Reduced ability to release adenosine by diabetic rat cardiac fibroblasts due to altered expression of nucleoside transporters

Abstract

Adenosine produced by cardiac cells is known to attenuate the proliferation of cardiac fibroblasts (CFs), inhibit collagen synthesis, and protect the myocardium against ischaemic and reperfusion injury. Diabetic patients' hearts exhibit ventricular hypertrophy and demonstrate reduced tolerance to hypoxia or ischaemia. In this study, we characterize the effects of glucose and insulin on processes that determine the release of adenosine from CFs. We showed that during ATP depletion, rat CFs cultured in the absence of insulin release significantly less adenosine compared to cells grown in the presence of insulin. Moreover, under both conditions the quantity of released adenosine depends on glucose concentration. We demonstrate that this is due to altered expression of nucleoside transporters. High glucose (25 mm) induced 85% decrease in nucleoside transporter ENT1 mRNA levels. Decrease of the insulin level below 10(-11) m resulted in over 3-fold increase in the nucleoside transporter CNT2 mRNA content. Measurements of adenosine transport in CFs cultured in the presence of 5 mm glucose and 10 nm insulin showed that the bidirectional equilibrative adenosine transport accounted for 70% of the overall adenosine uptake. However, cells grown in the presence of high glucose (25 mm) demonstrated 65% decrease of the bidirectional equilibrative adenosine transport. Experiments on CFs cultured in the absence of insulin showed that the unidirectional Na(+)-dependent adenosine uptake rose in these cells more than 4-fold. These results indicate that the development of diabetes may result in an increased uptake of interstitial adenosine by CFs, and reduction of the ability of these cells to release adenosine during ATP deprivation.

Figure 1. Outflow of purine nucleosides and bases from cardiac fibroblasts during ATP depletion

A, the level of ATP in cultured rat cardiac fibroblasts exposed to glycolytic and oxidative inhibitors. At ∼70% confluence the culture medium was changed to Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, and 5 mm glucose and 10 nm insulin (5 mm gluc + Ins), or 5 mm glucose and no insulin (5 mm gluc), or 25 mm glucose and no insulin (25 mm gluc) or 25 mm glucose and 10 nm insulin (25 mm gluc + Ins). No insulin refers to a concentration of 10⁻¹¹m. On the third day, the cells were labelled with [8-¹⁴C]adenine, washed, suspended in glucose-free DMEM and ATP depletion was induced by addition of 2-deoxyglucose (10 mm) and antimycin A (0.1 μg ml⁻¹). The data represent the mean ± s.d. from four independent experiments. *P < 0.05. B, the level of purine nucleosides in the cell culture medium during ATP depletion was determined by thin-layer chromatography as described in the Methods. The concentrations of glucose and insulin at which the cells were cultured before induction of ATP depletion were as described in A and are indicated on top of each graph. The data represent the mean ± s.d. from four independent experiments. *P < 0.05 versus (25 mm glucose); #P < 0.05 versus (5 mm glucose); §P < 0.05 versus (5 mm glucose + 10 nm insulin); †P < 0.05 versus (25 mm glucose + 10 nm insulin).

Figure 2. The effect of nitrobenzylthioinosine (NBTI) on adenosine outflow from cardiac fibroblasts during ATP depletion

The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 5 mm glucose and no insulin (A), or 5 mm glucose and 10 nm insulin (B), or 25 mm glucose and no insulin (C), or 25 mm glucose and 10 nm insulin (D). No insulin refers to a concentration of 10⁻¹¹m. Cells were labelled with [8-¹⁴C]adenine, and deprived of ATP as described in the legend to Fig. 1, except for the presence (•) or absence (○) of 1 μm NBTI in the glucose-free DMEM. The data represent the mean ± s.d. from three independent experiments. *P < 0.05 versus no NBTI.

Figure 3. The expression level of nucleoside transporters (NTs) in rat cardiac fibroblasts

The levels of NT mRNAs were determined in cardiac fibroblasts isolated from normal rats before (not cultured) and after 6 days of culture (cultured) in Dulbecco's modified Eagle's medium (DMEM) containing 5 mm glucose and 10 nm insulin. The expression level of each NT was normalized to β-actin and is expressed as NT/β-actin ratio. The data represent the mean ± s.d. from five independent experiments.

Figure 4. Diabetes-induced changes in expression of nucleoside transporters (NTs) in cardiac fibroblasts

The levels of NT mRNAs were determined in cardiac fibroblasts isolated from STZ-induced diabetic and normal rats. The expression level of each NT was normalized to β-actin and is expressed as NT/β-actin ratio. The data represent the mean ± s.d. from four independent experiments. *P < 0.05 versus normal.

Figure 5. Differential effect of glucose and insulin on expression levels of nucleoside transporters in cultured cardiac fibroblasts

Cardiac fibroblasts were isolated from normal rat and cultured to reach 60% confluence (sixth day) as described in the Methods. The culture medium was changed to Dulbecco's modified Eagle's medium containing 10% fetal bovine albumin and 5 mm glucose and 10 nm insulin (5 mm gluc + Ins), or 5 mm glucose and no insulin (5 mm gluc), or 25 mm glucose and no insulin (25 mm gluc), or 25 mm glucose and 10 nm insulin (25 mm gluc + Ins). No insulin refers to a concentration of 10⁻¹¹m. On the third day of culture, the cells were harvested and the NT mRNAs levels were determined. The expression level of each NT was normalized to β-actin and is expressed as NT/β-actin ratio. The data represent the mean ± s.d. from five independent experiments. *P < 0.05.

Figure 6. Differential effects of glucose and insulin on adenosine transport in cultured cardiac fibroblasts

Uptake of 10 μm adenosine was measured in cardiac fibroblasts cultured in Dulbecco's modified Eagle's medium containing10% fetal bovine albumin and 5 mm glucose and 10 nm insulin (5 mm gluc +Ins), or 5 mm glucose and no insulin (5 mm gluc), or 25 mm glucose and no insulin (25 mm gluc), or 25 mm glucose and 10 nm insulin (25 mm gluc + Ins). No insulin refers to a concentration of 10⁻¹¹m. The data represent the mean ± s.d. from four independent experiments. *P < 0.05.