AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 286: H627-H632, 2004. First published October 9, 2003; doi:10.1152/ajpheart.00639.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/2/H627    most recent
00639.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shuralyova, I.
Right arrow Articles by Coe, I. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shuralyova, I.
Right arrow Articles by Coe, I. R.

Inhibition of glucose uptake in murine cardiomyocyte cell line HL-1 by cardioprotective drugs dilazep and dipyridamole

Irina Shuralyova,1 Panteha Tajmir,1 Philip J. Bilan,2 Gary Sweeney,1 and Imogen R. Coe1

1Department of Biology, York University, Toronto M3J 1P3; and 2Department of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8

Submitted 7 July 2003 ; accepted in final form 1 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibition of adenosine reuptake by nucleoside transport inhibitors, such as dipyridamole and dilazep, is proposed to increase extracellular levels of adenosine and thereby potentiate adenosine receptor-dependent pathways that promote cardiovascular health. Thus adenosine can act as a paracrine and/or autocrine hormone, which has been shown to regulate glucose uptake in some cell types. However, the role of adenosine in modulating glucose transport in cardiomyocytes is not clear. Therefore, we investigated whether exogenously applied adenosine or inhibition of adenosine transport by S-(4-nitrobenzyl)-6-thioinosine (NBTI), dipyridamole, or dilazep modulated basal and insulin-stimulated glucose uptake in the murine cardiomyocyte cell line HL-1. HL-1 cell lysates were subjected to SDS-PAGE and immunoblotting to determine which GLUT isoforms are present. Glucose uptake was measured in the presence of dipyridamole (3–300 µM), dilazep (1–100 µM), NBTI (10–500 nM), and adenosine (50–250 µM) or the nonmetabolizable adenosine analog 2-chloro-adenosine (250 µM). Our results demonstrated that HL-1 cells possess GLUT1 and GLUT4, the isoforms typically present in cardiomyocytes. We found no evidence for adenosine-dependent regulation of basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes. However, we did observe a dose-dependent inhibition of glucose transport by dipyridamole (basal, IC50 = 12.2 µM, insulin stimulated, IC50 = 13.09 µM) and dilazep (basal, IC50 = 5.7 µM, insulin stimulated, IC50 = 19 µM) but not NBTI. Thus our data suggest that dipyridamole and dilazep, which are widely used to specifically inhibit nucleoside transport, have a broader spectrum of transport inhibition than previously described. Moreover, these data may explain previous observations, in which dipyridamole was noted to be proischemic at high doses.

adenosine; equilibrative nucleoside transporters; regulation

THE PURINE NUCLEOSIDE adenosine is well established as an autocrine and paracrine hormone in the cardiovascular system (25, 33). Adenosine promotes compensatory cellular responses to stress and enhances the ability of cells to recover from severe cellular insult (17, 25, 33). Adenosine is not lipophilic; therefore, the movement of adenosine across the cell membrane occurs via equilibrative nucleoside transporters (ENTs), which facilitate diffusion of adenosine down its concentration gradient. ENTs are highly expressed in the heart and are likely to play an important, but poorly understood, role in adenosine-dependent cellular physiology. Adenosine concentrations in the heart fluctuate enormously depending on cellular metabolic demands and activity of enzymes involved in adenosine metabolism (17–19). However, the influence of ENTs on adenosine flux across cardiomyocyte cell membranes is unknown. Clinically, adenosine has been used as an antiarrhythmia drug (41) and adenosine analogs are used, or have been proposed for clinical use, to treat various types of cardiovascular disease (e.g., 12, 33, 44). Moreover, several clinically important cardiovasodilatory and cardioprotectant drugs, such as dipyridamole, inhibit ENTs, and their use is based on the premise that preventing reuptake of adenosine will potentiate adenosine receptor activation.

Adenosine acts via G protein-coupled receptors linked to a multitude of signaling cascades that have been proposed to regulate activity of other membrane proteins such as ion channels and transporters (2, 49).

