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Cardiovascular Research 2004 61(4):780-788; doi:10.1016/j.cardiores.2003.11.031
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Copyright © 2004, European Society of Cardiology

Hypoxia regulates the adenosine transporter, mENT1, in the murine cardiomyocyte cell line, HL-1

Naz Chaudary, Zlatina Naydenova, Irina Shuralyova and Imogen R Coe*

Department of Biology, York University, 4700 Keele St., Toronto, ON, Canada M3J 1P3

* Corresponding author. Tel.: +1-416-736-2100x30825; fax: +1-416-736-5698. coe{at}yorku.ca

Received 1 August 2003; revised 23 October 2003; accepted 25 November 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Adenosine is an important paracrine hormone in the cardiovascular system. Adenosine flux across cardiomyocyte membranes occurs mainly via equilibrative nucleoside transporters (ENTs). The role of the ENTs in adenosine physiology is poorly understood, particularly in response to metabolic stress such as hypoxia. Therefore, we investigated the effects of chronic hypoxia on ENT1, the predominant ENT isoform in cardiomyocytes. Methods: HL-1 cells (immortalized murine cardiomyocytes) were exposed to hypoxia (2% O2) for 0–20 h. Cell viability, lactate dehydrogenase (LDH) release, glucose uptake, GLUT1 and GLUT4 protein, adenosine uptake, PKC activity, translocation profiles of PKC{delta} and {varepsilon}, nitrobenzylthioinosine (NBTI) binding and mENT1 mRNA levels were measured. The role of PKC in regulating mENT1 was further investigated using phorbol ester (100 nM, 18 h) and a dominant negative PKC{varepsilon} construct, pSVK3PKC{varepsilon}1-401. Results: HL-1 cells have typical cardiomyocyte responses to hypoxia based on cell viability, LDH release, glucose uptake and GLUT protein levels. Hypoxia (8–20 h) down-regulates mENT1-dependent adenosine uptake, NBTI-binding and PKC{varepsilon} but not PKC{delta} in HL-1 cells. Abrogation of PKC{varepsilon} activity using chronic phorbol ester or a dominant negative PKC{varepsilon} mimicked the effect of hypoxia on adenosine uptake suggesting that PKC{varepsilon} is involved in regulation of mENT1. Hypoxia (4 h) decreases mENT1 mRNA, which returns to basal levels by 20 h. Conclusions: Chronic hypoxia down-regulates mENT1 activity possibly via PKC{varepsilon}. Hypoxia and PKC also regulate mENT1 RNA levels. Cardiomyocytes may regulate mENT1 (via PKC{varepsilon}) to modulate release and/or uptake of adenosine. However, the relationship between mENT1 mRNA levels, protein levels and functional transport is complex.

KEYWORDS Adenosine transporter; mENT1; Regulation; PKC; HL-1; Cardiomyocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiac hypoxia can occur in a number of contexts such as myocardial infarction, congestive heart failure, atherosclerosis, coronary occlusion or surgery. Hypoxic exposure can extend from short periods of virtually no oxygen to long periods of low oxygen and can result in arrhythmia, cellular damage, and ultimately cell death [1]. There is a need for a better understanding of the causes and consequences of ischemia/hypoxia, and for the development of strategies to combat resultant damage.

The purine nucleoside, adenosine, is a cardioprotective metabolite [2–4]. Hypoxia and ischemia lead to a large increase in extracellular adenosine, which is released by cardiomyocytes. Extracellular adenosine activates G-protein coupled adenosine receptors linked to various signalling pathways, which initiate compensatory responses [5,6]. Intracellular and extracellular levels of adenosine fluctuate considerably depending on the metabolic state of the heart and flux of adenosine down its concentration gradient, across the cardiomyocyte cell membrane, is facilitated by integral membrane transport proteins known as equilibrative nucleoside transporters (ENTs).

ENTs are highly expressed in the cardiovascular system [7] but very little is known about their role in cardiomyocyte physiology. ENTs are bidirectional, allowing adenosine to be released from cells (to act as an autocrine/paracrine hormone) or transported into the cell (to terminate receptor activation, or restore adenosine metabolite pools). Thus, cardiomyocyte adenosine physiology is dependent on adenosine receptor profile, in addition to presence and activity of the ENTs. In a clinical setting, pharmacological inhibition of ENTs, using drugs such as dipyridamole, dilazep and draflazine, is used to promote cardiovascular health. However, despite the clinical relevance of ENTs as drug targets, very little is known about them. Recently, ENTs have been shown to be important in modulating the effects of adenosine in human epithelial cells [8]. Moreover, there is a correlation between ENT1 and A1 adenosine receptor distribution in the brain suggesting potential interactions and/or feedback between receptors and transporters [9].

