Brush Border Angiotensin Converting Enzyme Research Articles

The Serum and Glucocorticoid Inducible Kinases SGK1-3 Stimulate the Neutral Amino Acid Transporter SLC6A19

The neutral amino acid transporter SLC6A19 (B⁰AT1) plays a decisive role in transport of neutral amino acids in the kidney and intestine. Recently, mutations in SLC6A19 were identified that result in severe neutral aminoaciduria known as Hartnup disorder. SLC6A19 expression and function is controlled by the brush-border angiotensin-converting enzyme 2 (ACE2). Beyond that the mechanisms regulating SLC6A19 function are unknown. The SLC6A19 sequence contains a conserved putative phosphorylation site for the serum and glucocorticoid inducible kinase isoforms SGK1-3, kinases known to regulate a variety of channels and transporters. The present study explored the role of SGK1-3 in the regulation of SLC6A19. As shown by two-electrode voltage clamp in the Xenopus oocyte expression system, leucine-induced currents in SLC6A19 expressing oocytes were activated by the protein kinases SGK1-3. The putative phosphorylation site on the transporter is not essential for SLC6A19 regulation by the kinases. As determined by quantitative immunoassay and electrophysiology, the kinases increase SLC6A19 currents by increasing the cell surface expression of the protein without altering the affinity of the carrier. Following inhibition of carrier insertion into the cell membrane by treatment with brefeldin A (BFA), the leucine-induced current declined significantly slower in Xenopus oocytes expressing SLC6A19 together with SGK1 than in oocytes expressing SLC6A19 alone, a finding pointing to SGK-mediated transporter stabilization in the plasma membrane. Coexpression of ACE2 markedly increased leucine-induced currents in SLC6A19 expressing oocytes that were further enhanced by SGK1-3 kinases. In conclusion, SGK isoforms are novel potent stimulators of SLC6A19 and may thus participate in the regulation of neutral amino acid transport in vivo.

Endothelin-1 upregulation in the kidney of uninephrectomized spontaneously hypertensive rats and its modification by the angiotensin-converting enzyme inhibitor quinapril.

Endothelin (ET-1) is a potent vasoconstrictor that plays an important role in the control of renal circulation and tubular function. The contribution of this peptide to the pathogenesis of systemic hypertension and renal failure remains largely undefined. In spontaneously hypertensive rats (SHR) uninephrectomized at 20 weeks of age (UNX-SHR) and followed until 45 weeks of age, we determined ET-1 gene expression in renal tissue by reverse transcription-polymerase chain reaction and its localization by in situ hybridization in paraffin-embedded kidney sections. Age-matched SHR and normotensive Wistar-Kyoto (WKY) rats were chosen as controls. At the end of the follow-up, UNX-SHR had high systolic blood pressure, intense proteinuria, mesangial expansion, focal and segmental glomerular sclerosis, and tubulointerstitial lesions. In relation to WKY and SHR, UNX-SHR exhibited an increase in ET-1 gene expression in renal cortex and medulla. By in situ hybridization and immunoperoxidase staining, an overexpression of ET-1 gene and protein were seen in mesangial and glomerular epithelial cells and in some proximal tubules and vessels. Angiotensin-converting enzyme (ACE) activity was significantly increased in the renal brush border. Since in mesangial cells, angiotensin II induces ET-1 synthesis, a group of UNX-SHR received the ACE inhibitor quinapril from the time of UNX. These animals had a decrease in blood pressure, proteinuria, and serum and brush border ACE activity and in the expression and synthesis of ET-1 in all renal areas. On the whole, these data show that UNX-SHR have an upregulation of ET-1 gene and protein in several structures of the kidney compared with SHR and WKY rats. Quinapril diminished ACE activity and ET-1 expression and synthesis coincidentally with an improvement in proteinuria and morphological lesions. The beneficial effects of ACE inhibitors may be due to the diminution of both angiotensin II and ET-1 generation.

Effects of triiodothyronine and dexamethasone on plasma and tissue angiotensin converting enzyme in the rat.

In order to identify tissue specific regulation of angiotensin converting enzyme (ACE), the effects of dexamethasone (0.04 mg sc per day for 7 days) and triiodothyronine (T3) (0.5 mg/kg sc per day for 10 days) on ACE activity were investigated in different tissues in male Wistar rats. ACE activity was measured by fluorimetry in the plasma, heart, lung and kidney. In the kidney, ACE activity was measured in the medulla, cortex and brush border of proximal tubular cells and 3H-ramiprilat binding was used to characterise the changes in brush border ACE activity. Dexamethasone elicited a significant increase in lung ACE activity and a significant decrease in plasma ACE activity, but did not alter enzyme activity in the other tissues studied. T3 produced a significant decrease in lung ACE activity and an increase in ACE activity in the plasma and heart. In the kidney, ACE activity was not modified in the medulla whereas in the cortex and brush border ACE activity was doubled. This increase in ACE activity corresponded to a similar increase in the maximum number of binding sites of 3H-ramiprilat, suggesting that the increase in activity corresponded to an increase in the ACE level. The increased heart and kidney ACE activity in response to T3 may contribute to the cardiovascular effects of thyroid hormones through increased local angiotensin II generation. These results show that under dexamethasone or T3, ACE activity can vary from one tissue to another, suggesting that the ACE regulatory mechanism acts differently in each tissue.

