High resolution localization of angiotensin II receptors in rat renal medulla.

The cellular localization of angiotensin II (Ang II) receptors in the inner stripe of the outer medulla of the rat kidney was investigated by using high resolution light and electron microscopic autoradiography. Fresh tissue blocks from the inner stripe of the outer medulla were incubated with 125I-[Sar1, Ile8] Ang II and prepared for microscopic autoradiography. At the light microscopic level, 125I-[Sar1, Ile8] Ang II was found to penetrate into the tissue and to bind specifically to sites outlining renal tubules and vasa recta bundles. Electron microscopic autoradiography revealed that silver grains were detected over interstitial cells located between the tubules and components of the vasa recta bundles, but no silver grains were detected overlying the cells of the thin descending or thick ascending limbs of the loop of Henle, the collecting ducts, the vasa recta, or other blood vessels. These interstitial cells contained abundant endoplasmic reticulum, microfilaments, occasional lipid droplets and extensive cytoplasmic processes which closely related to the basement membranes of the vasa recta and loops of Henle. The cells therefore closely resemble type 1 interstitial cells. Since Ang II binding sites are absent in the inner medulla, the cells labelled by this technique must be a subset of type 1 interstitial cells, distinct from the typical lipid-laden interstitial cells most abundant in the inner medulla. These findings demonstrate that type 1 interstitial cells are the primary sites for a high density of Ang II receptors located in the inner stripe of the outer medulla.     

Mechanism of the antihypertensive effect of K depletion in the spontaneously hypertensive rat

K depletion reverses hypertension in the SHR (systolic blood pressure: K deplete 122 +/- 5 vs. K replete 164 +/- 4 mm Hg, P less than 0.001). To determine the role of the renin angiotensin system in the protective effect of K depletion, we performed studies of vascular reactivity in intact SHR and of angiotensin II (Ang II) binding to mesenteric artery particles and vascular smooth muscle cells (VSMC) in culture from SHR. Pressor sensitivity to Ang II (+/- converting enzyme inhibition) but not norepinephrine was reduced in K depleted SHR. Thus, the decreased vascular reactivity in K depletion was specific for Ang II rather than a generalized defect. Ang II binding and receptor number (Bmax) (156 +/- 20 vs. 81 +/- 5 fmol/mg of protein, P less than 0.01) were increased in K depleted mesenteric artery particles. Since K depletion and increases in Ang II have both been associated with increased Ang II binding, Ang II binding was measured after bilateral nephrectomy. Despite reduction of plasma renin activity, total binding and Bmax were still increased in nephrectomized K depleted SHR. To determine the specific effect of K depletion independent of Ang II on Ang II binding, studies were performed in mesenteric artery VSMC from SHR grown in culture. VSMC from K replete SHR were grown to confluency in K replete medium and then were incubated in K depleted medium for 24 hours. Binding was saturable, time and temperature-dependent in K replete and K depleted cells. Total binding and Bmax (139 +/- 13 vs. 93 +/- 7 fmol/mg protein, P less than 0.01) were increased in K depleted cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The luminal membrane of rat thick limb expresses AT1 receptor and aminopeptidase activities

BACKGROUND: Endogenous intratubular angiotensin II (Ang II) supports an autocrine tonic stimulation of NaCl absorption in the proximal tubule, and its production may be regulated independently of circulating Ang II. In addition, endogenous Ang II activity may be regulated at the brush border membrane (BBM), by the rate of aminopeptidase A and N (APA and APN) activities and the rate of Ca2+-independent phospholipase A2 (PLA2-dependent endocytosis and recycling of the complex Ang II subtype 1 (AT1) receptor (AT1-R). The aim of the present study was to look for subcellular localization of AT1-R, and APA and APN activities in the medullary thick ascending limb of Henle (mTAL), as well as search for an asymmetric coupling of AT1-R to signal transduction pathways. METHODS: Preparations of isolated basolateral membrane (BLMV) and luminal (LMV) membrane vesicles from rat mTAL were used to localize first, AT1-R by 125I-[Sar1, Ile8] Ang II binding studies and immunoblot experiments with a specific AT1-R antibody, and second, APA and APN activities. Microfluorometric monitoring of cytosolic Ca2+ with a Fura-2 probe was performed in mTAL microperfused in vitro, after apical or basolateral application of Ang II. RESULTS: AT1-R were present in both LMV and BLMV, with a similar Kd (nmol/L range) and Bmax. Accordingly, BLMV and LMV preparations similarly stained specific AT1-R antibody. APA and APN activities were selectively localized in LMV, although to a lesser extent than those measured in BBM. In the in vitro microperfused mTAL, basolateral but not apical Ang II induced a transient increase in cytosolic [Ca2+]. CONCLUSIONS: Besides the presence of basolateral AT1-R in mTAL coupled to the classical Ca2+-dependent transduction pathways, AT1-R are present in LMV, not coupled with Ca2+ signaling, and co-localized with APA and APN activities. Thus, apical APA and APN may play an important role in modulating endogenous Ang II activity on NaCl reabsorption in mTAL.     