Although there is an extensive literature on adenosine and adenosine receptor physiology in the cardiovasculature, very little is known about ENTs [10]. We are interested in the understanding the role of ENTs in adenosine physiology of the heart and have found the murine cardiomyocyte cell line HL-1, [11] to be particularly amenable for these studies. HL-1 cells represent an adult cardiomyocyte cell type [11–13] and have cardiomyocyte-specific hypoxic-inducible genes [14]. This sensitivity to hypoxia makes them attractive for hypoxic studies, in contrast to neonatal primary cultures, [15] (Chaudary and Coe, unpublished observations).

Since adenosine is such an important element in cardiomyocyte responses to reduced oxygen, an increased understanding of how hypoxia influences the flux of adenosine across plasma membrane via ENTs is needed. Therefore, we investigated hypoxic regulation of ENTs in cardiomyocytes.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
The HL-1 cells are a cardiac muscle cell line, derived from the AT-1 mouse, atrial cardiomyocyte tumor lineage and were maintained as previously described [12].

2.2 Induction of hypoxia
HL-1 cells were grown under standard conditions to approximately 80% confluency then transferred into degassed medium. Oxygen concentrations in the chamber were measured using a FYRITE Oxygen Analyzer. Oxygen concentrations in the media were measured using an oxygen electrode. Hypoxic cells were maintained in a Plexiglas GasPak container at 37 °C with a humidified hypoxic atmosphere of 2% O2, 5% CO2, 93% N2. Controls were maintained at 5% CO2 and 95% air at 37 °C. Cellular stress due to hypoxia was determined by lactate dehydrogenase (LDH) release [25] using an LDH assay kit (Sigma) according to manufacturer's instructions. Cell viability following hypoxia was determined using a standard trypan blue exclusion assay.

2.3 Uptake and binding assays
Glucose and adenosine uptake and NBTI binding assays were performed as previously described [12]. Transport values are calculated as picomoles per milligram protein.

2.4 Analysis of GLUT1 and GLUT4 protein
HL-1 cells (100 mm dishes, ~80% confluent) were exposed to normoxia or hypoxia then placed on ice and washed with ice-cold phosphate-buffered saline. Cells were scraped into 1–2 ml of lysis buffer (10 mM Tris–HCl pH 7.5; 1 mM EDTA; 0.1 mM NaCl; 1 Complete protease inhibitor cocktail tablet/10 ml) and homogenized by passing through a 26-gauge needle. Homogenate was centrifuged (10 min, 1000 x g, 4 °C) to remove cell debris and the supernatant was either used immediately for isolation of the membrane and cytosolic fractions or frozen at –80 °C for later analysis. Membranes were isolated by high-speed centrifugation (90 min, 54,000 x g, 4 °C). Supernatant was designated the cytosolic fraction. Membrane pellets were resuspended in solubilizing buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) SDS, 1 Complete protease inhibitor cocktail tablet/10 ml), using approximately 10–20 µl/pellet. Protein concentrations were determined by a modified Lowry protein assay. Samples (50 µg) were subjected to SDS-PAGE on 10% (w/v) acrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes for 2 h at 100 mV. Membranes were incubated (1 h, room temperature) in blocking solution (Tris-buffered saline (TBS); 150 mM NaCl, 50 mM Tris–HCl, pH 7.5 plus 3% (w/v) BSA) with 0.1% (v/v) Tween-20 and 0.1% (v/v) NP-40. Membranes were incubated (overnight, 4 °C) with primary antibodies (rabbit) against GLUT 1 and GLUT 4 (1:1000 dilution in TBS; 1% (v/v) BSA; 0.1% (w/v) NaN3), washed (TBS, 5 x 10 min), incubated (1.5 h, room temperature) with horseradish peroxidase-conjugated goat-anti rabbit secondary antibody (1:10000 dilution in TBS; 1% (v/v) BSA) and washed five times (TBS; 0.1%(v/v) Tween-20; 0.1% (v/v) NP-40). Antigen reactivity was detected using a LumiGLO Chemiluminescent Substrate Kit.