Effects of dietary protein and uninephrectomy on renal angiotensin converting enzyme activity in the rat

This study examines the effects of dietary protein and of uninephrectomy on angiotensin converting enzyme (ACE) in the normotensive rat, with particular regard to the kidney. Male Wistar Kyoto rats were fed isocaloric diets containing 5, 16 or 50% protein for three weeks. Other groups of rats were subjected to either left unilateral nephrectomy or sham operations, and the rats were killed eight days after surgery. ACE activity was measured in the kidney medulla, cortex, proximal tubule brush border membrane and in the plasma, heart and lung. Renal cortex and brush border ACE activity increased in parallel with protein intake, whereas plasma and lung ACE activity decreased; heart and kidney medulla ACE activity did not vary significantly. Uninephrectomy also led to a high increase in brush border ACE activity in the contralateral kidney, with no effect in the renal medulla or in the other tissues. The increase in ACE activity in the brush border membrane corresponded to a similar increase in the maximum number of binding sites of 3H-ramiprilat. This suggested that the increase in ACE activity corresponded to an increase in ACE concentration. The increase in renal tubular ACE activity could result in higher angiotensin II levels, and could consequently play a role in the modification of sodium reabsorption and cellular growth which occurs in the proximal tubule in these experimental models.

Effects of one-hour and one-week treatment with ramipril on plasma and renal brush border angiotensin converting enzyme in the rat

Prolonged treatment with an angiotensin converting enzyme inhibitor produces an induction of plasma angiotensin converting enzyme. Induction of angiotensin converting enzyme in tissues during prolonged treatment with an angiotensin converting enzyme inhibitor is less well documented. We compared the effects of 1 h and 1 week treatment with ramipril (0.1, 0.3, 1 mg/kg) on angiotensin converting enzyme activity in the plasma, renal cortex and renal brush border membrane of Wistar rats. As an increase in activity could be masked by the inhibition due to the presence of ramiprilat which is the active form of ramipril, we eliminated the ramiprilat present in renal cortex homogenates with EGTA during brush border preparation. The 1-h treatment with ramipril induced a dose-dependent inhibition of plasma and renal cortex angiotensin converting enzyme activity. The 1-week treatment with ramipril produced an increase in plasma angiotensin converting enzyme activity, whereas renal cortex angiotensin converting enzyme activity decreased. The decrease in angiotensin converting enzyme activity persisted in the brush border membrane after elimination of residual ramiprilat with EGTA. Our results show that prolonged ramipril treatment produces opposite responses in plasma and renal cortex angiotensin converting enzyme activity, suggesting that plasma and epithelial angiotensin converting enzymes are subject to specific local regulatory factors.

Human intestinal brush border angiotensin-converting enzyme activity and its inhibition by antihypertensive Ramipril

Angiotensin-converting enzyme (ACE) has been identified as a prominent brush border membrane-bound enzyme of human jejunum. In this study, we purified brush border membrane vesicles enriched in ACE, and characterized the ACE with regard to (a) its stability in the membrane, (b) substrate hydrolysis kinetics compared with pulmonary endothelial ACE, and (c) pharmacologic interaction with Ramipril. These investigations resulted in the following findings. The uninhibited enzyme is stable in native membranes in vitro, with a half-life of 195 ± 7 h. Kinetic analysis of ACE hydrolysis activity revealed the presence of a single enzyme species, which yielded a high Vmax and displayed a Km similar to purified ACE from lung endothelium. Brush border ACE was inhibited by Ramipril, one of the most specific and potent orally administered ACE inhibitors indicated for hypertension. We determined the brush border ACE value of IC50 = 3 × 10−9 M Ramipril-diacid, which is the same value for serum and lung ACE. Brush border ACE remains 100% inhibited by 10 μM Ramipril during at least 8 days in vitro. The data indicate that ACE is a prominent jejunal brush border enzyme that behaves pharmacologically and kinetically like its peripheral circulation counterpart. This study suggests that high doses of orally administered ACE inhibitors may affect intestinal epithelial function.

Ramipril inhibition of rabbit ( Oryctolagus cuniculus) small intestinal brush border membrane angiotensin converting enzyme

1. 1. Rabbit small intestinal brush border membranes possessed prominent angiotensin converting enzyme (ACE) activity. 2. 2. Intestinal ACE was located on the lumen surface, as verified by ACE co-enrichment with brush border membrane marker enzymes. 3. 3. Hydrolysis kinetics of rabbit intestinal ACE were comparable to the lung, utilizing the substrate (N-[3-(2- furyl) acryloyl]- l- phenylalanylglycylglycine ; the V max = 543 ± 51 μmol/min/g and K m = 0.62 ± 0.09 mmol/l. 4. 4. Intestinal brush border ACE activity was strongly inhibited by the antihypertensive drug Ramipril, which yielded an ic 50 value of 5 nmol/l; the ACE activity remained completely inhibited during 15 days after a single dose of 10 μmol/l Ramipril.

Contribution of renal angiotensin converting enzyme (ACE) to blood pressure regulation: possible role of brush border ACE.

The role of renal angiotensin converting enzyme (ACE) in blood pressure regulation is not well understood. In our studies, both acute and chronic treatment of hypertensive rats SHR and SHRSP with ACE inhibitors Enalapril and SA446 had a blood pressure lowering effect that coincided with an inhibition of renal cortical and aortic ACE, but not plasma ACE. Further, ACE activities in the renal cortex and aorta were found to increase with aging of the SHRSP, therefore concomitantly with hypertension development. In the kidney, brush border membranes (BBM) contained abundant ACE. We found that the activities of ACE in the renal cortex closely correlated to the activities in isolated BBM, in Wistar Kyoto rats and in the SHRSP. Thus, renal cortical ACE activity and blood pressure correlated in cases of ACE inhibition and hypertension development. Since the ACE activity in the renal cortex appeared to reflect the enzyme activity in BBM, the brush border ACE may have to be taken into account, in view of the relationship between renal ACE and blood pressure.