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P311 is an 8-kDa intracellular protein that is highly conserved across species and is expressed in the nervous system as well as in vascular and visceral smooth muscle cells. P311-null [...]

Angiotensin II (ANG II) contributes to hypertension, cardiac hypertrophy, fibrosis, and dysfunction; however, it is difficult to separate the cardiac effect of ANG II from its hemodynamic action in vivo. To overcome the limitations, we used transgenic mice with cardiac-specific expression of a transgene fusion protein that releases ANG II from cardiomyocytes (Tg-ANG II) and treated them with deoxycorticosterone acetate (DOCA)-salt to suppress their systemic renin-angiotensin system. Using this unique model, we tested the hypothesis that cardiac ANG II, acting on the angiotensin type 1 receptor ([AT.sub.1]R), increases inflammation, oxidative stress, and apoptosis, accelerating cardiac hypertrophy and fibrosis. Male Tg-ANG II mice and their non-transgenic littermates (n-Tg) were uninephrectomized and divided into the following three groups: 1) vehicle-treated normotensive controls; 2) DOCA-salt; and 3) DOCA-salt + valsartan ([AT.sub.1]R blocker). Under basal conditions, systolic blood pressure (SBP) and cardiac phenotypes were similar between strains. In DOCA-salt hypertension, SBP increased similarly in both n-Tg and Tg-ANG II, and cardiac function did not differ between strains; however, Tg-ANG II had 1) greater ventricular hypertrophy as well as interstitial and perivascular fibrosis; 2) a higher number of deoxynucleotidyl-transferase-mediated dUTP nick end labeling-positive cells and infiltrating macrophages; 3) increased protein expression of NADPH oxidase 2 and transforming growth factor-[[beta].sub.1]; and 4) downregulation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (Akt) phosphorylation. Valsartan partially reversed these effects in Tg-ANG II but not in n-Tg. We conclude that, when hemodynamic loading conditions remain unchanged, cardiac ANG II does not alter heart size or cardiac functions. However, in animals with hypertension, cardiac ANG II, acting via [AT.sub.1]R, enhances inflammation, oxidative stress, and cell death (most likely via downregulation of PI 3-kinase and Akt), contributing to cardiac hypertrophy and fibrosis. inflammation; oxidative stress; cardiac hypertrophy; fibrosis doi: 10.1152/ajpheart.00538.2010.