We are interested in the role of ENTs in modulating the effects of adenosine in cardiomyocytes and the role of adenosine in regulating transport processes in cardiomyocytes. Recently, we showed that the cardiomyocyte cell line HL-1 (10) is an excellent model in which to dissect adenosine-dependent pathways involved in cardiomyocyte cellular physiology (9). HL-1 cells are similar to cardiomyocytes in terms of their adenosine and glucose transport characteristics (9) and have been previously used for studies on cardiomyocyte physiology (e.g., 15, 38, 39, 47). It has been widely reported that adenosine potentiates insulin-stimulated glucose transport (via specific adenosine receptor-activated pathways) in skeletal muscle (e.g., 24, 52, 55). However, the role of adenosine in regulating insulin-stimulated glucose transport in cardiomyocytes is less clear, with reports (1, 20, 22, 32, 34) both supporting and discounting a role for adenosine in modulating glucose uptake. Therefore, we asked whether inhibition of ENTs, by the nucleoside transport inhibitors dipyridamole, dilazep, or S-(4-nitrobenzyl)-6-thioinosine (NBTI) to increase extracellular adenosine or treatment with adenosine directly, modulates basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Adenosine, 2-chloro-adenosine, antibiotic/antimycotic solution, bovine pancreas insulin, cytochalasin B, nonradiolabeled 2-deoxy-D-glucose, dilazep, dipyridamole, fetal bovine serum, fibronectin, NBTI, norepinephrine, and retinoic acid were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). The nonessential amino acids and gelatin were purchased from Canadian Life Technologies (Burlington, Ontario, Canada). The Mycoplasma Detection Kit and Complete protease inhibitor cocktail tablets were obtained from Roche Molecular Biochemicals (Quebec, Canada). Radiolabeled 2-[3H]deoxy-D-glucose was purchased from American Radiolabeled Chemicals (St. Louis, MO). Ex-Cell 320 and Claycomb media were obtained from JRH Biosciences (Lenexa, KS). Secondary (goat anti-rabbit horseradish peroxidase conjugated) antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Polyclonal rabbit antibodies, specific for GLUT1 and GLUT4 (25, 26), were the kind gift of Dr. Amira Klip, Hospital for Sick Children (Toronto, Ontario, Canada).

Cell culture. The HL-1 cells represent a cardiac muscle cell line derived from AT-1 mouse atrial cardiomyocyte tumor lineage and which keeps in vitro phenotypic characteristics of the adult cardiomyocyte (10). HL-1 cells were obtained from Dr. W. Claycomb (Louisiana State University Medical Center, New Orleans, LA) and were grown as previously described (9). Briefly, cells were grown as a monolayer (37°C, 5% CO2) in culture flasks, dishes, and plates precoated with 1 µg/cm2 fibronectin-0.02% (wt/vol) gelatin solution. HL-1 cells were maintained in Claycomb medium supplemented with 10% (vol/vol) fetal bovine serum, 4 mM L-glutamine, 100 µM norepinephrine, and 1x antibiotic/antimycotic solution. Cells were confirmed to be mycoplasma free on a regular basis.

Glucose transport studies. Glucose (2-deoxy-D-glucose) uptake assay was performed as previously described (57). Briefly, HL-1 cells were grown in Ex-Cell 320 medium containing parts A and B (9:1) with 10% (vol/vol) fetal bovine serum in 12- or 24-well plates until 80–90% confluent. Cells were then serum starved for 16–18 h. Cells were preincubated (37°C, 15–30 min) with media alone or with insulin (300 nM), adenosine (50 µM, 250 µM), 2-chloroadenosine (250 µM), or the nucleoside transport inhibitors dipyridamole (3–300 µM), dilazep (1–100 µM), and NBTI (10–500 nM). Cells were then washed twice with glucose-free HEPES-buffered saline solution [which contained (in mM) 140 NaCl, 5 KCl, 2.5 MgSO4, 1 CaCl2, and 20 HEPES, pH 7.4]. Carrier-mediated glucose uptake was determined after incubation (5 min) with 2-[3H]deoxy-D-glucose (10 µM, 60 Ci/mmol, and 1 µCi/µl) in the above solution (in the presence or absence of inhibitor) at room temperature. Uptake was terminated by rinsing the cells three times with ice-cold saline solution [0.9% (wt/vol) NaCl], followed by cell disruption with 0.05 N NaOH. Protein concentration was measured by the modified Lowry protein assay. Uptake-associated radioactivity was determined by scintillation counting. Nonspecific 2-deoxy-D-glucose uptake was determined in the presence of 10 µM cytochalasin B, and this value was subtracted from all measurements performed at least in quadruplicate for each condition. Specific uptake was expressed as picomoles per milligram of protein per minute.

Analysis of GLUT isoforms. Crude lysates of HL-1 or L6 (positive control) cells were prepared to determine which GLUT isoforms were present. HL-1 cells (100-mm dishes, 90–95% confluent) were placed on ice and washed with ice-cold phosphate-buffered saline. Cells were then scraped into 1 to 2 ml of lysis buffer composed of (in mM) 10 Tris·HCl, pH 7.5, 1 EDTA, 0.1 NaCl, and 1 Complete protease inhibitor cocktail tablet (10 ml) and homogenized by being passed through a 26-gauge needle. The homogenate was centrifuged (10 min, 1,000 g, 4°C) to remove cell debris and the supernatant was frozen at –80°C for later analysis by SDS-PAGE and immunoblotting. The protein concentration of the crude lysate was determined by a modified Lowry protein assay. Protein samples were subjected to SDS-PAGE on 7.5% (wt/vol) acrylamide gel and transferred to polyvinylidene difluoride membranes for 2 h at 100 mV. Membranes were incubated (1 h at room temperature) in blocking solution composed of Tris-buffered saline (TBS) containing (in mM) 150 NaCl and 50 Tris·HCl, pH 7.5, plus 3% (wt/vol) BSA with 0.1% (vol/vol) Tween 20 and 0.1% (vol/vol) Nonidet P-40 (NP-40). Membranes were then incubated (overnight, 4°C) with primary antibodies (rabbit) against GLUT 1 and GLUT 4 [1:1,000 dilution in TBS; 1% (vol/vol) BSA; 0.1% (wt/vol) NaN3]. Membranes were washed (TBS, 5 x 10 min) and then incubated (1.5 h, room temperature) with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody [1:10,000 dilution in TBS; 1% (vol/vol) BSA] and washed five times [TBS; 0.1%(vol/vol) Tween 20; 0.1% (vol/vol) NP-40]. Antigen reactivity was detected with the use of a LumiGLO Chemiluminescent Substrate Kit. GLUT1 and GLUT4 are typically seen as bands of 46–48 kDa (28, 46) using this experimental protocol.

