Expression of Glutamate Transporters in Rat Cardiomyocytes and Their Localization in the T-tubular System
Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany
Correspondence to: Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstr. 6, D-97070 Würzburg, Germany. E-mail: peter.kugler{at}mail.uni-wuerzburg.de
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Summary |
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(J Histochem Cytochem 52:13851392, 2004)
Key Words: glutamate transporters GLT1 variant EAAC1 cardiomyocytes transverse (T)-tubules
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Introduction |
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The concentrations of glutamate and aspartate in cardiomyocytes are 50- to 100-fold greater than the concentrations found in the cardiac circulation (Suleiman and Chapman 1993
; Dinkelborg et al. 1995
; Suleiman et al. 1997
). The cellular uptake of glutamate and aspartate may be enabled by glutamate transporters localized in the sarcolemma of cardiomyocytes. It is known that the mRNA for the glutamate transporters EAAC1 (detected by Northern blotting analysis; Kanai and Hediger 1992
) and GLT1 variant (GLT1v, detected by RT-PCR; Schmitt et al. 2002
) are present in the heart. Furthermore, glutamate (Dinkelborg et al. 1995
,1996
) and aspartate transport rates have been characterized and EAAC1 protein was detected in sarcolemmal vesicles in isolated cells from rat heart (King et al. 2001
). However, no studies are available about the cellular expression of glutamate transporters in the heart.
In this study, I used a highly sensitive method that provides clear cellular resolution, i.e., non-radioactive in situ hybridization (ISH) using cRNA probes (Kugler and Schmitt 1999; Schmitt et al. 2002
) to detect GLT1v and EAAC1 in rat heart. The distributions of GLT1v and EAAC1 proteins were detected by immunocytochemistry applying affinity-purified polyclonal antibodies against synthetic C-terminal transporter peptides to semithin plastic sections of myocardium (Kugler and Schmitt 1999
; Schmitt et al. 2002
). For confirmation of transporter protein detection in the heart I used Western blotting.
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Materials and Methods |
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In Situ Hybridization
The hearts removed were frozen in liquid nitrogencooled propane. Twelve-µm-thick sections were cut in a cryostat (20C; Frigocut 2800 E, Reichert-Jung, Nussloch, Germany). The sections were mounted on precoated glass slides (Superfrost Plus; Menzel, Braunschweig, Germany) and thawed. The sections were fixed for 5 min in freshly prepared 4% formaldehyde in 0.1 M PBS, pH 7.4. The fixed sections were transferred to absolute ethanol and stored at 4C for 12 days and then processed for ISH.
The ISH procedure used was previously described in more detail (Kugler and Schmitt 1999). Briefly, the sections were removed from ethanol, rehydrated in a graded series of ethanol, transferred to 2 x standard saline citrate (SSC), and treated with 0.05 N HCl for 30 min. After washing with 2 x SSC, the sections were incubated with freshly prepared 0.25% acidic anhydride, washed again with 2 x SSC, and covered with the hybridization solution containing the digoxigenin (DIG)-labeled antisense (sense) cRNA probe (final concentration 89 ng/µl) and 550 µg/ml salmon testis DNA (Sigma; Deisenhofen, Germany) in 4 x SSC, 1 x Denhardt's solution (Sambrook et al. 1989
), 10% dextran sulfate, and 50% deionized formamide at 60C for 1618 hr. Posthybridization washes were done stepwise at room temperature with 2 x SSC, 1 x SSC, 50% formamide, and then again with 2 x SSC. Next, the sections were treated with 40 µg/ml RNase A (50 Kunitz U/mg; Boehringer, Mannheim, Germany) in a solution containing 500 mM NaCl, 10 mM Tris-HCl (pH 8), and 1 mM EDTA at 37C for 30 min to remove unhybridized single-strand RNAs. After this treatment, the sections were incubated with the same buffer without RNase A at 60C for 30 min.
The sections were then rinsed in Tris-buffered saline (TBS; 100 mM Tris and 150 mM NaCl, pH 7.5) for 5 min, incubated with TBS containing 0.5% blocking reagent (DIG Nucleic Acid Detection Kit; Boehringer) for 30 min, followed by 0.3% Triton X-100 in TBS for 20 min. After incubation with 1.5 U/ml sheep anti-DIG-aP (alkaline phosphatase) conjugated (Boehringer) in TBS containing 0.3% Triton X-100 for 60 min, the sections were washed in TBS and transferred to a 0.1 M Tris buffer (pH 9.5) containing 100 mM NaCl and 50 mM MgCl2 for 2 min before the aP visualization (see below).