2.5 Analysis of PKC protein and activation
Presence and level of activation of PKC isoforms ({delta}/{varepsilon}) were determined by standard SDS-PAGE of cytosolic and membrane fractions of HL-1 cells exposed to normoxia, hypoxia (20 h) or PMA (100 nM, 18 h) as previously described [12]. PKC activity was measured using a SignaTECT PKC assay kit according to the manufacturer's instructions. Primary antibody was used at a dilution of 1:500 and secondary antibody at a dilution of 1:5000. Antigen reactivity was detected as described above and signals were quantified using NIH Image 1.62.

2.6 Northern analysis of mENT1
Northern analysis was conducted as previously described [12] following 4, 8 and 20 h of hypoxia or PMA (18 h, 100 nM). Paired control cells were exposed to normoxia for equivalent times. A radiolabelled PCR product (600 bp) of mENT1 was used as a hybridization probe. Hybridization and washing protocols were optimized for analysis of mENT1 only (i.e. no cross-reaction with mENT2). A Hewlatt Packard Instant Imager was used for quantification of signals and blots were then exposed to autoradiographic film. Loading and signals were normalized by re-probing blots with a 18s rRNA cDNA.

2.7 Transfection of HL-1 cells
Transient transfections were performed similarly to that previously described [16]. Cells were cultured in 12-well plates, to 90% confluency, in Claycomb medium. Media was changed to fresh Claycomb media (400 µl/well) 1 h before transfection. Cells were transfected with a dominant-negative PKC{varepsilon} plasmid, pSVK3PKC{varepsilon}1-401 [17] (5 µg) in transfection medium (final volume, 200 µl) consisting of LipofectAMINE (6 µl) and DMEM without serum and antibiotics (94 µl). Controls indicated that LipofectAMINE had no effect on transport activity up to 24 h post-transfection. Transfection efficiency was assessed using pEGFP-N1 (Clontech, Palo Alto, CA, USA) and was routinely 75–80% after 18–24 h. Adenosine uptake (10 s) was measured at 18 and 24 h after transfection.

2.8 Chemicals
LDH assay kit, trypan blue dye, antibiotic/antimycotic solution (100 x), norepinephrine, fetal bovine serum, S-(4-nitrobenzyl)-6-thioinosine (NBTI), fibronectin, and nonradiolabeled adenosine, were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). Radiolabeled [3H] adenosine and [3H] NBTI were purchased from Moravek Biochemicals (CA, USA). Claycomb media and L-glutamine was purchased from JRH Biosciences (KS, USA). The SignaTECT PKC assay kit was from Promega (CA, USA). LipofectAMINE was from Invitrogen Life Technologies (Burlington, Ontario, Canada). The PKC{varepsilon} dominant negative construct, pSVK3PKC{varepsilon}1-401, was a gift from Dr. Geoffrey Cooper, Boston University, MA, USA. Primary antibodies (polyclonal, rabbit) against PKC{delta} and PKC{varepsilon} were from Santa Cruz Biotechnologies (CA, USA). Secondary antibodies (goat-anti-rabbit) were from Kirkegaard and Perry Laboratories (MD, USA). Antibodies against GLUT1 and GLUT4 were gifts from Dr. Amira Klip, Hospital for Sick Children, Toronto, ON, Canada.

2.9 Statistical analysis
Statistical analyses were done using Student's unpaired two-tailed t-test (comparison of two variables), or analysis of variance test (one-way ANOVA) for comparison of more than two variables, followed by post-hoc Student–Newman–Keuls test. All statistical analyses were done using Graphpad Prism 3.0 for Macintosh (Graphpad Software, San Diego, CA, USA). A P value of less than 0.05 was considered statistically significant. Values are expressed as mean±S.D. or mean±S.E.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
HL-1 cardiomyocytes have previously been used in hypoxic studies and show equivalent responses to adult primary cardiomyocytes in terms of expression of hypoxic-inducible genes [14]. To ensure that our hypoxic protocols resulted in typical cardiomyocyte responses to hypoxic stress, we measured LDH release and cell viability (e.g. Refs. [18,19]). In addition, since hypoxic effects on membrane transport processes have not been investigated in HL-1, we measured glucose transport since glucose uptake increases several fold in response to hypoxia in cardiomyocytes (e.g. Refs. [20,21]). LDH release can be used as an indicator of cellular damage [25] in advance of decreases in cell viability and can be detected in the extracellular medium following relatively short periods of hypoxic challenge [18]. After 4 h of hypoxic exposure, some cellular injury was present in HL-1 cells. However, typical cellular responses to hypoxia became increasingly evident at 8 and 20 h. We observed fold-increases in LDH levels (indicating cellular stress), which preceded decreases in cell viability indicating cell death (Fig. 1), although cells died rapidly following more than 20 h hypoxia (data not shown). Based on these observations, we used 8–20 h of hypoxic exposure for further experiments.