Data analysis. Data were analyzed using Student's paired t-test to compare the mean of differences between two conditions, such as control and insulin treated. A value of P < 0.05 was considered to be significant. Dose-response curves and inhibition constants were generated using nonlinear regression analyses in GraphPad Prism 3.0 for Macintosh.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GLUT isoforms in HL-1 cells. Whereas basal and insulin-stimulated glucose uptake has been previously demonstrated in HL-1 cells (9), the identities of the GLUT isoforms in this cell type have not been determined. GLUT1 and GLUT4 have been shown to be responsible for both basal and insulin-stimulated glucose uptake in rodent cardiomyocytes (3). SDS-PAGE of HL-1 cell lysates and immunoblotting using well-characterized antibodies (31, 48) and control L6 cell lysates demonstrated that both GLUT1 and GLUT4 isoforms are present in HL-1 cells (Fig. 1). GLUT4 protein is characterized as a relatively diffuse band of ~46–48 kDa. Both proteins were identified by comparison with the known locations of these isoforms in L6 cell lysates (48).



View larger version (60K):
[in this window]
[in a new window]
  		Fig. 1. GLUT1 (A) and GLUT4 (B) are present in HL-1 cells. Lane 1 is a positive control of protein (20 µg) from L6 rat skeletal muscle cells. Lane 2 shows protein (20 µg) from HL-1. Lane 3 shows protein (100 µg) from HL-1. GLUT proteins are 46–48 kDa in size. Representative experiment is shown, repeated at least four times with similar results.

 

Inhibition of glucose uptake by dipyridamole and dilazep but not NBTI. Dipyridamole and dilazep have been widely used to promote adenosine-dependent processes in the cardiovascular system based on their inhibition of reuptake of endogenous extracellular adenosine by ENTs and potentiation of adenosine receptor activation (21, 23, 35, 53, 54). Dilazep and dipyridamole (at micromolar concentrations) will effectively inhibit both ENT1 and ENT2 isoforms in HL-1 cells (9). In contrast, the nucleoside analog NBTI is a tight-binding high-affinity inhibitor for ENT1 and will inhibit adenosine uptake via ENT1 at nanomolar concentrations. ENT1 is responsible for the majority of adenosine uptake in HL-1 cells (9). Therefore, to determine whether inhibition of adenosine uptake by ENT1, ENT2, or both modulates glucose transport, we measured uptake of 2-[3H]deoxy-D-glucose in the presence of various concentrations of dipyridamole (3–300 µM), dilazep (1–100 µM), and NBTI (10–500 nM). We found a dose-dependent decrease in glucose transport with dipyridamole and dilazep but no effect on basal levels of glucose transport with NBTI (Fig. 2). Glucose uptake was reduced at least 50% with dipyridamole and dilazep at concentrations we have previously determined (9) to fully inhibit adenosine transport (by ENT1 and ENT2). However, inhibition of ENT1 by NBTI has no effect on glucose uptake. These data suggested that inhibition of adenosine reuptake (leading to an increase in extracellular adenosine and activation of receptors) does not modulate glucose transport. Rather, these data suggest that dipyridamole and dilazep inhibit glucose uptake, possibly in a dose-dependent manner.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2. 2-[3H]deoxy-D-glucose (2DG; 10 µM) uptake in HL-1 cells is inhibited by dipyridamole (DIP) and dilazep (DIL) but not S-(4-nitrobenzyl)-6-thiosinine (NBTI). Cells were serum deprived (18 h) and preincubated (15 min) with inhibitor before uptake. Inhibitor was also present in permeant solution. Values of representative transport assay (uptake at 5 min) are means ± SE; n = 5.