TGF-{beta}- and angiotensin-II-induced mesangial matrix protein secretion is mediated by protein kinase C

Background: Glomerulonephritis is characterized by the accumulation of extracellular matrix protein within the glomerulus. This process, when allowed to proceed unimpeded, leads to glomerulosclerosis and eventually to cessation of glomerular filtration. There is evidence that protein kinase C (PKC) activation plays an important role in mediating at least some of the effects of TGF-{beta} in vascular smooth-muscle cells. The current study was undertaken to determine whether PKC activity is required for both TGF-{beta} and angiotensin II (Ang II) to induce mesangial cell matrix protein secretion. Methods: PKC was inhibited by two separate methods, and [³H]thymidine incorporation was assessed in both the presence and the absence of PKC inhibition. Conditioned medium from cells stimulated with TGF-{beta} or Ang II was collected and analysed for secreted matrix proteins and sulphated proteins by SDS-polyacrylamide gel electrophoresis and western blotting. Results: Twenty-four-hour incubation of rat mesangial cells with phorbol-12-myristate-13-acetate (PMA) reduced total PKC activity to basal levels. Both TGF-{beta} and Ang II were mitogenic in mesangial cells, and chronic PMA pre-incubation inhibited this DNA synthesis. TGF-{beta} and Ang II-induced sulphated protein secretion into conditioned medium was markedly attenuated in PKC-downregulated cells. Secretion of the specific matrix proteins laminin and fibronectin by mesangial cells stimulated with either TGF-{beta} or Ang II was also diminished in PKC-downregulated cells and in cells pre-incubated with the specific PKC inhibitor, chelerythrine. There was no evidence of generalized cell toxicity or decreased non-specific protein synthesis caused by these PKC inhibitors. Conclusions: PKC is a key intermediary in the process by which TGF-{beta} and Ang II cause DNA synthesis and mesangial cell matrix protein production. Thus, PKC inhibitors deserve further study as potential therapeutic agents for a variety of glomerular diseases.     

Tubular expression of angiotensin II receptors and their regulation in IgA nephropathy

Enhanced renal expression for the renin-angiotensin system (RAS) is detected in IgA nephropathy (IgAN). Previous data showed an altered glomerular expression of angiotensin II type 1 receptor (AT1R), suggesting a regulatory response to high intrarenal angiotensin II (Ang II) concentration in IgAN. In this study, the expression and regulation of Ang II receptors were examined in human proximal tubular epithelial cells (PTEC) in IgAN. Tubular expression of AT1R and Ang II type 2 receptor (AT2R) was increased in IgAN. In vitro culture experiment showed that the upregulation of Ang II receptors was not due to the direct effect of IgA but the indirect effect after IgA deposition on human mesangial cell. When PTEC were cultured with conditioned culture medium from human mesangial cells activated with IgA, Ang II production was upregulated, leading to inflammation and apoptosis via the AT1R and AT2R, respectively. Sequential expression of Ang II receptors determined the injury of PTEC induced by mediators in the conditioned medium. The initial interaction between Ang II and AT1R activated both protein kinase C and mitogen-activated protein kinase pathways, leading to inflammatory responses. This early AT1R-dependent event was followed by upregulation of AT2R expression and continued Ang II release. The interaction between Ang II and AT2R subsequently led to expression of cleaved poly[ADP-ribose] polymerase through downregulation of the mitogen-activated protein kinase pathway. The data suggest that appropriate control of Ang II receptor activities in PTEC may ameliorate tubulointerstitial injury in IgAN.     

