- Email: [email protected]
Tissue specific membrane association of α1T, a truncated form of the α1 subunit of the Na pump
FEBS Letters 337 (1994) 285-288
ELSEVIER
LETTERS
FEBS 13526
Tissue specific membrane association of alT, a truncated form of the al subunit of the Na pump Julius C. Allen”,*, Thomas A. Pressleyb, Timothy Odebunmi”, Russell M. Medford’ “Section of Cardiovascular Sciences, Department of Medicine, Baylor College of Medicine, Houston, TX 77030-3498, USA bUniversity of Texas, Health Sciences Center Houston, Department of Physiology, Houston, TX, USA ‘Emory University College of Medicine, Division of Cardiology, Atlanta, GA, USA Received 23 November 1993
Abstract
We have assessed the Na pump a-subunit isoform content utilizing site directed antibodies in two vascular smooth muscle (VSM) preparations known to contain functional Na pump sites, VSM microsomal fractions (Na’,K’-ATPase) and intact primary confluent cells (ouabain inhibited 86Rb uptake). A comparison of isoform content was made with kidney microsomes. Both VSM and kidney microsomes contained a full length al subunit (I 100 kDa) as well as a truncated subunit, alT (- 66 kDa). SDS treatment of VSM microsomes effected an increase in Na’,K’-ATPase and a retention of alT. SDS treated kidney microsomes retained the al isofonn and Na’,K’-ATPase. Confluent VSM cells showed no detectable al, only alT. In the absence of detectable full length al, the alT protein may represent a functional Na pump component in canine VSM. Key words: Na pump;
a Subunit
isoform;
Vascular
smooth
muscle
1. Introduction The Na’,K’-ATPase, the enzymatic manifestation of the transmembrane Na’ pump found in virtually all animal tissue has been well characterized in recent years. It is generally thought to consist of at least three isoforms of the larger 100 kDa a-subunit (al, ~2, ~3) complexed with one of at least three smaller (- 50 kDa) /? subunit isoforms. However, the specific nature of the complex, its control at all levels, and the function of the isoforms, are still under intense investigation [1,2]. We have recently demonstrated that instead of the expected - 100 kDa Na,K-ATPase al subunit, canine vascular smooth muscle expresses a - 66 kDa alT (truncated) isoform protein derived from the a1 gene by alternative RNA processing [3]. This protein is identical to the full length al through Gly 554. There is then an abrupt divergence in sequence, with the encoding of 27 amino acids from the retained intron sequence, completing the carboxy end of the molecule. The truncated protein contains the same key sites as the full length protein: the FITC binding site, phosphorylation sites, and the ouabain binding site. Furthermore, polymerase chain reaction analysis of the same tissue fails to detect mRNA for the a2 or a3 isoforms, although small amounts of al mRNA are present [4]. In the absence of a detectable, ‘classical’ a subunit, these results would suggest that alT may
*Correspondingauthor. Fax: (1) (713) 790 0681.
function as a transport protein in vascular smooth muscle. There are a variety of approaches available to determine if this alT can function as a pump protein. We have chosen to utilize existing models of VSM membranes and intact cultured cells to assess this possible role. Specifically we determined the isoform content of these two well known preparations each of which contains what is accepted as Na pump mediated functions: (i) the Na’,K+-ATPase activities of SL membrane fractions [5], and (ii) ouabain inhibited *‘jRb uptake of confluent canine VSM cells [6,7]. If the ollT isoform is functional in VSM, it should be readily demonstrable in these two model systems.