 

Dose dependence of glucose uptake inhibition by dipyridamole and dilazep. To further analyze the inhibition of glucose uptake by dipyridamole and dilazep, we conducted dose-response assays with a wider range of concentration of inhibitors and calculated the IC50 values for inhibition of glucose uptake by these compounds (Fig. 3). The IC50 for dipyridamole was 12.2 µM (pooled data, n = 5, each experiment conducted in quintuplicate) and 5.7 µM for dilazep (pooled data, n = 6, each experiment conducted in quintuplicate). Insulin stimulation leads to a recruitment of GLUT4 to the plasma membrane in cardiomyocytes (3). Therefore, we also determined inhibition profiles of dipyridamole and dilazep on glucose uptake in insulin-stimulated HL-1 cells in an attempt to determine whether one isoform was being preferentially inhibited. We found virtually no difference in the IC50 of dipyridamole-inhibited glucose uptake between basal and insulin-stimulated states (12.22 vs. 13.09 µM). There was an increase in the IC50 of dilazep-inhibited glucose uptake (5.6 vs. 19 µM) representing a slightly less effective inhibition of this drug on insulin-stimulated cells. In contrast to dipyridamole and dilazep, there was no effect of NBTI (100 nM) on insulin-stimulated glucose uptake in HL-1, which correlates with our observation of no effect on basal glucose uptake (n = 2, data not shown).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3. Dose-response analyses of dipyridamole (A) and dilazep (B) on 2DG (10 µM) transport in HL-1 cells. HL-1 cells were incubated with varying doses of dipyridamole or dilazep in the presence or absence of insulin as described in Fig. 2. Transport was measured at 5 min. Representative experiments are presented. Each concentration was conducted in quintuplicate (means ± SE are shown). Experiments were repeated several times (basal, n = 5 and insulin stimulated, n = 2) with similar results. Data are shown as a log dose curve (r2 >= 0.98 for all curves). Nonlinear regression analysis and IC50 values were determined with the use of GraphPad Prism version 3.0 for Macintosh.

 

Effect of adenosine on basal and insulin-stimulated glucose uptake. Clinical use of dipyridamole and dilazep is based on the premise that these drugs increase extracellular levels of adenosine (by preventing reuptake of adenosine), which, in turn, activates adenosine receptor-dependent pathways promoting cardiovascular well being. Because dipyridamole and dilazep inhibit glucose uptake directly, using them to study adenosine receptor-dependent regulation of glucose uptake is problematic. Extracellular levels of endogenous adenosine are generally relatively low (under normal conditions) and the half-life of adenosine is relatively short because it is rapidly deaminated to inosine (56). Therefore, to determine whether an increase in extracellular adenosine could modulate glucose transport, we treated cells directly with adenosine [in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to prevent degradation] and measured both basal and insulin-stimulated glucose uptake. We found no statistically significant difference between basal and insulin-stimulated levels of glucose uptake in the presence or absence of EHNA (data not shown). Glucose uptake in HL-1 cardiomyocytes was stimulated by insulin approximately threefold (Fig. 4A). However, in both basal and insulin-stimulated cells, we found no statistically significant difference in glucose uptake relative to control in the presence of varying concentrations of adenosine or 2-chloro-adenosine (Fig. 4B), suggesting that adenosine does not modulate basal or insulin-stimulated glucose uptake in HL-1 cardiomyocytes.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4. Adenosine shows no effect on basal or insulin-stimulated [3H]2DG transport in HL-1 cells. Cells were serum deprived (18 h) and preincubated in adenosine [0, 50, or 250 µM in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine] or 2-chloroadenosine (2-chloroA; 250 µM) for 15 min. Cells were then treated in the presence (A) or absence (B) of insulin (300 nM, 15 min) and glucose transport was measured at 5 min. Data are expressed as means ± SD, n = 4, each condition conducted in quintuplicate.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Adenosine is a remarkable molecule that has a diversity of actions both inside and outside of cells. The relationship between adenosine and regulation of glucose transport has been widely investigated and appears to be critically important in skeletal muscle (16, 55) and in insulin-stimulated glucose transport in adipocytes (30, 50). The evidence supporting a role for adenosine in the regulation of glucose transport in heart is less convincing. Our findings correlate with data from several recent studies in rat cardiomyocytes (11, 20, 22) suggesting that adenosine does not modulate glucose transport in rodent cardiomyocytes. Indeed, it has been suggested that earlier reports (1, 32, 34) of regulation by adenosine of glucose uptake in cardiomyocytes were due to the effects on metabolism rather than transport. Therefore, it would appear that there are significant cell type or tissue-specific differences in the role of adenosine in modulation of glucose transport. Indeed, in cardiomyocytes, nucleotides such as cGMP and ATP, rather than nucleosides, have been shown to modulate glucose transport (7, 22).

An unexpected result of our study was the observation of a dose-dependent inhibition of glucose transport (both basal and insulin stimulated) by the nucleoside transport inhibitors dipyridamole and dilazep but not NBTI. NBTI is nucleoside analog and differs considerably in structure compared with dipyridamole, a pyrimidopyrimidine and dilazep, an alkyldiamine aromatic ester (see Fig. 5). The nature of the molecular interactions between these inhibitors and ENTs is not yet known, although dipyridamole and dilazep are proposed to interact at the same site on the human ENT1 protein, which overlaps with, but may not be identical to, the binding site for NBTI (26). Moreover, dipyridamole and dilazep inhibit both ENT1 and ENT2 isoforms, whereas NBTI inhibits only ENT1, suggesting that dipyridamole and dilazep possess similar molecular characteristics of interaction with ENTs compared with NBTI. This correlates with our observations of similar effects of dipyridamole and dilazep but distinct (i.e., no) effects of NBTI on glucose uptake.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5. Chemical structures of nucleoside transporter inhibitors used in this study.