Spontaneously hypertensive rats (SHRs) have normal glomerular capillary pressure even though renal perfusion pressure is higher, suggesting that preglomerular vessels exhibit abnormally high resistance. This may be due to increased superoxide ([O.sup.-.sub.2]) production, which contributes to the vasoconstriction in hypertension. We tested the hypothesis that the myogenic response of the afferent arteriole (Af-Art) is exaggerated in SHRs because of increased levels of reactive oxygen species (ROS). Single Af-Arts were microdissected from kidneys of SHRs and Wistar-Kyoto (WKY) rats and microperfused in vitro. When perfusion pressure in the Af-Art was increased stepwise from 60 to 140 mmHg, the luminal diameter decreased by 8.4 [+ or -] 2.9% in WKY Af-Arts but fell by 29.3 [+ or -] 5.6% in SHR Af-Arts. To test whether ROS production is enhanced during myogenic response in SHRs, we measured chloromethyl-dichlorodihydrofluorescein diacetate acetyl ester (CM-[H.sub.2]DCFDA) florescence before and after increasing intraluminal pressure from 60 to 140 mmHg. Pressure-induced increases in ROS were fourfold greater in SHR Af-Arts compared with WKY Af-Arts (SHR, 48.0 [+ or -] 2.2%; and WKY, 12.2 [+ or -] 0.3%). To test whether [O.sup.-.sub.2] contributes to the myogenic response in SHRs, either the membrane-permeant [O.sup.-sub.2] scavenger Tempol or the nox2-based NADPH oxidase (NOX2) inhibitor gp91ds-tat were added to the Af-Art lumen and bath and the myogenic response was tested before and after treatment. Both Tempol ([10.sup.-4] M) and gp91ds-tat ([10.sup.-5] M) significantly attenuated the pressure-induced constriction in SHR Af-Arts but not in WKY Af-Arts. We conclude that 1) pressure-induced constriction is exaggerated in SHR Af-Arts, 2) NOX2-derived [O.sup.-.sub.2] may contribute to the enhanced myogenic response, and 3) [O.sup.-.sub.2] exerts little influence on the myogenic response under normotensive conditions. hypertension; superoxide doi: 10.1152/ajpheart.00537.2009.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits collagen production and cell proliferation in cultured rat cardiac fibroblasts, but its effect on differentiation of fibroblasts into myofibroblasts is not known. High amounts of transforming growth factor-[beta]1 (TGF-[beta]1) have been found in fibrotic cardiac tissue. TGF-[beta]1 converts fibroblasts into myofibroblasts, which produce more extracellular matrix proteins than fibroblasts. We hypothesized that 1) Ac-SDKP inhibits TGF-[beta]1-induced differentiation of fibroblasts into myofibroblasts; and 2) this effect is mediated in part by blocking phosphorylation of small-mothers-against-decapentaplegic (Smad) 2 and extracellular signal-regulated kinase (ERK) 1/2. For this study, we used human fetal cardiac fibroblasts (HCFs), which do not spontaneously become myofibroblasts when cultured at low passages. We investigated the effect of Ac-SDKP on TGF-[beta]1-induced HCF transformation into myofibroblasts, Smad2 and ERK1/2 phosphorylation, Smad7 expression, cell proliferation, and collagen production. We also investigated TGF-[beta]1 production by HCFs stimulated with endothelin-1 (ET-1). As expected, HCFs treated with TGF-[beta]1 transformed into myofibroblasts as indicated by increased expression of [alpha]-smooth muscle actin and a higher proportion of the embryonic isoform of smooth muscle myosin compared with untreated cells. TGF-[beta]1 also increased Smad2 and ERK1/2 phosphorylation but did not affect Smad7 expression. In addition. TGF-[beta]1 stimulated HCF proliferation as indicated by an increase in mitochondrial dehydrogenase activity and collagen production (hydroxyproline assay). AcSDKP significantly inhibited all of the effects of TGF-[beta]1. It also inhibited ET-l-stimulated TGF-[beta]1 production. We concluded that Ac-SDKP markedly suppresses differentiation of human cardiac fibroblasts into myofibroblasts, probably by inhibiting the TGF-[beta]/ Smad/ERK1/2 signaling pathway, and thus mediating its anti-fibrotic effects. transforming growth factor-[beta]1; human cardiac myofibroblasts; N-acetyl-seryl-aspartyl-lysyl-proline; collagen; signal transduction; fibroblast differentiation doi: 10.1152/ajpheart.00464.2009.

December Galectin-3 (Gal-3) is secreted by activated macrophages. In hypertension, Gal-3 is a marker for hypertrophic hearts prone to develop heart failure. Gal-3 infused in pericardial sac leads to cardiac inflammation, remodeling, and dysfunction. N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a naturally occurring tetrapeptide, prevents and reverses inflammation and collagen deposition in the heart in hypertension and heart failure postmyocardial infarction. In the present study, we hypothesize that Ac-SDKP prevents Gal-3-induced cardiac inflammation, remodeling, and dysfunction, and these effects are mediated by the transforming growth factor (TGF)-[beta]/Smad3 signaling pathway. Adult male rats were divided into four groups and received the following intrapericardial infusion for 4 wk: 1) vehicle (saline, n = 8); 2) Ac-SDKP (800 [micro]g x [kg.sup.-1] x [day.sup.-1], n = 8); 3) Gal-3 (12 [micro]g/day, n = 7); and 4) Ac-SDKP + Gal-3 (n = 7). Left ventricular ejection fraction, cardiac output, and transmittal velocity were measured by echocardiography; inflammatory cell infiltration, cardiomyocyte hypertrophy, and collagen deposition in the heart by histological and immunohistochemical staining; and TGF-[beta] expression and Smad3 phosphorylation by Western blot. We found that, in the left ventricle, Gal-3 1) enhanced macrophage and mast cell infiltration, increased cardiac interstitial and perivascular fibrosis, and causes cardiac hypertrophy; 2) increased TGF-[beta] expression and Smad3 phosphorylation; and 3) decreased negative change in pressure over time response to isoproterenol challenge, ratio of early left ventricular filling phase to atrial contraction phase, and left ventricular ejection fraction. Ac-SDKP partially or completely prevented these effects. We conclude that Ac-SDKP prevents Gal-3-induced cardiac inflammation, fibrosis, hypertrophy, and dysfunction, possibly via inhibition of the TGF-[betya]/Smad3 signaling pathway. N-acetyl-seryl-aspartyl-lysyl-proline; cardiac dysfunction; fibrosis; inflammation