The DIG-labeled antisense (sense) cRNA probes used were generated exactly as described previously (Kugler and Schmitt 1999; Schmitt et al. 2002
). The probes were complementary to nucleotide (nt) 16542043 of the GLT1v sequence (GLT1v cRNA; Schmitt et al. 2002
) and to nt 2471706 of the EAAC1 sequence (EAAC1 cRNA; Kugler and Schmitt 1999
).
The procedure used for detection of aP was described recently (Asan and Kugler 1995). The incubation medium contained 0.4 mM 5-bromo-4-chloro-3-indolylphosphate (BCIP; Boehringer), 100 mM NaCl, 50 mM MgCl2, 0.4 mM tetranitroblue tetrazoliumchloride (Serva; Heidelberg, Germany) in 100 mM Tris-HCl buffer at pH 9.5.
For a control, substitution of the antisense cRNA probe by an equivalent amount of labeled sense cRNA probe led to a complete lack of staining (Figure 1b) . Omission of labeled cRNA probes from the respective hybridization mixtures resulted in completely unstained sections.
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Immunoblotting
For immunoblotting, the hearts and whole brains (for a positive control) removed were homogenized at 4C in 10 mM NaH2PO4 (pH 7.2) containing 2 mM MgCl2, aprotinin (5 µg/ml), leupeptin (2 µg/ml), pepstatin (2 µg/ml), and phenylmethylsulfonyl fluoride (100 µg/ml). The homogenate was centrifuged at 1000 x g for 10 min, and the resulting supernatant was centrifuged at 150,000 x g for 1 hr. The protein content of the 150,000 x g pellet was determined by the Bio-Rad protein assay (Bio-Rad; Munich, Germany) and used for immunoblotting. Proteins (60 µg per lane) were electrophoretically separated on 10% gels by SDS-PAGE. Then the proteins were transferred electrophoretically to nitrocellulose membranes (Burnette 1981). Strips of the nitrocellulose membranes were incubated for 24 hr at 4C with the affinity-purified transporter antibodies (
1.8 µg/ml). Bound immunoglobulins were visualized using peroxidase-conjugated goat anti-rabbit IgG (1:3000; Bio-Rad, Richmond, Canada; blotting grade) and the enhanced luminol chemiluminescence technique (Amersham; Braunschweig, Germany). Antibodies previously absorbed with an excess of the peptides used for immunization served as control. These immunoblots showed no labeling (Figure 2)
.
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The tissue sections were preincubated for 3 hr at room temperature with 2% bovine serum albumin, 10% normal goat serum, and 0.05% Tween-20 (Ferrak; Berlin, Germany) in PBS, pH 7.4. Then the sections were incubated for 2448 hr at 4C with the primary antibody diluted in the preincubation solution (anti-glutamate transporters 15 µg/ml; anti-type IV collagen 1:50; anti-NCX 1:5 to 1:20). After several washes with PBS, the semithin plastic sections were incubated for 90 min at room temperature with indocarbocyanin (Cy3)-labeled secondary antibody (1:800; goat anti-rabbit IgG; Dianova, Hamburg, Germany) and/or with carbocyanin (Cy2)-labeled secondary antibody (1:50; goat anti-mouse IgG; Dianova). Controls were performed with primary antibody, previously absorbed with an excess of the corresponding glutamate transporter peptide used for immunization or without primary antibody. These sections were unstained (Figures 4b and 4d). The sections were examined with a Zeiss laser-scanning microscope (Zeiss LSM 510) equipped with an argon laser (488 nm) for excitation of Cy2, an HeNe laser (543 nm) for excitation of Cy3, and appropriate filter combinations for visualization of Cy2 and Cy3 emissions.
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Results |
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Western Blotting and Immunostaining
In immunoblots of the 150,000 x g pellet of heart homogenate, the affinity-purified antibodies generated in rabbit and mouse against GLT1v peptide labeled a band at 60 kD (Figure 2), showing a somewhat higher electrophoretic mobility than in brain homogenates (Figure 2; cf. Schmitt et al. 2002
). This may be caused by variant glycosylation of GLT1v in heart and brain. The affinity-purified antibody against EAAC1 peptide (from rabbit) labeled a band at
60 kD (Figure 2), which is in accordance with previous results of immunoblotting analyses in heart (King et al. 2001
) and brain (Figure 2; cf. Kugler and Schmitt 1999
; Kugler and Beyer 2003
).