Figure 1
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  		Fig. 1 Cell viability decreases and LDH release increases following hypoxic exposure. (a) Cell viability was determined using trypan blue assay (pooled data, mean±S.D., n = 3). (b) LDH release was measured as outlined in Materials and methods. Values are expressed as fold increase relative to control (normoxia), (pooled data, mean±S.E., n = 3), ** indicates significant difference compared to control.

 
Glucose uptake increased twofold following hypoxic challenge (Fig. 2a), which was correlated with a dramatic increase in GLUT1 and GLUT4 protein in the membrane of HL-1 cells (Fig. 2b). This increase was not due to translocation from cytosolic pools (as previously described for acute responses to ischemia in rat cardiomyocytes [42]) since very little GLUT protein was present in the cytosol. GLUT protein levels increased substantially more than transport rates (e.g. GLUT1 protein levels increase 11-fold at 8 h and 28-fold at 20 h) suggesting that there is a correlative increase in both transport and amount of protein but that magnitude of increase differs significantly.


Figure 2
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  		Fig. 2 Chronic hypoxia leads to an increase in glucose uptake and GLUT protein in HL-1 cells. (a) Uptake of [3H]-2-deoxy-glucose (10 µM) was measured after 8 and 20 h of hypoxic exposure. Values are expressed as uptake (%) relative to control (pooled data, mean±S.D., n = 2, each experiment conducted in triplicate, ** indicates significant difference compared to control). (b) Western analysis of GLUT1 and GLUT4 in cytosolic and membrane fractions (50 µg protein per lane) from HL-1 cells treated as in panel a). Representative experiment shown, repeated twice with similar results.

 
Taken together, these data established that HL-1 cells respond to hypoxia in a way that is consistent with that previously described for primary cultures of adult cardiomyocytes and supported further use of HL-1 for the study of hypoxic regulation of ENTs.

We next determined how hypoxia regulates ENT-dependent adenosine transport. We compared adenosine transport in the presence and absence of NBTI (which inhibits ENT1 only) after hypoxia and found that adenosine transport via ENTs decreased by 20% after 8 h hypoxia and 50% after 20 h (Fig. 3a). Approximately 20% of the adenosine uptake in HL-1 cells is due to mENT2 [12]. Therefore, to determine if the decrease in total uptake was due to a change in the proportion of uptake via mENT1 compared to mENT2, we measured the NBTI-insensitive component of adenosine uptake at both time points. We found that the mENT2-dependent component of adenosine uptake remained constant; NBTI-insensitive uptake was 21.88±2.13% after 8 h of hypoxic exposure and 19.6±2.40% after 20 h compared to paired normoxic controls (mean±S.D., n = 2, each experiment conducted in triplicate). These data suggest that both mENT1 and mENT2 are down-regulated by hypoxia. Since the major proportion of adenosine flux in HL-1 cells is via the ENT1 isoform (and mENT2 may be more important in nucleobase flux [43]), we focused the remainder of the study on mENT1 exclusively.


Figure 3
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  		Fig. 3 Chronic hypoxia leads to a decrease in adenosine uptake and NBTI binding. (a) HL-1 cells were exposed to 8 and 20 h of hypoxia and [3H]-adenosine uptake was measured (10 s). Uptake shown as a proportion of normoxic controls (pooled data, mean±S.E.M., n = 4, each condition conducted in triplicate, ** indicates significant difference compared to control). (b) NBTI binding in HL-1 cells following 20 h hypoxia. Representative experiment, each value is the mean of two replicates. Experiment was repeated three times with similar results.

