in aldosterone-infused rats. These results suggest a role for endothelin-1 in BP elevation and structural alterations of large and small vessels in aldosterone and salt-induced hypertension.

Role of Extracellular Signal-Regulated Kinases in Angiotensin II–Stimulated Contraction of Smooth Muscle Cells From Human Resistance Arteries

BACKGROUNDnWe assessed the role of extracellular signal-regulated kinases (ERKs) in Ang II-stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries.nnnMETHODS AND RESULTSnVSMCs derived from resistance arteries (<300 microm in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II-stimulated [Ca2+]i, pHi, and contractile responses. [Ca2+]i and pHi were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II-stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT1 and AT2) were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD123319 (a selective AT2 antagonist). Ang II dose-dependently increased [Ca2+]i (pD2=8.4+/-0.36, Emax=541+/-55 nmol/L), pHi (pD2=9. 4+/-0.29, Emax=7.19+/-0.01), and contraction (pD2=9.2+/-0.21, Emax=36+/-2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II-stimulated second messengers, and PD98059 reduced Ang II-induced contraction by >50%. [Sar1,Ile8]Ang II and losartan, but not PD123319, blocked Ang II-stimulated responses.nnnCONCLUSIONSnThese data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT1 subtype are coupled to signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II-stimulated contraction of human VSMCs.

Deoxycorticosterone Acetate Plus Salt Induces Overexpression of Vascular Endothelin-1 and Severe Vascular Hypertrophy in Spontaneously Hypertensive Rats

Endothelin-1 gene expression is enhanced in the aorta and mesenteric arteries, and possibly other vessels, of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In contrast, endothelin-1 gene expression is normal or reduced in spontaneously hypertensive rats (SHR). Severe vascular hypertrophy is present in DOCA-salt hypertensive rats but not in SHR. In this study we investigated whether treatment of SHR with DOCA and salt would result in enhanced endothelin-1 expression and at the same time in severe vascular hypertrophy. Increased abundance of endothelin-1 mRNA was found in the aorta and the mesenteric arterial bed of SHR treated simultaneously with DOCA and salt but not when rats were treated with either separately. The wet weight of the aorta and of the mesenteric arterial bed, media thickness, media cross-sectional area, and media-to-lumen ratio of mesenteric small arteries of DOCA-salt-treated SHR were exaggerated beyond what could be explained by the elevation of blood pressure, relative to SHR treated with salt or with DOCA, which did not overexpress vascular endothelin-1. In conclusion, SHR may exhibit enhanced expression of the endothelin-1 gene in blood vessels when treated with DOCA and salt, and associated with this there is severe vascular hypertrophy. These data support the hypothesis of a role of endothelin-1 in vascular hypertrophy.

In Vivo Study of AT1 and AT2 Angiotensin Receptors in Apoptosis in Rat Blood Vessels

In vitro experiments suggest that angiotensin II (Ang II) may cause growth via angiotensin type 1 (AT(1)) receptors and apoptosis via angiotensin type 2 (AT(2)) receptors. To answer the question of whether AT(1) or AT(2) receptor activation could induce apoptosis in the vasculature in vivo, Wistar rats were infused for 7 days with Ang II (120 ng. kg(-1). min(-1) subcutaneously) and treated with the AT(2) receptor antagonist PD 123319 (30 mg. kg(-1). d(-1) subcutaneously) or the AT(1) receptor antagonist losartan (10 mg. kg(-1). d(-1) orally). Apoptosis in thoracic aorta was quantified by radiolabeled DNA laddering and by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling. The expression of p53, bax, bcl-2, and caspase-3, which play critical roles in apoptotic signaling, was examined by Western blot analysis. The mRNA expression of AT(1) and AT(2) receptors was determined by reverse transcription-polymerase chain reaction. The increase in systolic blood pressure and aortic growth induced by Ang II infusion was completely prevented by losartan alone or losartan given with PD 123319, whereas PD 123319 resulted in a greater increase in systolic blood pressure and aortic growth than Ang II alone. Radiolabeled DNA laddering showed that Ang II infusion+/-losartan or PD 123319 significantly increased apoptosis (147+/-8%, 178+/-20%, and 238+/-41%, respectively, P<0.05 compared with control). Expression of bax and active forms of caspase-3 was increased in the Ang II+PD 123319 group, whereas the expression of p53 and bcl-2 was not significantly different in all groups. The expression of AT(1) and AT(2) receptor mRNA was downregulated by losartan and PD 123319, respectively. Thus, when AT(1) or AT(2) receptors are stimulated in vivo, apoptosis is enhanced in the media of blood vessels. In the case of AT(1) receptor stimulation, this may occur secondary to vascular growth and modulate the latter. Both bax and caspase-3 participate in the pathways of apoptosis triggered by in vivo AT(1) receptor stimulation.

