Mizuo Mifune

Angiotensin II Type 2 Receptors Stimulate Collagen Synthesis in Cultured Vascular Smooth Muscle Cells

Previously, we and others have shown that angiotensin II enhances vascular smooth muscle cell extracellular matrix synthesis via stimulation of the angiotensin II type 1 (AT1) receptor. Recently, expression of the type 2 (AT2) receptor has been confirmed in the adult vasculature, but its role has not yet been fully defined. The aim of the present study was to examine the effects of stimulation of AT2 receptors on collagen synthesis in vascular smooth muscle cells. Retroviral gene transfer was used to supplement adult vascular smooth muscle cells with AT2 receptors to mimic the vasculature in vivo. The treatment of these cells with the AT2 receptor agonist CGP42212A (10−7 mol/L) alone did not cause a significant change in p42/p44 MAP kinase activity but caused a modest (30% to 50%) decrease in protein tyrosine phosphatase activity. Treatment with CGP42112A also caused a dose- and time-dependent increase in both cell-associated and secretory collagen synthesis (148±17% of control at 48 hours, P <0.05), which was completely inhibited by the AT2 receptor antagonist PD123319, unaffected by the AT1 receptor antagonist losartan, and attenuated by treatment with pertussis toxin or G&agr;i antisense oligonucleotides. Interestingly, studies in other cell lines demonstrated that CGP42112A caused similar results in transfected mesangial cells but had essentially opposite effects in fibroblasts (NIH-3T3-AT2). These results suggest that AT2 receptor stimulation can increase collagen synthesis in vascular smooth muscle cells via a G&agr;i-mediated mechanism and provide evidence for heterogeneity in the effects of AT2 receptor stimulation in different tissues.

Insulin-Induced Akt Activation Is Inhibited by Angiotensin II in the Vasculature Through Protein Kinase C-α

Abstract—Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, the specific role of insulin resistance in the etiology of these diseases is poorly understood. Angiotensin (Ang) II is a potent vasculotrophic and vasoconstricting factor. We hypothesize that in vascular smooth muscle cells (VSMCs), Ang II interferes with insulin action by inhibiting Akt, a major signaling molecule implicated in the biological actions of insulin. By immunoblotting with a phospho-specific antibody for Akt, we found that Ang II inhibits insulin-induced Akt phosphorylation in a time- and concentration-dependent manner. The inhibitory effect of Ang II was blocked by a Ang II type 1 receptor antagonist, RNH6270. A protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, also inhibited insulin-induced Akt phosphorylation. PKC inhibitors, including Go6976 (specific for &agr;- and &bgr;-isoforms), blocked the Ang II– and PMA-induced inhibition of Akt phosphorylation by insulin. Moreover, overexpression of PKC-&agr; but not PKC-&bgr; isoform by adenovirus inhibited insulin-induced Akt phosphorylation. By contrast, an epidermal growth factor receptor inhibitor (AG1478), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 598,059), and a p38 MAPK inhibitor (SB 203,580) did not block the Ang II–induced inhibition of Akt phosphorylation. From these data, we conclude that Ang II negatively regulates the insulin signal, Akt, in the vasculature specifically through PKC-&agr; activation, providing an alternative molecular mechanism that may explain the association of hyperinsulinemia with cardiovascular diseases.

Examination of angiotensin II type 1 and type 2 receptor expression in human kidneys by immunohistochemistry.

Recent studies have suggested that both the angiotensin II type 1 (AT1) and type 2 (AT2) receptors may be involved in the control of renal function in rodents. The aim of this study was to examine the distribution of these receptors in normal and diseased human kidneys. Kidney samples were obtained from 21 patients with and without glomerular lesions (3 control kidney samples from patients undergoing nephrectomy, 4 patients with minimal change disease, 6 patients with IgA nephropathy, and 8 patients with membranous glomerulonephritis). AT1 receptor immunohistochemical staining was examined and found to be most prominent in blood vessels, but staining of the tubules and glomeruli was also seen. In the case of the AT2 receptor, mild-moderate immunohistochemical staining was seen in the blood vessels, with weaker staining in the glomeruli. A similar distribution was seen in the patients with glomerulopathy. These results suggest that both AT1 and AT2 receptors are expressed in the normal human kidney, as well as in patients with glomerular disease. The histological distribution of these receptors supports the notion that both receptors may have a physiological role in normal and diseased kidneys in humans.