Tubuloglomerular feedback (TGF) is the mechanism by which the macula densa (MD) senses increases in luminal NaCl concentration and sends a signal to constrict the afferent arteriole (Af-Art). The kidney expresses constitutively heine oxygenase-2 (HO-2) and low levels of HO-1. HOs release carbon monoxide (CO), biliverdin, and free iron. We hypothesized that renal HOs inhibit TGF via release of CO and biliverdin. Rabbit Af-Arts and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the MD perfusate. When HO activity was inhibited by adding stannous mesoporphyrin (SnMP) to the MD perfusate, the TGF response increased from 2.1 [+ or -] 0.2 to 4.1 [+ or -] 0.4 [micro]m (P = 0.003, control vs. SnMP, n = 7). When a CO-releasing molecule, (CORM-3; 50 [micro]M), was added to the MD perfusate, the TGF response decreased by 41%, from 3.6 [+ or -] 0.3 to 2.1 [+ or -] 0.2 [micro]m (P < 0.001, control vs. CORM-3, n = 12). When CORM-3 at 100 [micro]M was added to the perfusate, it completely blocked the TGF response, from 4.2 [+ or -] 0.4 to -0.2 [+ or -] 0.3 [micro]m (P < 0.001, control vs. CORM-3, n = 6). When biliverdin was added to the perfusate, the TGF response decreased by 79%, from 3.4 [+ or -] 0.3 to 0.7 [+ or -] 0.4 [micro]m (P = 0.001, control vs. biliverdin, n = 6). The effects of SnMP and CORM-3 were not blocked by inhibition of nitric oxide synthase. We concluded that renal HO inhibits TGF probably via release of CO and biliverdin. HO regulation of TGF is a novel mechanism that could lead to a better understanding of the control of renal microcirculation and function. carbon monoxide; biliverdin; afferent arteriole; macula densa

We hypothesized that elevated macula densa intracellular pH ([pH.sub.i]) during tubuloglomerular feedback enhances [O.sup.-.sub.2] production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, [O.sup.-.sub.2] production increased from 0.53 [+ or -] 0.09 to 2.62 [+ or -] 0.54 U/min (P < 0.01). To determine whether inhibiting the Na/H exchanger blocks [O.sup.-.sub.2] production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, [O.sup.-.sub.2] production induced by NaCl was blunted by 40%. To study the effect of [pH.sub.i] on [O.sup.-.sub.2] in intact macula densa cells, we measured [O.sup.-.sub.2] while pHi was changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, [O.sup.-.sub.2] production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of [O.sup.-.sub.2], we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin ([10.sup.-5] M), [O.sup.-.sub.2] production induced by elevating pHi was blocked. Finally, we measured the optimum pH for [O.sup.-.sub.2] production by the macula densa and found optimum extracellular pH is at 7.7 and optimum [pH.sub.] is ~8 for [O.sup.-.sub.2] production. We found that elevated [pH.sub.i] enhances [O.sup.-.sub.2] production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and [O.sup.-.sub.2] production is pH sensitive, with an optimum [pH.sub.i] of 8. tubuloglomerular feedback; pH optimum; Na/H exchange; reactive oxygen species

Fibrosis is an important component of large conduit artery disease in hypertension. The endogenous tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory and antifibrotic effects in the heart and kidney. However, it is not known whether Ac-SDKP has an anti-inflammatory and antifibrotic effect on conduit arteries such as the aorta. We hypothesize that in ANG II-induced hypertension Ac-SDKP prevents aortic fibrosis and that this effect is associated with decreased protein kinase C (PKC) activation, leading to reduced oxidative stress and inflammation and a decrease in the profibrotic cytokine transforming growth factor-[beta]1 (TGF-[beta]1) and phosphorylation of its second messenger Smad2. To test this hypothesis we used rats with ANG II-induced hypertension and treated them with either vehicle or Ac-SDKP. In this hypertensive model we found an increased collagen deposition and collagen type I and III mRNA expression in the aorta. These changes were associated with increased PKC activation, oxidative stress, intercellular adhesion molecule (ICAM)-1 mRNA expression, and macrophage infiltration. TGF-[beta]1 expression and Smad2 phosphorylation also increased. Ac-SDKP prevented these effects without decreasing blood pressure or aortic hypertrophy. Ac-SDKP also enhanced expression of inhibitory Smad7. These data indicate that in ANG II-induced hypertension Ac-SDKP has an aortic antifibrotic effect. This effect may be due in part to inhibition of PKC activation, which in turn could reduce oxidative stress, ICAM-1 expression, and macrophage infiltration. Part of the effect of Ac-SDKP could also be due to reduced expression of the profibrotic cytokine TGF-[beta]1 and inhibition of Smad2 phosphorylation. arteriosclerosis; vascular hypertrophy; inflammation; protein kinase C; transforming growth factor-[beta]1/Smad