Angiotensin II Regulates Vascular Smooth Muscle Cell pH, Contraction, and Growth Via Tyrosine Kinase–Dependent Signaling Pathways

Angiotensin II (Ang II), a potent vasoactive peptide with mitogenic potential, influences vascular smooth muscle cell contraction and growth through receptor-linked pathways that increase intracellular free Ca2+ concentration ([Ca2+]i) and pH (pHi). Activation of these second messengers by Ang II may involve tyrosine kinase-dependent signaling pathways. This study determined the role of tyrosine kinases in Ang II-stimulated pHi, and in simultaneously measured contractile and [Ca2+]i responses, as well as growth in cultured vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto rats. pHi was determined by fluorescent digital imaging using 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Vascular smooth muscle cell [Ca2+]i and contractile responses were assessed simultaneously by fura 2 methodology and by photomicroscopy in cells grown on rat tail collagen gels. Cell growth was determined by DNA and protein synthesis as measured by [3H]thymidine and [3H]leucine incorporation, respectively. The Ang II receptor subtypes (AT1 or AT2) through which Ang II mediates effects were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD 123319 (a selective AT2 antagonist). To determine whether tyrosine kinases influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (a specific tyrosine kinase inhibitor). Ang II increased pHi in a dose-dependent manner (pD2, 9.2+/-0.2) and significantly increased vascular smooth muscle cell contraction (30%) and [Ca2+]i (pD2, 7.4+/-0.1). Ang II (10(-7) mol/L) increased DNA ([3H]thymidine incorporation) and protein synthesis ([3H]leucine incorporation). [Sar1,Ile8]Ang II and losartan but not PD 123319 abolished Ang II-elicited responses. Tyrphostin A-23 significantly attenuated Ang II-stimulated pHi responses; it also inhibited [Ca2+]i and contractile responses and cell growth. The inactive analogue tyrphostin A-1 did not alter Ang II-stimulated actions. These results provide novel evidence for a role of tyrosine kinases in Ang II-mediated pHi responses in vascular smooth muscle cells and indicate that tyrosine kinases participate in the regulation of signal transduction associated with AT1 receptor subtype-mediated contraction and growth.

Cytosolic Calcium Changes Induced by Angiotensin II in Neonatal Rat Atrial and Ventricular Cardiomyocytes Are Mediated via Angiotensin II Subtype 1 Receptors

We determined the effects of angiotensin II (Ang II) on cytosolic free calcium concentrations ([Ca2+]i) in the absence and presence of the selective angiotensin subtype 1 (AT1) receptor antagonist losartan or the selective AT2 antagonist PD 123319 in cultured neonatal rat atrial and ventricular cardiomyocytes. We also Ang II receptor density, affinity, and mRNA expression. [Ca2+]i was measured in single cells microphotometrically and by fluorescent digital imaging with fura 2 methodology. Receptor parameters were assessed by competitive binding studies with 125I-[Sar1,Ile8]Ang II in the presence of increasing concentrations of [Sar1,Ile8]Ang II, losartan, and PD 123319. AT1 receptor (types AT1A and AT1B) mRNA abundance was measured by reverse transcription-polymerase chain reaction. Ang II produced concentration-dependent increases in [Ca2+]i values in atrial and ventricular cells were similar but Ang II (10-9 mol/L)-induced [Ca2+]i changes were significantly greater in atrial compared with ventricular cells Ang II responses were blocked by losartan (10-7 mol/L) but not PD 123319 (10-7 mol/L). Binding studies demonstrated a single class of high-affinity. Ang II binding sites on cardiomyocyte membranes (Kd = 0.71 +/- 0.11 mumol/L). 125I-[Sar1,Ile8]Ang II was displaced by losartan but not by PD 123319. AT1 receptor mRNA was detected by reverse transcription-polymerase chain reaction in cells from atria and ventricles. In atrial cardiomyocytes, both AT1A and AT1B receptor genes were expressed, whereas in ventricular cardiomyocytes, only the AT1A receptor gene was expressed. These data demonstrate that neonatal cardiomyocytes possess Ang II receptors of the AT1 receptor subtype that are linked to [Ca2+]i signaling pathways. The different Ang II-induced [Ca2+]i responses between atrial and ventricular cells may be related to differences in the distribution of AT1 receptor subtype subvariants.