 

Dipyridamole and dilazep have been widely used as cardioprotectants because of their properties as nucleoside transport inhibitors, which prevent reuptake of adenosine, thereby enhancing extracellular adenosine levels and potentiating adenosine receptor-activated processes, which compensate for cellular stresses, such as hypoxia and ischemia (e.g., 21, 26). Dipyridamole is also used clinically as an antiplatelet agent in prevention of secondary stroke (e.g., 13, 51). However, dipyridamole has previously been reported to inhibit the organic cation transporter (OCT) (6), with an IC50 value (7.7 µM) that is very similar to our findings. GLUT1 and GLUT4 are both present at the cardiomyocyte cell membrane under basal conditions, although the proportion of GLUT4 at the plasma membrane increases in response to insulin (3). On the basis of these observations, a significant shift or change in shape of inhibition profiles under insulin-stimulation could have been indicative of preferential inhibition of one isoform (predominantly GLUT1 under basal conditions) to the other (predominantly GLUT4 under insulin-stimulated conditions). However, our data are inconclusive with regard to whether one isoform is preferentially inhibited compared with the other, but our findings do demonstrate that inhibition occurs in both physiologically relevant conditions.

The inhibition of three different transporter families (GLUT, OCT, and ENT) by dipyridamole (Table 1) suggest that these proteins possess common structural motifs. Indeed, the GLUT and OCT families of proteins are considered members of the sugar porter superfamily of transport proteins (40) and both GLUT1 and OCT1 possess significant sequence similarity at the amino acid level. The relationship of the ENTs to the sugar porter superfamily is not clear but a more detailed analysis of common amino acids and motifs within these different proteins may provide insight into some aspects of tertiary structure of transporters. Indeed, both computational and in vivo approaches, based on transporter substrate (inhibitor) binding studies, are now being used to elucidate transporter structures (4, 59).


View this table:
[in this window]
[in a new window]
  		Table 1. Comparison of inhibition of transport processes by NBTI, dipyridamole, and dilazep

 

Clinically, the use of dipyridamole to promote cardiovascular health is complicated by the observation that high doses are actually proischemic (23, 42). It has been proposed that high doses of dipyridamole lead to high concentrations of extracellular adenosine and activation of low-affinity (A2a) adenosine receptors. A2a receptor activation is further proposed to result in cellular and vascular responses, which compromise the cardioprotectant effects of low-dose dipyridamole (which results in activation of high-affinity A1 adenosine receptors only) (23, 42). Our data suggest that direct inhibition of glucose uptake may be an additional mechanism responsible for the proischemic effects of high concentrations of dipyridamole. Lower concentrations of dipyridamole are anti-ischemic because only adenosine reuptake is inhibited and adenosine receptor activation is potentiated (see Table 1). In humans, the IC50 value of the ENTs to inhibition of nucleoside uptake with dipyridamole is in the nanomolar range (43). However, dipyridamole is typically administered orally (e.g., 5, 23), and, whereas plasma levels of adenosine have been found to rise after administration, the concentration of dipyridamole present at the cardiomyocyte membrane is not clear. Moreover, the observation that administration of glucose in combination with dipyridamole treatment reduces ischemic injury in an animal model (8) suggests that monitoring of plasma levels of dipyridamole in patients may be advantageous to ensure that levels do not rise to the point where glucose (or organic cation) uptake is compromised. In addition, our data suggest that extended treatment with dipyridamole or dilazep should be avoided because it may promote development of unexpected side effects. Unanticipated complications have been described for indinavir, a protease inhibitor widely used in anti-human immunodeficiency virus treatment, which has recently been found to also inhibit GLUT4, thereby contributing to the insulin resistance seen in patients using this drug (27, 36).

In addition to transport inhibition, other cellular effects have been described for both dipyridamole [inhibition of cGMP phosphodiesterase (14)] and dilazep (antioxidant free radical scavenger) (37, 45). Thus these observations need to be taken into account in interpretation of data from experiments where dipyridamole and dilazep have been used exclusively as nucleoside transport inhibitors.

In summary, we have found that extracellular adenosine does not modulate glucose uptake in cardiomyocytes. However, the compounds dipyridamole and dilazep, which are used clinically to inhibit adenosine transport and enhance extracellular adenosine, may themselves affect glucose uptake by directly inhibiting transport glucose via the GLUTs under both basal and insulin-stimulated conditions.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Amira Klip for assistance with the Western blot analysis of the GLUTs and L6 cell particulate fractions.

GRANTS

This work was funded by Canadian Institutes of Health Research Grant MOP-38013 (to I. R. Coe).


