Abstract
The effect of forskolin, an activator of adenylate cyclase, was investigated on glucose transport in human erythrocytes. Forskolin was found to be a potent inhibitor of 3-O-methylglucose (3-O-MG) influx in human erythrocytes. The inhibition of 3-O-MG transport was instantaneous and reversible. The inhibitory effect of forskolin was concentration-dependent, having an IC50 value of 7.5 microM. Forskolin caused a decrease in Vmax of carrier-mediated 3-O-MG transport from 35.32 to 1.56 mumol/ml of cell X min in the presence of 50 microM forskolin. Inhibition of influx was not reversed at high concentrations of 3-O-MG. In addition, forskolin inhibited the influx of other carbohydrates including galactose, ribose, and fructose. In contrast, forskolin was without effect on adenosine transport. To unravel the underlying mechanism responsible for the inhibitory action of forskolin, the possible involvement of cyclic AMP in controlling glucose transport was examined. Erythrocytes treated with 50 microM forskolin exhibited an increase in cyclic AMP content from the basal levels of 258 fmol/ml of cell to 334 fmol/ml of cell within 10 s after forskolin exposure. However, erythrocytes in which cyclic AMP was allowed to accumulate in excess of 10,000 times the basal level, by means of preincubation with exogenous cyclic AMP, displayed 3-O-MG transport indistinguishable from that of cyclic AMP-poor control cells. In view of the finding that cyclic AMP plays no discernible role in the erythrocyte 3-O-MG transport, it is suggested that the forskolin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity. Moreover, forskolin appears to directly inactivate the 3-O-MG transport system since glucose-sensitive cytochalasin B binding to erythrocyte membranes is virtually abolished by 50 microM forskolin.
Highlights
As in adipocytes, human erythrocyte membranes possess adenylate cyclase activity [4,5]
In view of the finding that cyclic AMP plays no discernible role in the erythrocyte3-0-MG transport, itis suggested that the forskoIin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity
Transport of 3-0-MG by forskolin-treated cellswasfully restored after thorough washing
Summary
Hibited an increase in cyclic AMP content from the Chemicals-Forskolinwas purchased from Calbiochem-Behring. basal levels of 258 fmol/ml of cell to 334 fmol/ml of ["C]3-O-Methylglucose, ['4C]galactose, and [3H]cyt~chalasiBn were cell within 10 s after forskolin exposure. ['4C]Fructose,["CC]ribose, and erythrocytes in which cyclic AMP was allowed to ac- [3H]adenosine were purchased from ICN. Washed cells were suspended a t room temperature to 20% hematocrit in a balanced salt solution (BSS) composed of 118mM NaCl, 5 mM KC1, Human erythrocytes possess a hexose transport system. Erythrocytes, unlike adipocytes, exhibit no discerniblechange in glucose transport in responseto hormones. The influx of 3-0°C was stopped by the addition of 1ml ofcold stoppingsolution, composed of 2 mM HgCl,, 2 mM KI, and 154 mM NaCl, followed by rapid pelleting of cells by centrifugation (Brinkmann, Instruments, Model 3200).The resulting supernatant was discarded by aspiration, transport is altered by a number of agents including @-adre- and cells were washed with 1ml of cold stopping solution and pelleted again.
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