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-[alpha] by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-[alpha] release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 [+ or -] 1.06 mean fluorescent intensity for vehicle and 10.63 [+ or -] 0.35 for Ac-SDKP: P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, AcSDKP decreased secretion of TNF-[alpha] by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension. macrophages; inflammation: activation; angiotensin II

ANG II has a clear role in development of cardiac hypertrophy, fibrosis, and dysfunction. It has been difficult, however, to determine whether these actions are direct or consequences of its systemic hemodynamic effects in vivo. To overcome this limitation, we used transgenic mice with cardiacspecific expression of a transgene fusion protein that releases ANG II from cardiomyocytes (Tg-ANG II-cardiac) without involvement of the systemic renin-angiotensin system and tested whether increased cardiac ANG II accelerates remodeling and dysfunction postmyocardial infarction (MI), whereas those mice show no evidence of cardiac hypertrophy under the basal condition. Male 12- to 14-wk-old Tg-ANG H-cardiac mice and their wild-type littermates (WT) were subjected to sham-MI or MI by ligating the left anterior descending coronary artery tbr 8 wk. Cardiac ANG II levels were ~10-fold higher in Tg-ANG H-cardiac mice than their WT, whereas ANG II levels in plasma and other tissues did not differ between strains. Systolic blood pressure and heart rate were similar between groups with or without MI. In sham-MI, Tg-ANG II-cardiac mice had increased collagen deposition and decreased capillary density. The differences between strains became more pronounced after MI. Although cardiac function was well preserved in the Tg-ANG II-cardiac mice with sham-MI, cardiac remodeling and dysfunction post-MI were more severe than WT. Our results demonstrate that, independent of systemic hemodynamic effects, cardiac ANG II may act locally in the heart, causing interstitial fibrosis in sham-MI and accelerating deterioration of cardiac dysfunction and remodeling post-MI. angiotensin II; myocardial infarction; heart failure; transgenic mice

Superoxide ([O.sup.-.sub.2]) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of [O.sup.-.sub.2] production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces [O.sup.-.sub.2] production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect [O.sup.-.sub.2] production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, [O.sup.-.sub.2] production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in [O.sup.-.sub.2], we used tempol ([10.sup.-4] M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of [O.sup.-.sub.2], we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, [O.sup.-.sub.2] production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, [O.sup.-.sub.2] production was blocked. To test whether depolarization of the macula densa induces [O.sup.-.sub.2] production, we artificially induced depolarization by adding valinomycin ([10.sup.-6] M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases [O.sup.-.sub.2] production. We conclude that increasing luminal NaC1 induces [O.sup.-.sub.2] production during tubuloglomerular feedback. [O.sup.-.sub.2] generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization. tubuloglomerular feedback doi:10.1152/ajprenal.00515.2006

We have shown that the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibited endothelin-1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone (salt)- and ANG II-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts antifibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized by a specific radioligand, [sup.125]I-labeled 3-(p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP. [sup.125]I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentration- and time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (maximal binding: 1,704 [+ or -] 198 fmol/mg protein; dissociation constant: 3.3 [+ or -] 0.6 nM). [sup.125]I-labeled Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (inhibition constant: 0.69 [+ or -] 0.15 nM) and the analog Hpp-Aca-SDKP (inhibition constant: 10.4 [+ or -] 0.2 nM) but not the unrelated peptide ANG II or ET-1 (10 [micro]M). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 [+ or -] 0.4, ET-1: 19.9 [+ or -] 1.2, ET-1+SDKP: 7.7 [+ or -] 0.4, ET-1+Hpp-Aca-SDKP: 9.7 [+ or -] 0.1 [micro]g/mg protein; P < 0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and MAPK ERK1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific [sup.125]I-labeled Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitially. We believe that these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites and that both Ac-SDKP and Hpp-Aca-SDKP exert antifibrotic effects by binding to Ac-SDKP receptors in rat CFs. collagen synthesis; heart; autoradiography; receptor

Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG H is taken up by proximal tubule cells (PTCs) through A[T.sub.1] receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase[dependent mechanisms, and 3) A[T.sub.1] receptor-mediated endocytosis of ANG H has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [[sup.125]I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and 'colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the A[T.sub.1] receptor iblocker losartan (16.5 [+ or -] 4.6%, P < 0.01 vs. ANG II, 78.3 [+ or -] 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 [+ or -] 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by ~58% (basal, 229.8 [+ or -] 11.4 vs. ANG II, 361.3 [+ or -] 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan (P < '0.01), the cytoskeleton microtubule inhibitor colchicine (P < 0.05), and PAO (P < 0.01), whereas depletion of clathrin-coated pits with hyperosmotic sucrose had no effect (356.1 [+ or -] 25.5 pg ANG II/mg protein, not 'significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 [+ or -] 2.8 vs. ANG II, 9.1 [+ or -] 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 [+ or -] 8.6 vs. forskolin + ANG II, 42.8 [+ or -] 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through A[T.sub.1] receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling. kidney; receptor-mediated endocytosis doi:10.1152/ajprenal.00405.2005

Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) and, if so, elucidated the cellular mechanisms involved. Changes in [[Ca.sup.2+]]i responses were studied by fluorescence imaging using the [Ca.sup.2+] indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (A[T.sub.1]) receptors were blocked by losartan (10 [micro]M). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [[[Ca.sup.2+]].sub.i] that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [[[Ca.sup.2+]].sub.i] that peaked at 30 s (basal: 2.2 [+ or -] 0.3 vs. ANG II: 14.9 [+ or -] 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular [Ca.sup.2+] with EGTA (2 mM) did not alter microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses ([Ca.sup.2+] free + ANG II: 12.3 [+ or -] 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular [Ca.sup.2+] stores or with U-73122 to inhibit phospholipase C (1 [micro]M each) markedly attenuated microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses. Combined microinjection of ANG II and losartan abolished [[[Ca.sup.2+]].sub.i] responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [[[Ca.sup.2+]].sub.i] by stimulating intracellular A[T.sub.1] receptors and releases [Ca.sup.2+] from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function. intracellular calcium; kidney: microinjection; proximal tubules; receptor-mediated endocytosis

Using inducible nitric oxide (NO) synthase (iNOS) knockout mice (iNO[S.sup.-/-]), we tested the hypotheses that 1) lack of iNOS attenuates cardiac remodeling and dysfunction and improves cardiac reserve postmyocardial infarction (MI), an effect that is partially mediated by reduction of oxidative stress due to reduced interaction between NO and reactive oxygen species (ROS); and 2) the cardioprotection afforded by iNOS deletion is eliminated by [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) due to inhibition of endothelial NOS (eNOS) and neuronal NOS (nNOS). MI was induced by ligating the left anterior descending coronary artery. Male iNO[S.sup.-/-] mice and wild-type controls (WT, C57BL/6J) were divided into sham MI, MI+vehicle, and MI + l-NAME (100 mg.[kg.sup.-1]*[day.sup.-1] in drinking water for 8 wk). Cardiac function was evaluated by echocardiography. Left ventricular (LV) maximum rate of rise of ventricular pressure divided by pressure at the moment such maximum occurs (dP/dt/instant pressure) in response to isoproterenol (100 ng.[kg.sup.-1]*[min.sup.-1] iv) was measured with a Millar catheter. Collagen deposition, myocyte cross-sectional area, and expression of nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE), markers for ROS, were determined by histopathological and immunohistochemical staining. We found that the MI-induced increase in LV chamber dimension and the decrease in ejection fraction, an index of systolic function, were less severe in iNO[S.sup.-/-] compared with WT mice. l-NAME worsened LV remodeling and dysfunction further, and these detrimental effects were also attenuated in iNO[S.sup.-/-] mice, associated with better preservation of cardiac function. Lack of iNOS also reduced nitrotyrosine and 4-HNE expression after MI, indicating reduced oxidative stress. We conclude that iNOS does not seem to be a pathological mediator of heart failure; however, the lack of iNOS improves cardiac reserve post-MI, particularly when constitutive NOS isoforms are blocked. Decreased oxidative stress and other adaptive mechanisms independent of NOS may be partially responsible for such an effect, which needs to be studied further. oxidative stress; reactive oxygen species; ejection time