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. R. Coe, Dept. of Biology, York Univ., 4700 Keele St., Toronto, Ontario, Canada M3J 1P3 (E-mail: coe{at}yorku.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Angello DA, Berne RM, and Coddington NM. Adenosine and insulin mediate glucose uptake in normoxic rat hearts by different mechanisms. Am J Physiol Heart Circ Physiol 265: H880–H885, 1993.[Abstract/Free Full Text]
  2. Bailey A, Matthes H, Kieffer B, Slowe S, Hourani SMO, and Kitchen I. Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains and spinal cords of mice deficient in the µ-opioid receptor gene. Brain Res 943: 68–79, 2002.[CrossRef][ISI][Medline]
  3. Becker C, Sevilla L, Tomas E, Palacin M, Zorzano A, and Fischer Y. The endosomal compartment is an insulin-sensitive recruitment site for GLUT4 and GLUT1 glucose transporters in cardiac myocytes. Endocrinology 142: 5267–5276, 1989.
  4. Bednarczyk D, Ekins S, Wikel JH, and Wright SH. Influence of molecular structure on substrate binding to the human organic cation transporter, hOCT1. Mol Pharmacol 63: 489–498, 2003.[Abstract/Free Full Text]
  5. Belardinelli R, Belardinelli L, and Shyrock JC. Effects of dipyridamole on coronary collateralization and myocardial perfusion in patients with ischaemic cardiomyopathy. Eur Heart J 22: 1205–1213, 2001.[Abstract/Free Full Text]
  6. Bendayan R. Interaction of dipyridamole, a nucleoside transport inhibitor, with the renal transport of organic cations by LLCPK 1 cells. Can J Physiol Pharmacol 75: 52–56, 1997.[CrossRef][ISI][Medline]
  7. Bergmann C, Loken C, Becker C, Graf B, Hamidizadeh M, and Fischer Y. Inhibition of glucose transport by cyclic GMP in cardiomyocytes. Life Sci 69: 1391–1406, 2001.[CrossRef][ISI][Medline]
  8. Bertuglia S, Giusti A, Fedele S, and Picano E. Glucose-insulin-potassium treatment in combination with dipyridamole inhibits ischaemia-reperfusion-induced damage. Diabetologia 44: 2165–2170, 2001.[CrossRef][ISI][Medline]
  9. Chaudary N, Shuralyova I, Liron T, Sweeney G, and Coe IR. Transport characteristics of HL-1 cells: a new model for the study of adenosine physiology in cardiomyocytes. Biochem Cell Biol 80: 655–665, 2002.[CrossRef][ISI][Medline]
  10. Claycomb WC, Lanson NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, and Izzo NJ. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA 95: 2979–2984, 1998.[Abstract/Free Full Text]
  11. Dale WE, Hale CC, Kim HD, and Rovetto MJ. Myocardial glucose utilization: failure of adenosine to alter it and inhibition by the adenosine analogue N6-(L-2-phenylisopropyl) adenosine. Circ Res 69: 791–799, 1991.[Abstract/Free Full Text]
  12. De Jong JW, de Jonge R, Keijzer E, and Bradamante S. The role of adenosine in preconditioning. Pharmacol Ther 87: 141–149, 2000.[CrossRef][ISI][Medline]
  13. De SC, Algra A, and Van Gijn J. Dipyridamole for preventing stroke and other vascular events in patients with vascular disease (Cochrane Review). Cochrane Database Sys Rev CD001820 [GenBank] , 2003.
  14. De La Cruz JP, Blanco E, and Sanchez de la Cuesta F. Effect of dipyridamole and aspirin on the platelet neutrophil interaction via the nitric oxide pathway. Eur J Pharmacol 397: 35–40, 2000.[CrossRef][ISI][Medline]
  15. Deng XF, Rokosh DG, and Simpson PC. Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes: comparison with rat. Circ Res 87: 781–788, 2000.[Abstract/Free Full Text]
  16. Derave W and Hespel P. Role of adenosine in regulating glucose uptake during contractions and hypoxia in rat skeletal muscle. J Physiol 515: 255–263, 1999.[Abstract/Free Full Text]
  17. Deussen A. Cardiac adenosine metabolism: physiological implications. In: Purines and Myocardial Protection, edited by Wechsler ASA. Boston, MA: Kluwer, 1996.
  18. Deussen A. Metabolic flux rates of adenosine in the heart. Naunyn-Schmeidberg's Arch Pharmacol 362: 351–363, 2000.[CrossRef][ISI][Medline]
  19. Deussen A. Quantitative integration of different sites of adenosine metabolism in the heart. Ann Biomed Eng 28: 877–883, 2000.[CrossRef][ISI][Medline]
  20. Eblenkamp M, Bottcher U, Thomoas J, Loken C, Ionescu I, Rose H, Kammermeier H, and Fischer Y. The effect of anoxia on cardiomyocyte glucose transport does not involve an adenosine release or a change in energy state. Life Sci 59: 141–151, 1996.[CrossRef][ISI][Medline]