When the affinity-purified antibodies against GLT1v and EAAC1 were applied to semithin plastic sections (1 µm thick) of the myocardium (left and right ventricular walls), an identical staining pattern was observed intracellularly in cardiomyocytes (Figures 3, 4a, and 4c) . In longitudinal sections through cardiomyocytes, reaction product was deposited in small dots that were arranged in longitudinal parallel rows forming dotted strands (Figure 3a). In tangential longitudinal sections through the cell periphery of cardiomycytes, it was obvious that the dots of neighboring strands also formed transversely oriented dotted strands with regular distances (Figures 3a, 3d, 4a, and 4c). In transverse sections through cardiomyocytes, the fluorescent staining was also arranged in small dots and in short strands that sometimes formed circular profiles and reached to the surface of cells (Figures 3g and 3k). The sarcolemma of cells seemed to be almost unstained, but the dots of reaction product detected in the cell periphery could be also bound to the sarcolemma.
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To prove the likely colocalization of GLT1v and EAAC1, double immunolabeling was performed using a mouse antibody against GLT1v and a rabbit antibody against EAAC1. The resulting immunostaining showed that both transporters are equally codistributed throughout the T-tubular system (Figures 3n3s).
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Discussion |
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Applying immunocytochemistry on semithin plastic sections (1 µm thick), I was able to detect GLT1v and EAAC1 proteins colocalized in the T-tubular system of cardiomyocytes. The transporter proteins were highly enriched in this localization, and a possibly sarcolemmal spotty labeling could not be excluded with certainty at the light microscopic level. The T-system consists of tubular invaginations of the sarcolemma at the Z-band, extending deep into the cytoplasm and representing a specialized form of sarcolemma. The volume density of the T-system is up to 3.2%, and it represents about one third of the entire cell membrane area (Stewart and Page 1978; Severs et al.1985
). It is generally accepted that the T-system is necessary for the rapid propagation of the excitation impulse throughout the muscle fiber (Sommer 1995
).
Key proteins involved in excitationcontraction (EC) coupling and further membrane proteins are concentrated at the T-tubules, e.g., L-type Ca2+ channels (Carl et al. 1995; Takagishi et al. 2000
), Na+/Ca2+-exchange protein (Frank et al. 1992
; Thomas et al. 2003
), voltage-gated K+ channel, Kv 4.2 (Takeuchi et al. 2000
), and Na+-pump
-catalytic subunit isoforms (McDonough et al. 1996
). On stimulation, the insulin-regulated glucose transporter GLUT4 is found not only in the sarcolemma but also in the T-tubular system (Slot et al. 1991
). Moreover, we now can show that the glutamate transporters GLT1v and EAAC1 are also localized in this special domain of the sarcolemma. The deep invaginations of the T-tubules in the cytoplasm of cardiomyocytes contribute to the shortening of the cytoplasmatic diffusion distances of glutamate and aspartate transported through the T-tubule membranes. They also facilitate the intracellular enrichment of both amino acids for their extensive use in the intermediary metabolism.
The uptake process of both glutamate transporters is electrogenic and is driven by the ion gradients of K+ and Na+ (for review, see Danbolt 2001). Under ischemic conditions in the brain, glutamate is released mainly from neurons via reversed operation of glutamate transporters (Seki et al. 1999
; Rossi et al. 2000
; for review, see Danbolt 2001
). Provided that the glutamate transporters in the heart operate similarly to those in the brain, the same mechanism could be responsible for the severe loss of dicarboxylic amino acids during acute ischemia or anoxia of the myocardium (Song et al. 1996
). In brain slices, ischemia removes the ATP supply to the Na+/K+ pump, K+ accumulates in the extracellular space, depolarizing the cells, and the Na+ gradient driving glutamate uptake is reduced so that glutamate is moved not into the cells but out of them to the extracellular space (Rossi et al. 2000
). In hearts perfused by the Langendorff technique, the reversed operation of glutamate transporters is probably one of various mechanisms that lead to loss of these amino acids during acute ischemia (Song et al. 1998
).
Further studies are needed to show whether, in addition to GLT1v and EAAC1, further glutamate transporters are expressed in the myocardium. It is now proven (Y. Dehnes and N.C. Danbolt, unpublished data) by immunoprecipitation, subsequent SDS-PAGE, and immunoblotting that at least three different glutamate transporters are detectable in the heart (Danbolt 2001). The significance of the coexpression of different high-affinity glutamate transporters in the same cell type (also documented in the brain; Lehre et al. 1995
; Schmitt et al. 2002
; Kugler and Beyer 2003
) is not obvious.
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Acknowledgments |
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I am indebted to Erna Kleinschroth and Julia Medvedev for their excellent technical assistance.
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Footnotes |
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