Vascular aldosterone production at the pre-diabetic stage of young Otsuka Long-Evans Tokushima Fatty (OLETF) rats, compared with Long-Evans Tokushima Otsuka (LETO) rats

Abstract

We examined the ability of aortic smooth muscle cells (AoSMC) prepared from spontaneously diabetic rats to produce aldosterone (Aldo) and the regulatory mechanism that controls their Aldo production. AoSMC of 6 week-old Long-Evans Tokushima Otsuka (LETO: the control group) and 6 week-old Otsuka Long-Evans Tokushima Fatty (OLETF: the type 2 diabetes group) rats were used in the present experiments. CYP11B2 (Aldo synthetase) mRNA expression was detected in both the LETO and OLETF AoSMC. Basal Aldo production was significantly greater (4-5 fold higher) in the OLETF AoSMC culture medium than in the LETO AoSMC culture medium. When AoSMC were co-incubated with high-density lipoproteins (HDL), supplying cholesterol as a substrate for steroidogenesis in rats, angiotensin II (AII) significantly increased greater Aldo production in the OLETF AoSMC than in the LETO AoSMC. The present data suggested that future onset of diabetic vascular dysfunction is partly caused by excess Aldo production by AoSMC in young OLETF rats. Concomitant stimulation by HDL and AII resulted in elevated Aldo production in the OLETF and the LETO AoSMC, and also demonstrated that AII-induced Aldo production is greatly enhanced by HDL in OLETF, rather than in LETO. In conclusion, our data clearly demonstrated that Aldo production in the OLETF AoSMC was significantly higher than in the LETO AoSMC, suggesting possible future onset of vascular dysfunction in diabetes, induced by local Aldo production in the AoSMC.

Figure 1

RT-PCR analysis of the CYP11B2 mRNA expression of AoSMC prepared from OLETF diabetic rats and control LETO rats.The mRNA of the indicated molecules was reverse-transcribed, and the resultant cDNA was amplified with RT-PCR. Ø × 174 fragments that had been digested with Hae III were added to the right lane as a molecular weight marker.

Figure 2

Pregnenolone production by rat AoSMC. The cells were cultured with/without (Bu)2cAMP for 2 h and then incubated with 30 μM trilostane, which is an antagonist of 3βHSD. Pregnenolone concentrations were assessed using a specific radioimmunoassay. Results are expressed as the mean ± S.E.M. * p < 0.05 vs. control (no additives); ** p < 0.001 vs. control of LETO (no additives). ^a p < 0.01 vs. +(Bu)2cAMP of LETO, ^b p < 0.01 vs. control of OLETF (no additives), ^c p < 0.001 vs. +(Bu)2cAMP of LETO.

Figure 3

Effects of angiotensin II on aldosterone synthesis. The aldosterone production of rat AoSMC that had been cultured with or without angiotensin II for 2 h was determined as described in the text. The vertical bars represent the S.E.M. of the mean. * p < 0.01 vs. control (no additives); ** p < 0.001 vs. control (no additives). ^a p < 0.001 vs. control of LETO (no additives & +10⁻⁹ M AII ), ^b p < 0.01 vs. +10⁻¹¹ M AII of LETO, ^c p < 0.001 vs. +10⁻⁹ M AII of LETO, ^d p < 0.05 vs. +10⁻⁷ M AII of LETO.

Figure 4

Effect of losartan on basal aldosterone production by rat AoSMC. Rat AoSMC were incubated with or without 10⁻⁵ M losartan for 2 h, and then the culture media had their aldosterone concentrations determined, as described in the text. Data are presented as the mean of three different experiments. Each experiment was performed in triplicate. ^a p < 0.001 vs. control of LETO, * p < 0.001 vs. control of OLETF (none).

Figure 5

Effect of angiotensin II in the presence of 20 mg cholesterol/dL of HDL on aldosterone production by rat AoSMC. Data are expressed as percentage increases compared with the control. Data are presented as mean ± S.E.M. values of three different experiments. Each experiment was performed in triplicate, and aldosterone concentrations were determined using a radioimmunoassay. * p < 0.001 vs. LETO.

Figure 6

Effect of angiotensin II on AT1R protein expression. Rat AoSMC were incubated with “(+)” or without “(−)” 10⁻⁷ M angiotensin II for 2, 4 and 6 h. The pelleted cells were soon solubilized and subjected to SDS-PAGE using a linear gradient of 10%–20% acrylamide gel. All proteins were electrophoretically transferred to nitrocellulose membranes and analyzed using standard immunoblotting procedures. Molecular weight (MW) markers were run in the right lane.

Figure 7

Effect of angiotensin I on AT1R protein expression.Rat AoSMC were incubated with 10⁻⁷ M angiotensin I for 2, 4 and 6 h. The pelleted cells were soon solubilized and subjected to SDS-PAGE using a linear gradient of 10%–20% acrylamide gel. All proteins were electrophoretically transferred to nitrocellulose membranes and analyzed using standard immunoblotting procedures. Molecular weight (MW) markers were run in the right lane.