  21. Figueredo VM, Diamond I, Zhou HZ, and Camacho SA. Chronic dipyridamole therapy produces sustained protection against cardiac ischemic-reperfusion injury. Am J Physiol Heart Circ Physiol 277: H2091–H2097, 1999.[Abstract/Free Full Text]
  22. Fischer Y, Becker C, and Loken C. Purinergic inhibition of glucose transport in cardiomyocytes. J Biol Chem 274: 755–761, 1999.[Abstract/Free Full Text]
  23. Guideri F, Capecchi PL, Acampa M, Cuomo A, Lazzerini PE, Giorgi LD, and Pasini FL. Oral low-dose dipyridamole protects from intravenous high-dose dipyridamole-induced ischemia. A stress echocardiographic study. Int J Cardiol 83: 209–216, 2002.[CrossRef][ISI][Medline]
  24. Han DH, Hansen PA, Nolte LA, and Holloszy JO. Removal of adenosine decreases the responsiveness of muscle glucose transport to insulin and contractions. Diabetes 47: 1671–1675, 1998.[Abstract]
  25. Headrick JP. Ischemic preconditioning: bioenergetic and metabolic changes, and the role of endogenous adenosine. J Mol Cell Cardiol 27: 1041–1054, 1996.
  26. Hyde RJ, Cass CE, Young JD, and Baldwin SA. The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms. Mol Membr Biol 18: 53–63, 2001.[ISI][Medline]
  27. Hruz PW, Murata H, Qiu H, and Mueckler M. Indinavir induces acute and reversible peripheral insulin resistance in rats. Diabetes 51: 937–942.
  28. James DE, Strube M, and Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 338: 83–87, 1989.[CrossRef][Medline]
  29. Joost HG and Thorens B. The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequences characteristics, and potential function of its novel members. Mol Membr Biol 18: 247–256, 2001.[CrossRef][ISI][Medline]
  30. Joost HG, Weber TM, Cushman SW, and Simpson IA. Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine. J Biol Chem 261: 10033–10036, 1986.[Abstract/Free Full Text]
  31. Koivisto UM, Martinez-Valdez H, Bilan PJ, Burdett E, Ramlal T, and Klip A. Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6. J Biol Chem 266: 2615–2621, 1991.[Abstract/Free Full Text]
  32. Law WR and Raymond RM. Adenosine potentiates insulin-stimulated myocardial glucose uptake in vivo. Am J Physiol Heart Circ Physiol 254: H970–H975, 1988.[Abstract/Free Full Text]
  33. Liang BT, Swierkosz TA, Herrmann HC, Kimmel S, and Jacobsen KA. Adenosine and ischemic preconditioning. Curr Pharm Des 5: 1029–1041, 1999.[ISI][Medline]
  34. Mainwaring R, Lasley R, Rubio R, Wyatt DA, and Mentzer RM Jr. Adenosine stimulates glucose uptake in the isolated rat heart. Surgery 103: 445–449, 1998.
  35. Marzilli M, Trivella MG, Levantesi D, Pelosi G, Dalle Vacche M, Taddei L, and L'Abbate A. Effect of dilazep on coronary and systemic circulations. Pharmacology 31: 82–87, 1985.[ISI][Medline]
  36. Murata H, Hruza PW, and Mueckler M. Indinavir inhibits the glucose transporter isoform Glut4 at physiologic concentrations. AIDS 16: 859–863, 2002.[CrossRef][ISI][Medline]
  37. Nakamura K, Kojima K, Arai T, Shirai M, Usutani S, Akimoto H, Masaoka H, Nagase M, and Yamamoto M. Dipyridamole and dilazep suppress oxygen radicals in puromycin aminonucleoside nephrosis rats. Eur J Clin Invest 28: 877–888, 1998.[CrossRef][ISI][Medline]
  38. Neilan CL, Kenyon E, Kovach MA, Bowden K, Claycomb WC, Traynor JR, and Bolling SF. An immortalized myocyte cell line, HL-1, expresses a functional {delta}-opioid receptor. J Mol Cell Cardiol 32: 2187–2193, 2000.[CrossRef][ISI][Medline]
  39. Nguyen SU and Claycomb WC. Hypoxia regulates the expression of the adrenomedullin and HIF-1 genes in cultured HL-1 cardiomyocytes. Biochem Biophys Res Commun 265: 382–386, 1999.[CrossRef][ISI][Medline]
  40. Pao SS, Paulsen IT, and Saier MH Jr. Major facilitator superfamily. Microbiol Mol Biol Rev 62: 1–34, 1998.[Abstract/Free Full Text]
  41. Pelleg A, Pennock RS, and Kutalek SP. Proarrhythmic effects of adenosine: one decade of clinical data. Am J Ther 9: 141–147, 2002.[CrossRef][Medline]
  42. Picano E. Dipyridamole in myocardial ischemia: good Samaritan or terminator? Int J Cardiol 83: 215–216, 2002.[CrossRef]