2. Methods and materials 2. I. Microsomal preparations 2.1.1. Vascular smooth muscle (carotid artery or saphenous vein). Standard procedures were used. In brief, tissues were cleaned in the cold room (0_4”C), minced with a McIlwain Chopper, and dispersed in a Polytron, 3 times for 15-20 s in approximately 15 ml 0.25 M Sucrose, 10 mM Tris, pH 7.4. The supematant from a 2,000 xg 15 min spin was centrifuged at 100,OOOxg for 1 h. The resulting pellet was suspended in about 500~1 of homogenizing solution and protein concentration determined by the method of Bradford [9]. 2.1.2. Kidney medulla and cortex. Crude microsomes were prepared in a manner similar to the VSM microsomes. Instead of a McIlwain Chopper the tissue was minced with scissors. The initial spin was at 1,000 x g 20 min followed by a spin of 100,000 x g 60 min.
2.2. SDS treatment The microsomes were incubated for 45 min at room temperature with
0014-5793/94/$7.00 0 1994 Federation of European Biochemical Societies. All rights reserved. SSDI 0014-5793(93)El443-P
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J. C Allen et al. I FEBS Letters 337 (1994) 285-288
100 kDa65 kDa -
Fig. 2. Immunoblot of vascular microsomes, using anti-HSL antibody. Vascular microsomes were isolated and treated identically to the microsomes as outlined in Fig. 1.
assessed its content with ‘anti-HSL’, an antibody that reveals only that isoform, and not al full length. Fig. 2 shows an immunoblot of control and SDS-treated VSM microsomes using ‘anti-HSL’. No 100 kDa (al full length) band is present, and SDS treatment does not eliminate the band corresponding to alT. Thus detergent treatment of VSM eliminates cxl protein, while retaining ollT. The opposite is true for kidney microsomes which retain ~1 and eliminate alT upon detergent treatment. 3.2. VSA4 cell culture Since VSM microsomes retained the ollT and Na+,K+ATPase activity after SDS treatment, we wished to determine the major isoform expressed in cells that clearly demonstrate Na pump function. We have shown that [3H]ouabain binding and ouabain inhibited 86Rb uptake are present in a variety of cell cultures of canine VSM [6,7] and presume these to be representative of the Na pump. Fig. 3 shows an immunoblot of total protein from saphenous vein smooth muscle cells grown for 1, 4 and 5 days probed with ‘anti-LEAVE’ using canine kidney medulla for comparison. It can be seen that while both al and alT were present in kidney microsomes (see Fig. 1). only alT was detectable in proliferating VSM cells.
4. Discussion The data contained in this paper strongly suggest but do not prove that the 66 kDa, cllT protein may serve a transport function in VSM. We showed that SDS treatment of VSM microsomes enhanced the Na’,K’-ATPase activity of the preparation, eliminated the ccl protein, and retained alT protein. This is in sharp contrast to similar SDS treatment of kidney microsomes, which resulted in elimination of alT and retention of the al and Na’,K’-ATPase. Thus SDS treated VSM microsomes show Na’,K+-ATPase activity, ouabain binding, no detectable full-length al protein, and alT protein.
It appears that despite the presence of both al and a 1T isoforms in crude microsomal fractions from kidney and VSM, the proteins have differing membrane-associative characteristics. This is not surprising in view of their large structural differences and may also reflect different lipid profiles of the membranes of different tissues as well. Regardless of the reasons for such tissuespecific differences, we suggest that selective retention in the cell membrane of either of the 01 subunits reflects interaction with the ,f?subunit necessary to form a functional pump site. Since no detectable full-length al subunit is retained in VSM fractions this argues that the cllT subunit functions as a pump protein in this tissue and that al does not associate with /3. Furthermore, these data suggest that this truncated protein, lacking 40% of the -COOH terminal sequence, can be targeted to the membrane and fulfil1 a transport function. Some workers have suggested that glutamic acid residues 955 and 956 are important in the ion transport function of the full length protein [ 16,171. Since alT lacks this region, this would seem to preclude a transport role for this protein. However, more recent evidence utilizing site-directed mutagenesis of these predicted cation binding sites argues against the contribution of these sites and the carboxy terminus [18]. Thus specific designation of ion transport residues remains unclear and cllT may yet prove to retain the necessary motifs for Na’ and K’ transport. Since intact tissues (or at least microsomes) demonstrate both al and alT proteins, suggestions that alT serves a transport function cannot be made without cytochemical localization studies, or the determination of the actual molecular nature of the pump complex (i.e. (al)@?) or (ollT)(/$). For these reasons, we assessed the protein components contained in confluent cultures of canine vascular smooth muscle cells, which we know express functional Na pump sites [6,7]. These cells show a large component of alT and no detectable al. Clearly since microsomes show an al full-length protein before SDS treatment, the lack of detectable al in confluent
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Fig. 3. Immunoblot of saphenous vein smooth muscle cells, and canine kidney medullary microsomes. Saphenous vein cells were grown for indicated time on 100 mm plastic dishes, lysed with NP 40, and Western analysis performed as in Fig. 1 and Section 2. Canine kidney microsomes were assessed as described above.