Prepubertal Treatment with Angiotensin Receptor Blocker Causes Partial Attenuation of Hypertension and Renal Damage in Adult Dahl Salt-Sensitive Rats

Recently, we have shown that treatment of stroke-prone spontaneously hypertensive rats with angiotensin inhibitors for a limited time-window before puberty results in an attenuation of hypertensive nephrosclerosis in later life. The aim of this study was to examine the applicability of this therapeutic paradigm to a low-renin model. Dahl salt-sensitive (Dahl-S) rats were fed a high-salt diet from age 6 weeks. Some rats were treated with the angiotensin receptor blocker (ARB) candesartan cilexetil (2 mg/kg/d) from weaning to puberty (age 3–10 weeks), whereas other rats were treated continuously until overt renal damage was seen (age 3–16 weeks). Dahl-S rats on a high salt diet had increased blood pressure (207 ± 3 vs. 125 ± 2 mm Hg), proteinuria, and glomerular/vascular histological changes. The prepubertal treatment with ARB resulted in a continued suppression of blood pressure (153 ± 2 mm Hg) at 16 weeks. Both proteinuria and renal histological changes were significantly (p < 0.05) attenuated, but not completely prevented by the treatment. No significant differences in plasma renin activity, renin mRNA, or AT1/AT2 mRNA were seen between groups. These results suggest that prepubertal treatment affords sustained renoprotection, even in an animal model with a suppressed renin-angiotensin system, and support the notion that appropriate prepubertal intervention may lead to a partial attenuation in the susceptibility to inherited renal diseases.

Analysis of Gα protein recognition profiles of angiotensin II receptors using chimeric Gα proteins

Abstract Receptors with a heptahelical structure initiate signal transduction by interacting with specific Gα proteins. The aim of this study was to analyze the ability of type 1 (AT1) and type 2 (AT2) angiotensin receptors to recognize the receptor coupling regions of Gα proteins using our previously described technique (Ikezu, T., Okamoto, T., Komatsuzaki, K., Matsui, T., Martyn, J.A.J., Nishimoto, I., 1996. Negative transactivation of cAMP response element by familial Alzheimers mutants of APP. EMBO J. 15, 2468–2475; Komatsuzaki, K., Murayama, Y., Giambarella, U., Ogata, E., Seino, S., Nishimoto, I., 1996. A novel system that reports the G-proteins linked to a given receptor: a study of the type 3 somatostatin receptor. FEBS Lett. 406, 165–170). Chimeric Gαs protein constructs, whose receptor binding regions contained sequences from the four major families of Gα proteins (Gαq, Gαi, Gα12, Gαs), were cotransfected with AT1 or AT2 receptors in COS cells, then stimulated with angiotensin II (Ang II). Changes in cellular cAMP were assayed on cell lysates by enzyme immunoassay. In the case of the Gαq family, cotransfection of AT1 with Gα11/Gαs, Gα14/Gαs, Gα16/Gαs, elicited significant increases in cAMP after agonist stimulation. Confirmatory results were found using an independent [ 35 S]GTPγS binding assay. Further examination using chimeric G proteins for Gα12 proteins and Gαi family proteins provided evidence that the AT1 receptor can recognize sequences from Gα12, Gαi1/i2, Gαz, Gαo, while both receptors interacted with Gαi3. These results provide a Gα protein recognition database for both AT1 and AT2 receptors, which may be important for understanding the full spectrum of cellular responses mediated by the hormone Ang II.

Comparison of effects of low dose of spironolactone and a thiazide diuretic in patients with hypertension treated with an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker.

This study was performed to investigate the additional anti-hypertensive effects and safety of low-dose thiazide diuretic, trichlormethiazide (TCTZ), and a mineralocorticoid receptor blocker, spironolactone (SPI), as add-on therapy in 64 patients whose blood pressure (BP) at office were over 140/90 mmHg, while receiving anti-hypertensive medication including an angiotensin-converting enzyme inhibitor or angiotensin II type I receptor antagonist. After 6 months, we observed a decrease of office and home BP. Moreover, urinary albumin excretion (UAE) was reduced in SPI-treated group, but not in the TCTZ-treated group. No significant change in serum potassium, lipids, glucose, or uric acid was observed. In conclusion, low-dose thiazide diuretic or SPI provided a significant additional anti-hypertensive effect in patients in whom hypertension was not controlled by medication, and SPI-reduced UAE.