  43. Plagemann PGW and Woffendin C. Species differences in sensitivity of nucleoside transport in erythrocytes and cultured cells to inhibition by nitrobenzylthioinosine, dipyridamole, dilazep and lidoflazine. Biochim Biophys Acta 969: 1–8, 1988.[Medline]
  44. Pouzet B, Lecharny JB, Dehoux M, Paquin S, Kitazake M, Mantz J, and Menasche P. Is there a place for preconditioning during cardiac operations in humans? Ann Thorac Surg 73: 843–848, 2002.[Abstract/Free Full Text]
  45. Ruggiero AC, Neopmuceno MF, Jacob RF, Dorta DJ, and Tabak M. Antioxidant effect of dipyridamole (DIP) and its derivative RA 25 upon lipid peroxidation and hemolysis in red blood cells. Physiol Chem Phys Med NMR 32: 35–48, 2000.[Medline]
  46. Sargeant RJ and Paquet MR. Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3–L1 adipocytes. Biochem J 290: 913–919, 1993.
  47. Soltys CLM, Buchholz L, Gandhi M, Clanachan AS, Walsh K, and Dyck JRB. Phosphorylation of cardiac protein kinase B is regulated by palmitate. Am J Physiol Heart Circ Physiol 283: H1056–H1064, 2002.[Abstract/Free Full Text]
  48. Sumitani S, Ramlal T, Somwar R, Keller SR, and Klip A. Insulin regulation and selective segregation with glucose transporter-4 of the membrane aminopeptidase vp165 in rat skeletal muscle cells. Endocrinology 138: 1029–1034, 1997.[Abstract/Free Full Text]
  49. Szkotak AJ, Ng AML, Sawicka J, Baldwin SA, Man SFP, Cass CE, Young JD, and Duszyk M. Regulation of K+ current in human airway epithelial cells by exogenous and autocrine adenosine. Am J Physiol Cell Physiol 281: C1991–C2002, 2001.[Abstract/Free Full Text]
  50. Takasuga S, Katada T, Ui M, and Hazeku O. Enhancement by adenosine of insulin-induced activation of phosphoinositide 3-kinase and protein kinase B in rat adipocytes. J Biol Chem 274: 19545–19550, 1999.[Abstract/Free Full Text]
  51. Talbert RL and Crawford KM. Antiplatelet therapy in secondary stroke prevention. Exp Op Pharm 2: 1609–1613, 2001.[CrossRef]
  52. Thong FS and Graham TE. The putative roles of adenosine in insulin- and exercise-mediated regulation of glucose transport and glycogen metabolism in skeletal muscle. Can J Appl Physiol 27: 152–178, 2002.[ISI][Medline]
  53. Tommasi S, Carluccio E, Bentivoglio M, Corea L, and Picano E. Low-dose dipyridamole infusion acutely increases exercise capacity in angina pectoris: a double blind, placebo controlled crossover stress echocardiographic study. J Am Coll Cardiol 35: 83–88, 2000.[Abstract/Free Full Text]
  54. Torry RJ, O'Brien DM, Connell PM, and Tomanek RJ. Dipyridamole-induced capillary growth in normal and hypertrophic hearts. Am J Physiol Heart Circ Physiol 262: H980–H986, 1992.[Abstract/Free Full Text]
  55. Vergauwen L, Hespel P, and Richter EA. Adenosine receptors mediate synergistic stimulation of glucose uptake and transport by insulin and by contractions in rat skeletal muscle. J Clin Invest 93: 974–981, 1994.[ISI][Medline]
  56. Wakai A, Winter DC, Street JT, O'Sullivan RG, Wang JH, and Redmond HP. Inosine attenuates tourniquet-induced skeletal muscle reperfusion injury. J Surg Res 99: 311–315, 2001.[CrossRef][ISI][Medline]
  57. Walker PS, Ramlal T, Sarabia V, Koivisto UM, Bilan PJ, Pessin JF, and Klip A. Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription. J Biol Chem 265: 1516–1523, 1990.[Abstract/Free Full Text]
  58. Yamasaki K, Tanaka M, Yokota R, Fujiwara H, and Sasayama S. Dilazep dihydrochloride prolongs the infarct size-limiting effect of ischemic preconditioning in rabbits. Heart Vessels 15: 227–232, 2000.[CrossRef][ISI][Medline]
  59. Yao H, Kristensen DM, Mihalek I, Sowa ME, Shaw C, Kimmel M, Kavraki L, and Lichtarge O. An accurate, sensitive and scalable method to identify functional sites in protein structures. J Mol Biol 326: 255–261, 2003.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 PhysiologyHome page
J. B. Rose and I. R. Coe
Physiology of Nucleoside Transporters: Back to the Future. . . .
Physiology, February 1, 2008; 23(1): 41 - 48.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/2/H627    most recent
00639.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shuralyova, I.
Right arrow Articles by Coe, I. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shuralyova, I.
Right arrow Articles by Coe, I. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.