IGF-1-induced lipid accumulation impairs mesangial cell migration and contractile function

BACKGROUND: Chronic treatment of mesangial cells with insulin-like growth factor-1 (IGF-1) results in intracellular lipid accumulation. These mesangial cells resemble foam cells. METHODS: To determine whether this phenotype affects cell function, lipid-laden mesangial cells were tested for their ability to migrate in response to IGF-binding protein-5 (IGFBP-5) and to contract in response to angiotensin II (Ang II). IGFBP-5 binding and subsequent activation of the signal transduction cascade for migration were examined. To confirm that lipid accumulation was responsible for impaired contractility, lipid was removed from lipid-laden mesangial cells and the cells were re-tested for contractile response to Ang II. RESULTS: In comparison to control mesangial cells, lipid-laden cells failed to migrate in response to IGFBP-5. Although cellular binding of IGFBP-5 was not altered by lipid accumulation, IGFBP-5 failed to activate cdc42, a Rho GTPase required for IGFBP-5-mediated mesangial cell migration. These data indicate that lipid accumulation within the mesangial cell interferes with the signal transduction response to IGFBP-5. In addition, mesangial cells treated with IGF-1 had reduced contraction to Ang II. When lipid accumulation was exaggerated by adding cholesteryl esters to the culture medium, mesangial cells failed to contract in response to Ang II. Following removal of excess lipid from these mesangial cells, the contractile response to Ang II was restored. CONCLUSIONS: IGF-1 induces lipid accumulation in mesangial cells, which impairs their ability to respond to specific migratory and contractile stimuli. These observations are relevant to understanding functional abnormalities in diseases where mesangial foam cells occur, such as focal and segmental glomerulosclerosis and diabetic nephropathy.     

Regulation of human mesangial cell growth in culture by thromboxane A2 and prostacyclin.

Elevated eicosanoid biosynthesis characterizes certain forms of human and experimental glomerular proliferative disease. Thromboxane A2 (TxA2) and other prostaglandins (PG) act through specific receptors and mechanisms of intracellular signal transduction in human mesangial cells. We studied the actions of U-46619, a TxA2 mimetic which stimulates mesangial phospholipase C, and of the PGI2 analogue, Iloprost, a potent activator of adenylate cyclase, on proliferation of cultured human mesangial cells. When applied alone to quiescent cells, U-46619 had only weak mitogenic activity, as assessed by [3H]thymidine [( 3H]-TdR) incorporation and cell counts. On the other hand, addition of U-46619 10 minutes prior to stimulation of the cells with 1 to 17% fetal bovine serum (FBS) for 24 hours, potently and dose-dependently inhibited FBS-stimulated [3H]-TdR incorporation. Similarly, U-46619 inhibited the effects of 10 ng/ml platelet-derived growth factor (PDGF), epidermal growth factor or basic fibroblast growth factor on [3H]-TdR incorporation, by 55, 79 and 88%, respectively. The effects of U-46619 were not mimicked by another stimulus of phospholipase C, angiotensin II. Iloprost also inhibited FBS-activated proliferation. Neither eicosanoid inhibited the rise of cytosolic Ca2+ induced by FBS or PDGF. The actions of TxA2 and Iloprost in cultured cells point to multiple functional interactions between eicosanoids and growth factors in the control of mesangial cell proliferation.     

Vasoactive agents affect growth and protein synthesis of cultured rat mesangial cells.