J.C. Allen et al. IFEBS
288
cells may reflect the up-regulation of the alT, and downregulation of al. Such modulation is currently under investigation. Freshly dispersed cells do show a small amount of al, which seems to disappear during proliferation (data not shown). Thus strong circumstantial evidence suggests that alT may serve a transport function in VSM. We have shown that in the absence of detectable al, VSM microsomes show a Na’,K’-ATPase activity and [3H]ouabain binding. In addition, in VSM confluent cells, containing apparently normal Na pump function, al was undetectable and alT was clearly demonstrable. We have shown earlier that canine VSM contains both pl mRNA [4] and protein [15], but have not yet determined its associative characteristics with the alT in the sarcolemma. It does, however, remain attached to the sarcolemma after extensive purification attempts with detergents. Furthermore, the SDS data in this paper reveal nothing about the nature of the association of alT with the different membranes, or the reasons for such different associative characteristics. More extensive characterization with additional detergents is necessary to more clearly define these phenomena. Despite these limitations we can conclude that both al and alT exist in both canine kidney and VSM canine tissues but their membrane associative characteristics vary greatly. The al is resistant to SDS treatment in the kidney membrane, and the alT is more tightly retained in VSM. Since there is no detectable al in confluent cultured VSM, it is possible that alT serves as a pump subunit in VSM. In the absence of al in VSM, a.lT may complex with an appropriate /3, insert in the membrane and may form functional pump sites in the SL of this tissue. This would account for the Na’,K’-ATPase activity in VSM SL preparations. The manifestations of alT not associated with a p subunit, and not inserted in the membrane could be significantly different from alT complexed with B, and bound to a membrane. Acknowledgements: Supported by NIH Grants: HL-24585 (JCA) and
HL-39846 (TAP) and by AHA 90-1336 (RMM). TAP is the recipient of an RCDA (HL-002305). RMM is an Established Investtgator of the AHA.
Letters 337 (1994) 285-288
References
111Kaplan, J.H. and De Weer, P. (1991) The Sodium Pump: Structure, Mechanisms and Regulation, Rockefeller University Press, New York. 121Kaplan, J.H. and De Weer, P. (1991) The Sodium Pump: Recent Developments, Rockefeller University Press, New York. [31 Medford, R.M., Hyman, A.M., Allen, J.C., Pressley, T.A., Allen, P.D. and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 183088 18312. [41 Allen, J.C.. Medford, R.M.. Zhao, X. and Pressley. T.A. (1991) Circ. Res. 69. 3944. PI Allen, J.C.. Kahn, A.M., Navran, S.S. (1986) Am. J. Physiol. 250. C536C540. 161Allen, J.C., Navran, S.S., Seidel, C.L., Dennison, D.K., Amann, J.M. and Jemelka. S.J. (1989) Am. J. Physiol. 256, C786792. [71 Feltes, T.F., Seidel, C.L., Dennison, D.K., Amick, S. and Allen. J.C. (1993) Am. J. Physiol. 264, C169-C174. PI Jorgensen, P.L. (1974) in: Methods in Enzymology Volume XXX11 Biomembranes, Part B, pp. 2777290, Academic Press, New York. [91 Bradford, M.M. (1976) Anal. Biochem. 72, 248-259. UOI Pressley, T.A. (1992) Am. J. Phystol. 