N-acetyl-seryl-aspartyl-lysyl-proline stimulates angiogenesis in vitro and in vivo. Am J Physiol Heart Circ Physiol 287: H2099-H2105, 2004. First published July 15, 2004; doi: 10.1152/ajpheart.00592. 2004.--N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a natural inhibitor of pluripotent hematopoietic stem cell proliferation, has been suggested as capable of promoting an angiogenic response. We studied whether Ac-SDKP stimulates endothelial cell proliferation, migration, and tube formation; enhances angiogenic response in the rat cornea after implantation of a tumor spheroid; and increases capillary density in rat hearts with myocardial infarction (MI). In vitro, an immortal BALB/c mouse aortic endothelial 22106 cell line was used to determine the effects of Ac-SDKP on endothelial cell proliferation and migration and tube formation. In vivo, a 9L-gliosarcoma cell spheroid (250-300 [micro]m in diameter) was implanted in the rat cornea and vehicle or Ac-SDKP (800 [micro]g * [kg.sup.-1] * [day.sup.-1] ip) infused via osmotic minipump. Myocardial capillary density was studied in rats with MI given either vehicle or Ac-SDKP. We found that Ac-SDKP 1) stimulated endothelial cell proliferation and migration and tube formation in a dose-dependent manner, 2) enhanced corneal neovascularization, and 3) increased myocardial capillary density. Endothelial cell proliferation and angiogenesis stimulated by Ac-SDKP could be beneficial in cardiovascular diseases such as hypertension and MI. Furthermore, because Ac-SDKP is mainly cleaved by ACE, it may partially mediate the cardioprotective effect of ACE inhibitors. endothelial cell; capillaries; myocardial infarction

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1[beta], phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad. CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration. gene transfer; cardiac-specific promoter

Ren, Yilin, Hong Yu, Hong Wang, Oscar A. Carretero, and Jeffrey L. Garvin. Nystatin and valinomycin induce tubuloglomerular feedback. Am J Physiol Renal Physiol 281: F1102--F1108, 2001. First published September 21, 2001; 10.1152/ajprenal.00357.2001.--The macula densa expresses a luminal [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter and a basolateral [Cl.sup.-] conductance. Although it is known that cotransport of [Na.sup.+], [K.sup.+], and [Cl.sup.-] is the first step in tubuloglomerular feedback (TGF), subsequent steps are unclear. We hypothesized that [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] entry via the luminal [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter elevates intracellular [Cl.sup.-], increases electrogenic Clefflux across the basolateral membrane, and depolarizes the macula densa, initiating TGF. We perfused afferent arterioles with macula densa attached. The macula densa was perfused with solutions containing either 5 mM [Na.sup.+] and 3 mM [Cl.sup.-] (low NaCl) or 80 mM [Na.sup.+] and 77 mM [Cl.sup.-] (high NaCl). When the macula densa perfusate was changed from low to high NaCl, afferent arteriole diameter decreased from 15.8 [+ or -] 0.8 to 13.1 [+ or -] 0.7 mm (P < 0.05). Adding 10 [micro] M furosemide to the macula densa lumen blocked TGF. When nystatin, a group I cation ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl, it induced TGF (from 18.0 [+ or -] 1.5 to 15.6 [+ or -] 1.6 mm; P = 0.003). When valinomycin, a [K.sup.+]-selective ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl containing 5 mM [K.sup.+], it did not induce TGF. Subsequent addition of 50 mM KCl to the macula densa perfusate induced TGF (from 21.7 [+ or -] 0.8 to 17.5 [+ or -] 1.3 mm; P = 0.0047; n = 6). Adding 50 mM KCl without valinomycin did not induce TGF. When 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1 [micro] M), a [Cl.sup.-] channel blocker, was added to the bath, it blocked TGF induced by high NaCl, but did not block TGF induced by valinomycin plus 50 mM KCl. NPPB did not alter afferent arteriole constriction induced by norepinephrine. We concluded that increased NaCl in the lumen of the macula densa leads to influx of [Cl.sup.-] via the [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter. The accelerated transport increases intracellular [Cl.sup.-]. The subsequent exit of [Cl.sup.-] across the basolateral membrane via [Cl.sup.-] channels in turn leads to depolarization of the macula densa and thereby induces TGF. afferent arteriole; transport; chloride channel Received 1 December 2000; accepted in final form 16 July 2001

The cardiac output, left ventricle pressure and central hemodynamics of Lewis inbred rats with or without heart failure (HF) were analyzed to determine the effects of HF on cardiac performance. Analysis of hemodynamics in rat models of chronic heart failure by left coronary artery ligation indicated the presence of left ventricle dysfunction due to large infarct size. The model of chronic heart failure in Lewis inbred rats exhibited low mortality and developed cardiac dysfunction and chamber dilation one week after myocardial infarction.