Mesangial cell (MC) proliferation and extracellular matrix (ECM) formation are hallmarks of chronic glomerular disease. The present in vitro study examined the effects of the vasoactive agents angiotensin II (Ang II), arginine vasopressin (AVP), and serotonin (5-HT) on growth and protein biosynthesis of cultured rat MCs after 72 hours of incubation. AVP and 5-HT (10(-6) M) significantly increased DNA synthesis and growth of quiescent subconfluent MCs to levels of 25 and 45%, respectively, of the optimal stimulatory effect of 10% fetal calf serum (FCS) (both P less than 0.001). The mitogenic effect of Ang II was 10% of the 10% FCS effect (P less than 0.01). ECM production was studied by ELISA assay for fibronectin (FN) secreted into the culture medium (SeFN) and cell-associated FN, that is, intra- and pericellular FN (CaFN). In all incubations, highly significant negative linear relationships were found between the numbers of MCs per well and quantities of both SeFN and CaFN after normalization of the data by logarithmic transformation (SeFN: r values greater than -0.9705; CaFN: r greater than -0.9620; P less than 0.001). Thus, increasing cell densities progressively suppressed ECM formation by MCs. The ECM production was found to be independent of growth activity. AVP significantly increased SeFN (P less than 0.05) and decreased CaFN (P less than 0.001) in subconfluent cultures; Ang II and 5-HT had no effect. Metabolic labeling with 35S-methionine (18 hr, 200 microCi/ml medium) and 2-D electrophoresis of MC lysates resulted in resolution of greater than 500 different radiolabeled intracellular proteins in molecular weight from 110 to 20 Kd over an isoelectric interval of 5.0 to 7.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional endothelin 1 receptors on human glomerular podocytes and mesangial cells.

To determine if endothelin 1 (Et1) receptors are present in human glomeruli, and which glomerular cells possess these receptors, 125I Et1 binding to isolated glomeruli and cultured glomerular mesangial and epithelial cells was studied. The latter were identified as podocytes. We demonstrated that Et1 binds specifically and reversibly to isolated human glomeruli and to cultured glomerular mesangial and epithelial cells. Scatchard analysis of competitive inhibition of 125I Et1 binding gave the following results (m +/- SEM, n = 3): isolated glomeruli, Kd = 4.2 +/- 2.1 x 10(-10) M, Bmax = 8.1 +/- 1.2 x 10(10) sites/mg protein; mesangial cells, Kd = 5.2 +/- 1.5 x 10(-10) M, Bmax = 1.87 +/- 0.49 x 10(4) sites/cell; epithelial cells, Kd = 7.2 +/- 1.5 x 10(-10) M, Bmax = 2.46 +/- 0.15 x 10(4) sites/cell. These receptors seem to be functional, since in both mesangial and epithelial cells Et1 induces a rapid and transient increase in intracellular [Ca2+]i. All these results indicate that Et1 may regulate glomerular filtration rate through an autocrine-paracrine pathway on mesangial cells and on podocytes.     

Angiotensin type 2 receptor is expressed in the adult rat kidney and promotes cellular proliferation and apoptosis

BACKGROUND: Angiotensin II (Ang II) is associated with cell proliferation and apoptosis. The role of the angiotensin type 2 receptor (AT2R) in these processes remains controversial. Conventional radioligand binding of 125I-Sar1, Ile8 Ang II in adult kidney has failed to demonstrate the binding for the AT2R. METHODS: The presence of the AT2R was explored in adult rat kidney by in vitro and in vivo autoradiography using the selective AT2R radioligand 125I-CGP 42112B. The roles of the angiotensin type 1 receptor (AT1R) and the AT2R in mediating cellular proliferation and apoptosis were assessed using selective AT1R or AT2R antagonists in Ang II-infused Sprague-Dawley (SD) rats. RESULTS: 125I-CGP 42112B binding was demonstrated by in vitro and in vivo autoradiography techniques in the glomeruli and proximal tubules of SD rats. This binding could be displaced by Ang II and the AT2R antagonist PD123319 but not by the AT1R antagonist valsartan. Subcutaneous infusion of Ang II for 14 days in eight-week-old SD rats induced proliferation of proximal tubular epithelial cells, as assessed by a twofold increase in proliferating cell nuclear antigen (PCNA)-positive cells and apoptosis, as assessed by a threefold increase in terminal dUTP nick end labeling (TUNEL)-positive cells. The administration of the AT2R antagonist PD123319 or the AT1R antagonist valsartan was associated with attenuation of the increases in both PCNA- and TUNEL-positive cells following Ang II infusion. Ang II infusion was associated with increased osteopontin gene and protein expression, which could be reduced by treatment with either valsartan or PD123319. CONCLUSION: These findings indicate that there is significant expression of the AT2R in the adult kidney, and that the AT2R has a role in mediating Ang II-induced proliferation and apoptosis in proximal tubular epithelial cells and expression of osteopontin.     

