Uptake of glucose analogs reflects the rate of contraction of cultured myocytes

Abstract

The present study demonstrates that: a) adenosine and R-N6-(2-phenylisopropyl)-adenosine (R-PIA, A1 and A3 adenosine receptor agonist) inhibited [3H]deoxyglucose uptake or [3H]3-O-methyl-D-glucose uptake; b) sugar uptake reflects the rate of contraction in cardiac cultures; c) [3H]deoxyglucose uptake or [3H]3-O-methyl-D-glucose uptake are useful quantitative probes for beating rate evaluation. A 25-40% decrease in [3H]deoxyglucose uptake (p < 0.01) was obtained following 13-21 min treatment with 100 microM adenosine together with 1 microM dipyridamole or with 10 microM R-PIA, which inhibited spontaneous contractions. Adenosine (10 microM) attenuated spontaneous beating rate and inhibited approximately 55% of the [3H]deoxyglucose uptake following 22 h treatment (p < 0.01). 1 microM R-PIA also attenuated beating rate following either a short (1 min) or long (24 h) application and decreased [3H]deoxyglucose uptake by 20-30% (p < 0.01) during 0.5-24 h of treatment. A 157 +/- 9% and 205 +/- 11% increase (p < 0.01) in [3H]deoxyglucose uptake was obtained at 27 and 37 degrees C, respectively, compared with the uptake at 17 degrees C, which completely inhibited spontaneous contractions. Similar results [33 +/- 6% (p < 0.01) and 21 +/- 8% (p < 0.05) inhibition in [3H]deoxyglucose uptake] were obtained following 2 and 22 h of carbamylcholine treatment, respectively. This treatment also reduced spontaneous contractions. [3H] 3-O-Methyl-D-glucose uptake also decreased by 31 +/- 12% (p < 0.05) as a result of the arrest of contractions by adenosine. Elevations of 90 +/- 13% and 34 +/- 11% (p < 0.01) in [3H]deoxyglucose uptake were obtained following treatment with isoprenaline after 2 and 22 h application, respectively. It is concluded that adenosine and R-PIA inhibited [3H]deoxyglucose uptake or [3H] 3-O-methyl-D-glucose uptake in rat heart culture and that there is a linkage between the rate of cardiac contractions in culture and sugar uptake.

Fig. 1:

[³H]Deoxyglucose ([³H]DG) uptake as a function of time. Five day-old rat cardiocytes were incubated with 1 μCi/ml [³H]deoxyglucose at 37°C for the indicated time. The cells were then washed, solubilized and radioactivity was measured. Each point is the mean (± S.E.) of five determinations of a representative experiment out of three experiments that gave similar results.

Fig. 2:

Dose response of glucose on [³H]deoxyglucose ([³H]DG) uptake. A: Five day-old cardiocytes were subjected to the indicated glucose concentrations and were incubated in [³H]deoxyglucose uptake medium at 37°C for 15 min. B: Lineweaver-Burke representation of [³H]deoxyglucose uptake into the cardiocytes of Fig. A. Each point is the mean (± S.E.) of five determinations of a representative experiment out of three experiments.

Fig. 3:

The effect of adenosine or R-PIA on beating rate. Five day-old cardiocytes were treated with the indicated concentration of adenosine (ade) and dipyridamole (dip), or R-N⁶-(2-phenylisopropyl)-adenosine (R-PIA). Beating rate was measured by video optic system. The experiments were performed for 20 min (A) or for 24 h (B). Each point is the mean (° S.E.) of five determinations of a representative experiment out of three-four experiments. A significant difference (p<0.01) was detected between the various times of exposure to 0.1 μM R-PIA and the control.

Fig. 4:

The effect of adenosine or R-PIA on [³H]deoxyglucose ([³H]DG) uptake. Five day-old cardiocytes were treated with 100 μM adenosine and 1 μM dipyridamole (ade+dip), or 10 μM R-PIA for the indicated times. [³H]-Deoxyglucose uptake was then measured. Each point is the mean (± S.E.) of five determinations of a representative experiment out of three experiments. Significant differences (p<0.01) were obtained between [³H]deoxyglucose uptake in the control and ade+dip or R-PIA treatments (*). No significant differences were obtained at the time of 9 min treatment between the control and the ade+dip. No significant differences were obtained between the values of ade+dip and the corresponding R-PIA treatment, between 9–21 min of [³H]deoxyglucose uptake (unpaired t-test).

Fig. 5:

Time response of R-PIA on [³H]deoxvglucose uptake. Five day-old cardiocytes were subjected to 1 μM R-PIA for the indicated times. [³H]-Deoxyglucose uptake was then performed. Each point is the mean (± S.E.) of five determinations in percentage of control of a representative experiment out of four experiments. Significant differences (*p<0.01) were obtained between the control and treated cells.

Fig. 6:

The effect of adenosine, carbamylcholine and isoprenaline on [³H]deoxyglucose uptake. Five day-old cardiocytes were treated with 10 μM adenosine (ade), 100 μM carbamylcholine (carb) or 10 μM isoprenaline (iso) for 2 or 22 h. [³H]Deoxyglucose uptake was then measured. Each point is the mean (± S.E.) of five determinations in percentage of control of a representative experiment out of three experiments. Values were significantly different (*p<0.01) according to ANOVA-Dunnett’s test.

Fig. 7:

[³H]3-O-Methyl-D-glucose ([³H]3-OMG) uptake. A: Five day-old cardiocytes were incubated with [³H]3-OMG for the indicated time. Each point is the mean (± S.E.) of duplicate determinations of a representative experiment out of four experiments. B: Effect of adenosine on [³H]3-OMG uptake. Four day-old cardiocytes were treated with 100 μM adenosine and 1 μM dipyridamole for 1 h. [³H]3-OMG uptake was then performed for an initial 20 sec. Each point is the mean (± S.E.) of duplicate determinations of a representative experiment out of four experiments. Significant differences (*p<0.05) were obtained between the control (black) and the treated cells (stripes).