Characteristic changes in coronary artery at the early hyperglycaemic stage in a rat type 2 diabetes model and the effects of pravastatin

Abstract

Background and purpose: Diabetes is a risk factor for the development of coronary artery disease but it is not known whether the functions of endothelium-derived nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in coronary arteries are altered in the early stage of diabetes. Such alterations and the effects of pravastatin were examined in left anterior descending coronary arteries (LAD) from Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycaemic stage [vs. non-diabetic Long-Evans Tokushima Otsuka (LETO) rats].

Experimental approach: Isometric tension, membrane potential and superoxide production were measured, as were protein expression of NAD(P)H oxidase components and endothelial NO synthase (eNOS).

Key results: Superoxide production and the protein expressions of both the nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase components and eNOS were increased in OLETF rats. These changes were normalized by pravastatin administration. Not only acetylcholine (ACh)-induced endothelial NO production but also functions of endothelium-derived NO [from (i) the absolute tension induced by epithio-thromboxane A(2) (STA(2)) or high K(+); (ii) enhancement of the STA(2)-contraction by a nitric oxide synthase (NOS) inhibitor; and (iii) the ACh-induced endothelium-dependent relaxation of high K(+)-induced contraction] or EDHF [from (iv) ACh-induced endothelium-dependent smooth muscle cell hyperpolarization and relaxation in the presence of a NOS inhibitor] were similar between LETO and OLETF rats [whether or not the latter were pravastatin-treated or -untreated].

Conclusions and implications: Under conditions of increased vascular superoxide production, endothelial function is retained in LAD in OLETF rats at the early hyperglycaemic stage, partly due to enhanced endothelial NOS protein expression. Inhibition of superoxide production may contribute to the beneficial vascular effects of pravastatin.

Figure 1

Superoxide production in left anterior descending coronary artery. (A) Chemiluminescence intensity of the superoxide-sensitive dye L-012 and the effect of N^ω-nitro-L-arginine methyl ester (L-NAME) (3 mM). LETO Long-Evans Tokushima Otsuka rat; OLETF Otsuka Long-Evans Tokushima Fatty rat; OLETF + PRV, pravastatin-treated OLETF rat. Each column is the mean of data from four different preparations (from four different animals) with SEM. *P < 0.05 versus LETO. #P < 0.05 versus OLETF. ††P < 0.01 versus L-NAME (−). (Ba) Fluorescence intensity of the superoxide-sensitive dye dihydroethidium. SMC, smooth muscle cell; EC, endothelial cell. (Bb) Effect of L-NAME. Each column is the mean of data from five different sections (each from a different animal) with SEM. *P < 0.05 versus LETO. #P < 0.05 versus OLETF. ††P < 0.01 versus L-NAME (−).

Figure 3

Expressions of nicotinamide adenine dinucteotide (phosphate) [NAD(P)H] oxidase components (gp91^phox and p47^phox) and Cu/Zn-SOD proteins, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in left anterior descending coronary artery. (A) Protein expressions of gp91^phox (a) and p47^phox (b) were measured by western blot analysis. LETO Long-Evans Tokushima Otsuka rat; OLETF Otsuka Long-Evans Tokushima Fatty rat; OLETF + PRV, pravastatin-treated OLETF rat. (B) Expression of Cu/Zn- superoxide dismutase (SOD). (C) NADPH oxidase activity was measured in homogenized preparations. Each column is the mean of data from four different preparations (each from a different animal) with SEM. *P < 0.05, **P < 0.01 versus LETO. #P < 0.05 versus OLETF.

Figure 2

Immunostaining for p22^phox, endothelial NO synthase (eNOS) and angiotensin type 1 receptor (AT₁R) in left anterior descending coronary artery. Immunofluorescence staining against p22^phox (a), eNOS (b) and AT₁R (c) in preparations from a Long-Evans Tokushima Otsuka (LETO) rat (left column), an Otsuka Long-Evans Tokushima Fatty (OLETF) rat (middle column) and a pravastatin-treated OLETF (OLETF + PRV) rat (right column). EC, endothelial cell; IEL, internal elastic lamina. Arrowheads indicate endothelial cells. Similar observations were made in other sections obtained from five preparations, each from a different animal, in each group. (a4-c4) Summary of data from each group. Data are shown as mean ± SEM. *P < 0.05 versus LETO. *P < 0.05 versus LETO. #P < 0.05 versus OLETF.

Figure 4

ACh-induced nitric oxide (NO) production in endothelial cells in left anterior descending coronary artery. Intracellular concentrations of NO ([NO]_i), as estimated from fluorescence-intensity changes in the NO-sensitive dye DAF-2. [NO]_i is expressed as the ratio of F (fluorescence intensity at a given time after ACh-application) to F₀ (just before ACh-application). ACh (10 µM) was applied as indicated by the bar. Data are the mean from four different preparations (each from a different animal) with SEM. No significant differences were observed among the three groups of rats. LETO, Long-Evans Tokushima Otsuka; OLETF, Otsuka Long-Evans Tokushima Fatty, OLETF + PRV, pravastatin-treated OLETF.

Figure 5

Morphometric characteristics and the effects of high-K⁺ and 9,11-epithio-11,12-methano-thromboxane A₂ (STA₂) on mechanical activities. (A) Summaries of left anterior descending coronary artery (LAD) wall thickness (a1) and number of smooth muscle cells across the wall (a2) in Long-Evans Tokushima Otsuka (LETO) rats and Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Summary of data obtained for absolute tension induced by 80 mM K⁺ (B) and effect of the nitric oxide synthase inhibitor N^ω-nitro-L-arginine (L-NNA) on STA₂ (30 nM)-induced contractions in endothelium-intact strips of LAD (C). LETO: LETO rats; OLETF: OLETF rats; OLETF + PRV: pravastatin-treated OLETF rats. In (C), the maximum amplitude of contraction induced by 80 mM K⁺ before application of L-NNA was normalized as a relative tension of 1.0. Data are shown as mean ± SEM for four strips, each from a different animal, in each group. **P < 0.01 versus corresponding L-NNA (−).

Figure 6

ACh-induced relaxation of the contractions induced by high K⁺ and 9,11-epithio-11,12-methano-thromboxane A₂ (STA₂). Concentration-response relationships for ACh-induced relaxations in endothelium-intact strips of left anterior descending coronary artery from Long-Evans Tokushima Otsuka (LETO) rats, Otsuka Long-Evans Tokushima Fatty (OLETF) rats and pravastatin-treated OLETF rats (OLETF + PRV) in the presence of the cyclooxygenase inhibitor diclofenac, (A) on the contraction induced by 40 mM K⁺, (B) on the contraction induced by 30 nM STA₂, (C) on the contraction induced by 5 nM STA₂ in the presence of N^ω-nitro-L-arginine (0.1 mM). Means of 3–5 strips, each from a different animal, in each group, with SEM shown by vertical bar.

Figure 7

Effects of N^ω-nitro-L-arginine (L-NNA) and blockers of Ca²⁺-activated K⁺-channels on ACh-induced smooth muscle cell hyperpolarization. The experiments were performed in the presence of the cyclooxygenase inhibitor diclofenac. Representative tracings of the effects of L-NNA (0.1 mM), in the presence or absence of charybdotoxin (0.1 µM) + apamin (0.1 µM), on ACh-induced smooth muscle cell hyperpolarization in left anterior descending coronary artery from Long-Evans Tokushima Otsuka rat (A) and Otsuka Long-Evans Tokushima Fatty rat (B).