Changes in the biochemical profiles of mid-cervically located adenosine A1 receptors after repeated theophylline administration in adult rats

BACKGROUND/OBJECTIVE: Adenosine A1 receptors localized in the phrenic motoneurons (PMNs), where the axons of the descending bulbospinal respiratory make synaptic contacts, may be involved in theophylline-induced respiratory-related activity in rats. The objective of this study was to characterize the biochemical profiles of adenosine A1 receptors in 2 groups of rats: (a) na?ve and (b) theophylline-treated (3-day oral administration). METHODS: Biochemical binding characteristics of adenosine A1 receptors in the C3 to C5 (PMN) of adult rats were assessed in na?ve (n = 6) and theophylline-treated animals (n = 6) using [3H]-DPCPX (10 pmol/L to 30 nmol/L), the specific adenosine A1 receptor antagonist in saturation-binding assays. Competition assays used theophylline as the competing ligand (20 mmol/L to 20 pmol/L), and protein concentration was determined with the Bradford assay using a range of standards (0.016-1.0 mg/mL). RESULTS: In saturation-binding assays in na?ve animals, the A1 receptor was characterized by a single binding site with Bmax and Kd values of 256.00 +/- 32.13 fmol/mg protein and 2.89 +/- 0.45 nmol/L, respectively. Analysis of the isotherm in theophylline-treated animals showed 1 site with Bmax and Kd values of 219.00 +/- 26.3 fmol/mg protein and 0.60 +/- 0.21 nmol/L, respectively, and a second site characterized by Bmax and Kd values of 492.6 +/- 3.15 fmol/mg protein and 14.09 +/- 2.06 nmol/L, respectively. CONCLUSIONS: Theophylline administration revealed 2 binding sites on receptors (characterized by the specific adenosine A1 antagonist, [3H]-DPCPX) located in the vicinity of phrenic motoneurons (C3-C5). Alteration of the receptor profiles after theophylline may underlie the respiratory-related actions of the drug.     

Angiotensin II type 1 receptor activation increases microvascular hydraulic permeability

BACKGROUND: In addition to its vasoconstricting effects, angiotensin II (Ang II) has also demonstrated the ability to modulate microvessel permeability. We hypothesized that activation of the angiotensin II type 1 receptor (AT1) would increase hydraulic permeability. METHODS: Hydraulic permeability (L(p)) was measured in rat mesenteric venules using the Landis micro-occlusion technique. Paired measures of L(p) were obtained at baseline and after perfusion with the AT1 agonist, [Sar(1)]-angiotensin II, at 10 micromol/L (n=6) and 100 micromol/L (n=6). Activation of the AT1 receptor was also achieved by perfusion with 20 nmol/L Ang II plus the angiotensin II type 2 receptor (AT2) antagonist, PD123319. In these studies, 30 micromol/L (n=6) and 300 micromol/L (n=6) of PD123319 were used. RESULTS: [Sar(1)]-angiotensin II increased L(p) 2-fold with the 10 micromol/L dose (P=.04) and 4-fold with the 100 micromol/L dose (P < .001). The L(p) peak due to [Sar(1)]-angiotensin II occurred sooner than the peak observed with Ang II. PD123319 (30 micromol/L) plus 20 nmol/L Ang II increased L(p) 5-fold (P=.003), while PD123319 (300 micromol/L) plus 20 nmol/L Ang II increased L(p) 20-fold (P < .0001). The magnitude of the effect due to PD123319 (300 micromol/L) plus Ang II (20 nmol/L) was approximately twice the summation of effects due to PD123319 (300 micromol/L) alone and Ang II (20 nmol/L) alone. CONCLUSIONS: We conclude that endothelial cell Ang II receptors play an important role in modulating transendothelial fluid flux. Activating the AT1 receptor increases L(p); the AT2 receptor may operate to oppose this action. Pharmacologic manipulation of Ang II receptors may be beneficial during shock states to limit intravascular fluid loss.     