 
Changes in transport of substrate following hypoxia likely correlate with changes in the amount of transport protein at the membrane (as suggested by our GLUT data). However, antibodies against mENT1 are not currently available. Therefore, we used NBTI-binding as an indication of the presence of mENT1 proteins (Fig. 3b). After 20 h of hypoxic exposure we found a decrease of 25% in NBTI binding sites (control, Bmax=1.26±0.18 pmole/mg protein; hypoxia, Bmax=0.94±0.14 pmole/mg protein, mean±S.D., n = 2, each experiment conducted in duplicate). Affinity of mENT1 for NBTI remained virtually unchanged (control, Kd=0.18±0.02 nM; hypoxia, Kd=0.15±0.01 nM, mean±S.D., n = 2, each experiment conducted in duplicate). Since the decrease in NBTI-binding (25%) is not sufficient to account for the decrease in uptake (50%), we speculated that some of the mENT1 proteins are functionally inactive as previously proposed [22]. That is, NBTI-binding is an indication of the presence of mENT1 protein but not functional status. We have previously demonstrated that PKC regulates ENT1 activity in human cells [22]. Therefore, we measured PKC activity and found a significant decrease after extended hypoxia (Fig. 4a). These data suggest that chronic hypoxia down-regulates PKC, which correlates with the decrease in mENT1 adenosine uptake.


Figure 4
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  		Fig. 4 Chronic hypoxia down-regulates total PKC activity and PKC{varepsilon} specifically in HL-1 cells. (a) Total PKC activity (membrane plus cytosol) was measured after 20 h of hypoxia and is shown as a proportion of normoxic controls. (b) Western analysis of PKC{delta} and PKC{varepsilon} in cytosolic (C) and membrane (M) fractions from control and hypoxic cells (100 µg protein loaded per lane). Representative experiment shown, repeated twice with similar results. (c) Decrease in total amount of PKC{varepsilon} following chronic hypoxia but no overall change in total amount of PKC{delta} (mean±S.D., n = 2). ** indicates significant difference compared to control.

 
We have previously shown that the PKC isoforms {delta} and/or {varepsilon} regulate ENT1-dependent nucleoside uptake in human cells [22]. PKC{varepsilon} is well established to play a pivotal role in cardiomyocyte responses to hypoxic challenge [23]. Therefore, we determined if PKC{delta} and {varepsilon} were affected by hypoxia and found that, whereas PKC{delta} was unaffected, PKC{varepsilon} virtually disappeared from the particulate fraction (Fig. 4b). Moreover, the total amount of PKC{varepsilon} was reduced by approximately 50% (Fig. 4c). This significant decrease in PKC{varepsilon} correlates with a reduction in overall enzyme activity (Fig. 4a) and a decrease in mENT1 transport.

To confirm the involvement of PKC in regulation of mENT1 activity, we mimicked the hypoxic down-regulation of PKC pharmacologically, using phorbol ester as previously described [22]. This resulted in a significant decrease in adenosine uptake (Fig. 5a) though not to the same extent as seen with hypoxia, possibly due to less effective down-regulation of PKC{varepsilon}. Indeed, while there was a decrease in total PKC{varepsilon} (Fig. 5c), some enzyme remained activated (i.e. present in the particulate fraction, Fig. 5b). It is possible that longer exposures to PMA would result in a more efficient down-regulation of PKC{varepsilon} and greater decrease in adenosine uptake. However, PMA is non-specific and toxic to cells for long exposures so we did not attempt longer incubations. PKC{varepsilon} in HL-1 cells may also be resistant to phorbol ester down-regulation, as described for other cell lines such as NIH 3T3 [17]. Therefore, we used a more specific approach targeting PKC{varepsilon} with a dominant negative construct, pSVK3PKC{varepsilon}1-401. This construct consists of the N-terminal region of PKC{varepsilon}, containing the regulatory domain, which physically interacts with the pseudosubstrate domain of PKC{varepsilon} to block its catalytic activity. The C-terminal catalytic domain has been deleted in the construct. Transfection of the dominant negative PKC{varepsilon} into HL-1 resulted in approximately 50% decrease in adenosine transport after 24 h, mimicking the effects of hypoxia on adenosine transport (Fig. 6).


Figure 5
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  		Fig. 5 Chronic phorbol ester treatment down-regulates adenosine transport and PKC{varepsilon}. (a) HL-1 cells were serum deprived (6 h) and then treated with PMA (100 nM, 18 h) and adenosine uptake measured at 10 s. Uptake is shown as a proportion of normoxic controls (pooled data, mean±S.E., n = 3, each condition conducted in triplicate). (b) Western analysis of PKC{varepsilon} following treatment as described in panel a. Representative experiment shown, repeated three times with similar results (100 µg protein loaded per lane). (c) Decrease in total PKC{varepsilon} following PMA treatment (pooled data, mean±S.E., n = 3). ** indicates significant difference compared to control.