Specific receptors for beta-endorphin on mesangial cells.

beta-Endorphin is an endogenous opioid considered to be a modulator of immune injury. We studied the binding of [125I]beta-endorphin on cultured rat mesangial cells at 4 and 37 degrees C. The results were analyzed by computer program (Ligand). Incubation of rat mesangial cells with unlabeled beta-endorphin displaced [125I]beta-endorphin in a concentration-dependent manner. The binding of [125I]beta-endorphin was not affected by either opiate agonists or antagonists. Saturation studies at 37 degrees C revealed that beta-endorphin binding was time dependent. Binding studies revealed the presence of a single class of high-affinity binding sites with an apparent Kd of 15.3 nM. The number of receptor sites was calculated as 8.48 x 10(5) sites/cell. Mesangial cells exposed to beta-endorphin (10(-6) M) for 48 h showed enhanced incorporation of [3H]thymidine when compared to untreated cells (control, 23,228 +/- 2,778 cpm/well vs. beta-endorphin, 44,887 +/- 4,259 cpm/well; p less than 0.01). Our results show that mesangial cells carry a specific receptor for beta-endorphin which may be linked to proliferation of mesangial cells.     

Transactivation of ErbB1 and ErbB2 receptors by angiotensin II in normal human prostate stromal cells

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized primarily in the stromal compartment of the human prostate and may regulate stromal as well as epithelial cell growth and survival. The primary cognate HB-EGF receptor, ErbB1, has been shown recently to be transactivated by G-protein coupled receptors (GPCRs) through regulated proteolytic cleavage of the membrane-bound, precursor form of HB-EGF. Previous studies have demonstrated that human prostate tissue, especially tissue from benign prostatic hyperplasia (BPH), has high angiotensin converting enzyme activity, and a high density of angiotensin (Ang) receptors in periurethral stromal cells. Because the pressor peptide Ang II signals through GPCRs, we tested the possibility that Ang II could transactivate ErbB1/ErbB2 in human prostate stromal (hPS) cells. METHODS: Primary and early passage hPS cells were used as an in vitro model. Cells were stimulated by recombinant HB-EGF or Ang II and total cell lysates were harvested for immunoprecipitation and Western blot. Cell growth was measured by [(3)H]thymidine incorporation assay and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide (MTT) assay. RESULTS: Ang II receptors AT1 and AT2 were expressed in hPS cells. ErbB1 and ErbB2 receptors were activated by HB-EGF. Furthermore, Ang II was able to transactivate both ErbB1 and ErbB2, and this transactivation activity could be abolished by pretreatment with [Glu-52]-diphtheria toxin/CRM197, a specific inhibitor of HB-EGF bioactivity. Consistent with its transactivation activity, Ang II modestly promoted hPS cell growth and this effect was abolished by pretreatment with the ErbB1 antagonist AG1478. CONCLUSION: These experiments suggest a regulatory role for Ang II in the prostate stroma and implicate the endogenous stromal growth factor HB-EGF as a mediator of Ang II signaling in the prostate.     

Evidence of functional gastric inhibitory polypeptide (GIP) receptors in human insulinoma. Binding of synthetic human GIP 1-31 and activation of adenylate cyclase

Specific gastric inhibitory polypeptide (GIP) receptors were characterized in human benign insulinoma plasma membranes employing [mono-[125I]iodo-Tyr10]-GIP (125I-GIP) as the radioligand. GIP 1-42 inhibited 125I-GIP binding with an IC50 value of 10(-9) M. Scatchard analysis showed two classes of binding sites: a high-affinity site (Kd = 2.23 x 10(-10) M; Bmax = 24 fmol/mg protein) and a low-affinity site (Kd = 8.39 x 10(-9) M; Bmax = 118 fmol/mg protein). A synthetic replicate of human GIP 1-31 inhibited 125I-GIP binding with an IC50 value of 10(-8) M. The GIP binding sites of human insulinoma were coupled to adenylate cyclase stimulation. GIP 1-31 regulated the adenylate cyclase activity to the same extent as GIP 1-42. The concentrations of GIP required for maximal activity ranged from 10(-9) to 10(-8) M for either GIP 1-42 or GIP 1-31. The existence of functional GIP receptors in human insulinoma substantiates our recent reports demonstrating the presence of GIP binding sites in transplantable hamster insulinoma and indicates that GIP could exert a direct control of the beta-cell function in humans through a purely endocrine pathway.     