 

Figure 6
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  		Fig. 6 Expression of a dominant negative PKC{varepsilon} in HL-1 cells mimics the effects of chronic hypoxia. HL-1 cells were transiently transfected with the PKC{varepsilon} dominant negative construct, pSVKPKC{varepsilon}1-401. Adenosine uptake (at 10 s) was measured 18 and 24 h post transfection. Representative experiment shown (mean±S.D., each condition conducted in sextuplicate). Repeated twice with identical results.

 
To determine if the decrease in mENT1 protein (as suggested by reduced adenosine uptake and NBTI binding) is due to decreased transcriptional activity of the mENT1 gene, we measured changes mENT1 RNA levels in HL-1 cells following hypoxia. Levels of mENT1 mRNA were significantly lower after 4 h of hypoxia (compared to paired normoxic controls) but recovered to normal levels by 20 h (Fig. 7a and b). Down-regulation of PKC by PMA mimicked the effect of hypoxia on mENT1 mRNA levels (Fig. 7c). Paradoxically, the time course of changes in mENT1 RNA levels does not reflect the time course of changes in adenosine transport or NBTI binding. While there is a suggestion that a decrease in mENT1 mRNA precedes a decrease in protein and transport, overall mRNA and protein levels do not correlate precisely with each other nor with functional adenosine transport in HL-1 cells suggesting complex regulation.


Figure 7
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  		Fig. 7 Chronic hypoxia leads to a decrease in mENT1 mRNA levels. (a) HL-1 cells were exposed to normoxia or hypoxia for times indicated and total RNA analyzed (50 µg per lane) as described in Materials and methods. Expression levels were normalized with 18s cDNA. Representative blot shown, experiment repeated three times. Lower panel shows pooled data of mENT1 mRNA expression as a proportion of normoxic control (mean±S.E, n = 3). (b) HL-1 cells were treated as in Fig. 5. Total RNA (50 mg per lane) was extracted and analyzed as in panel a. Representative blot shown, experiment repeated three times with similar results. Lower panel shows pooled data (mean±S.E, n = 3). ** indicates significant difference compared to control.

 
These data suggest that hypoxia down-regulates mENT1 activity by a mechanism that involves PKC{varepsilon}. PKC may also be involved in the regulation of mENT1 RNA levels, either through regulation of transcription or mRNA stability. The total decrease in mENT1-dependent adenosine uptake by HL-1 cells following chronic hypoxia is likely due to a combination of overall decreased mENT1 protein in the cell, possibly as a consequence of a reduction in mENT1 mRNA levels, and decreased activity of those mENT1 proteins remaining at the cell membrane.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Chronic hypoxia in the cardiovasculature is a serious complication in a number of pathophysiological and clinical situations (e.g. coronary occlusion and surgery). In the heart, adenosine, acts as a paracrine and autocrine signal of imbalance between cellular oxygen availability and oxygen usage. Since adenosine is also an essential component of ATP synthesis, adenosine pools need to be carefully maintained in metabolically active cells such as cardiomyocytes. ENTs are responsible for the flux of adenosine across cardiomyocyte membranes and ENT1 is highly expressed in cardiovascular tissue [7]. Thus, ENT1 is an important, but poorly understood, component of adenosine physiology in cardiomyocytes.

HL-1 cells are a model system for the study of adenosine physiology [12] and we show here that they respond to hypoxia in a similar manner to that previously described for rodent cardiomyocytes [18,24,25]. Typical cellular responses to hypoxia include an increase in glucose transport [21,26,27], which is due to an increase in GLUT1 protein levels (rather than a translocation to the membrane [42]). Indeed, hypoxic induction of glut1 gene expression has been well described [21,28]. Paradoxically, GLUT1 protein levels increase substantially more than uptake suggesting that absolute amount of protein does not correlate exactly with transport activity. This mismatch between protein levels and transport also appears to the case for adenosine uptake, since chronic hypoxia resulted in a greater decrease in adenosine transport than NBTI binding. Although intracellular NBTI binding sites have been described [29,30], more recent immunocytochemical data suggest that ENT1 is located primarily in the plasma membrane [31–33]. Thus, fewer NBTI-binding sites are more likely to be due to less total cellular mENT1 protein rather than trafficking away from the membrane. Therefore, we propose that chronic hypoxia decreases mENT1dependent adenosine uptake as a consequence, at least in part, of a decrease in the amount of mENT1 protein at the membrane.

A decrease in mENT1 protein only partly explains the overall decrease in adenosine uptake. A number of transporters are regulated by kinases (e.g. Refs. [34,35]) and we have previously proposed that ENT1 is regulated by PKC{delta} and/or {varepsilon} such that both presence of ENT1 and kinase activation are necessary for maximal transport [10,22]. PKC{varepsilon} plays an important role in cardiomyocyte physiology [36], particularly in response to ischemic or hypoxic stress [37], and we have found that it is strongly affected by hypoxia in HL-1 cells. Therefore, we propose that PKC{varepsilon} regulates mENT1 activity in HL-1 cells and down regulation of PKC{varepsilon} during chronic hypoxia results in a down-regulation of mENT1. Pharmacological down-regulation of PKC{varepsilon}, by PMA (which regulates PKC{varepsilon} in a manner similar to physiological activators in cardiomyocytes [38]) or a dominant negative construct, mimics the effect of hypoxia supporting our model for mENT1 regulation.

The underlying mechanisms of PKC{varepsilon} regulation of ENT1 are not known but may involve phosphorylation of the protein, either by PKC{varepsilon} or a kinase regulated by PKC. Standard sequence analysis software (e.g. PROSITE) predicts a number of putative kinase target sites within intracellular loops of mENT1. Some of these sites are conserved in human ENT1 and rat ENT1, which is suggestive of functional relevance although clearly further studies are required.

Chronic hypoxia in HL-1 cells leads to a decrease in mENT1 protein (based on NBTI binding studies) and in mRNA levels. The decrease in mENT1 protein and mRNA cannot be ascribed to a decrease in overall cellular protein or RNA synthesis rates since GLUT protein levels increases substantially and PKC{delta} levels remain unaffected. In addition, while moderate hypoxia may lead to a reduced rate of energy production by cardiomyocytes, this is not associated with decreased rates of protein and RNA synthesis [39]. Unexpectedly, the effects of hypoxia on transcription and/or mRNA stability occur much sooner than effects seen at the protein level suggesting that there is a considerable lag time between hypoxic effects on ENT1 at the gene/mRNA level and the consequent response by the mENT1 protein at the cell membrane. Indeed, hypoxia appears to have diverse effects on transport proteins, stimulating transcription for glut1 but decreasing mRNA stability for the amino acid transporter, LAT1 [28]. Expression levels of ENT1 are highly variable [7] and do not appear to correlate directly with cellular toxicity of nucleoside analog drugs [40] suggesting that the relationship between transcriptional activity, mRNA stability, protein levels and functional transport is complex and still poorly understood.

To our knowledge, the only other study on the effects of hypoxia on ENTs used PC12, a rat line, which is a model for studies on neural cellular physiology [41]. In these cells, chronic hypoxia decreased rENT1 mRNA levels and adenosine uptake [41] in agreement with our findings. These data suggest that ENT1 gene expression may be sensitive to hypoxic regulation in both cardiac and neural tissue.

The ability of a cardiomyoycyte to regulate adenosine flux across the membrane, particularly if coupled to adenosine receptor activation, is physiologically useful in a number of situations. Hypoxic stress results in an imbalance between oxygen supply and consumption, and is usually accompanied by a rapid loss of adenosine from the cell via ENTs. Adenosine loss could be minimized by down-regulation of ENT activity, which might aid in restoration of ATP pools once the stress has been removed. Alternatively, an increase in ENT activity could lead to reuptake of extracellular adenosine to terminate receptor activation (as is the case for the neurotransmitter transporters) and/or help restore intracellular pools of adenosine. The precise nature of the relationship between adenosine transporters, adenosine receptors and intracellular signaling pathways remains to be determined.

These data are the first to show regulation of the adenosine transporter, mENT1, by hypoxia, in cardiomyocytes. Moreover, we demonstrate that PKC{varepsilon} is involved in regulation of mENT1 and propose a mechanism whereby chronic hypoxia regulates ENT1, both in terms of number of proteins and also in overall activity.


   Acknowledgements
 
The authors wish to thank Dr. Andre Bedard for assistance with use of the dominant negative PKC{varepsilon} construct and Dr. William Claycomb for useful suggestions. This work was funded by a grant to IRC from the Canadian Institutes from Health Research (CIHR; MOP-38013).


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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