Angiotensin II in child urinary bladder: functional and autoradiographic studies

OBJECTIVES: To investigate the receptors for angiotensin II (AII, reported to be a potent contractile agent in human urinary bladder), using functional and autoradiographic techniques in child and adult bladder specimens. Materials and methods Bladder specimens were obtained from 61 children (aged 4 months to 12 years) undergoing ureteric reimplantation for vesico-ureteric reflux, and from 10 adults undergoing cystectomy. After overnight storage, the mucosa was removed and isometric contractions obtained from detrusor muscle strips in the presence of phosphoramidon (10 micromol/L). Only one concentration of AII was added to each preparation because of tachyphylaxis. The response to KCl (124 mmol/L) was 43% of that to carbachol (100 micromol/L). Sections of child bladder were radio-labelled with the ligand [125I]Sar1,Ile8-AII and binding sites visualized using emulsion autoradio- graphy. RESULTS: The potency of AII was similar in child and adult detrusor strips, with mean (SEM) pD2 values of 6.9 (1.0) (n = 25) and 6.7 (0.2) (n = 9) respectively, and the maximum responses (to 10 micromol/L AII) rather low (39% and 49%, respectively, P > 0.05), compared with carbachol (100 micromol/L). There were no age- or gender-related differences. Responses to AII in strips from children under 3 years old were antagonized by the AT1 receptor antagonist losartan (1 micromol/L) but not by the AT2 receptor antagonist PD 123319 (1 micromol/L), indicating interaction with the AT1 receptor. Sections of child bladder radiolabelled with [125I]Sar1,Ile8-AII showed moderate specific binding over detrusor muscle and arterioles, with denser specific binding over subepithelial blood vessels. Specific binding was inhibited by co-incubation with losartan (10 micromol/L) but not with PD 123319 (10 micromol/L). CONCLUSION: AII was a weak contractile agent of detrusor strips, with no significant differences in potency between child and adult bladder samples. These data show the presence of functional AT1 but not AT2 receptors in child detrusor smooth muscle.     

Mechanism of increased angiotensin II levels in glomerular mesangial cells cultured in high glucose

Previous studies have shown that glucose increases angiotensin II (AngII) levels in rat glomerular mesangial cells and that AngII mediates the inhibitory effects of high glucose on matrix degradation in these cells. The present study addresses the following questions: (1) What are the mechanisms for the generation of AngII in mesangial cells? (2) What are the effects of glucose on AngII generation by these mechanisms? Experiments employed primary mesangial cells from normal Sprague-Dawley rats. The levels of immunoreactive angiotensinogen (AGT), angiotensin I (AngI), and angiotensin II (AngII) were measured by ELISA. AGT mRNA expression was determined by Northern blot analysis. Incubation of cells for 24 h in high glucose (30 mM) increased AGT levels by 1.5-fold and increased AGT mRNA expression; this was accompanied by a 1.5-fold increment in AngI and 1.7-fold increment in AngII levels. Renin activity (measured as AngI generation in the presence of excess AGT) and ACE levels and activity were not altered by high glucose. In further experiments, the effect of high glucose on formation of Ang peptides from exogenous AngI in mesangial cell extracts was examined using HPLC. Exogenous AngI was converted into various Ang peptides, including AngII, Ang(1-9), Ang(1-7), and Ang(3-8). A significant increase in formation of AngII from AngI was observed in cells incubated in high glucose. In addition, AngII production from exogenous Ang(1-9) in cell extracts was also stimulated by high glucose. These findings demonstrate that glucose increases mesangial AngII levels via an increase in AGT and AngI. In addition, this study provides new information that Ang(1-9) is produced by mesangial cells, can be converted to AngII, and that this conversion is also stimulated under high-glucose conditions.