262, C743-C751. 1111Eggermont, J.A., Vrolix, M.. Raemaekers, L., Wuytack. F. and Casteels, R. (1986) Circ. Res. 62, 26627812. WI Allen, J.C., Smith, D.J. and Gerthoffer, W.T. (1981) Life SCI. 28. 945-950. u31 Navran, S.S., Adair, S.E., Jemelka. S.K., Setdel. C.L. and Allen, J. (1988) Pharm. Exp. and Ther. 245, 6088613. [I41 Jorgenson, P.L. and Anderson, J.P. (1988) J. Membr. Biol. 103. 955120. USI Allen, J.C., Navran, S.S. and Jemelka, S.K. (1988) The functional role of Na pump modulation in vascular smooth muscle, in, Vascular Neuroeffector Mechanisms (Bevan, J.A., Majewskt, H., Maxwell, R.A. and Story, D.F.. Eds.) Vol. 10, pp. 217-224, ICSU Press. U61 Argello, J.M. and Kaplan, J.H. (1991) J. Btol. Chem. 266. 146277 14636. 1171Goldshleger, R.. Tal, D.M., Stem, W.D. and Karlish. S.J.D. ( 1992) Proc. Natl. Acad. Sci. USA 89, 6911-6915. P81 Van Huysse, J.W., Jewell, E.A. and Lingrel, J.B. (1993) Btochemistry 32, 819-826.
ELSEVIER
LETTERS
FEBS 13526
Tissue specific membrane association of alT, a truncated form of the al subunit of the Na pump Julius C. Allen”,*, Thomas A. Pressleyb, Timothy Odebunmi”, Russell M. Medford’ “Section of Cardiovascular Sciences, Department of Medicine, Baylor College of Medicine, Houston, TX 77030-3498, USA bUniversity of Texas, Health Sciences Center Houston, Department of Physiology, Houston, TX, USA ‘Emory University College of Medicine, Division of Cardiology, Atlanta, GA, USA Received 23 November 1993
Abstract
We have assessed the Na pump a-subunit isoform content utilizing site directed antibodies in two vascular smooth muscle (VSM) preparations known to contain functional Na pump sites, VSM microsomal fractions (Na’,K’-ATPase) and intact primary confluent cells (ouabain inhibited 86Rb uptake). A comparison of isoform content was made with kidney microsomes. Both VSM and kidney microsomes contained a full length al subunit (I 100 kDa) as well as a truncated subunit, alT (- 66 kDa). SDS treatment of VSM microsomes effected an increase in Na’,K’-ATPase and a retention of alT. SDS treated kidney microsomes retained the al isofonn and Na’,K’-ATPase. Confluent VSM cells showed no detectable al, only alT. In the absence of detectable full length al, the alT protein may represent a functional Na pump component in canine VSM. Key words: Na pump;
a Subunit
isoform;
Vascular
smooth
muscle
1. Introduction The Na’,K’-ATPase, the enzymatic manifestation of the transmembrane Na’ pump found in virtually all animal tissue has been well characterized in recent years. It is generally thought to consist of at least three isoforms of the larger 100 kDa a-subunit (al, ~2, ~3) complexed with one of at least three smaller (- 50 kDa) /? subunit isoforms. However, the specific nature of the complex, its control at all levels, and the function of the isoforms, are still under intense investigation [1,2]. We have recently demonstrated that instead of the expected - 100 kDa Na,K-ATPase al subunit, canine vascular smooth muscle expresses a - 66 kDa alT (truncated) isoform protein derived from the a1 gene by alternative RNA processing [3]. This protein is identical to the full length al through Gly 554. There is then an abrupt divergence in sequence, with the encoding of 27 amino acids from the retained intron sequence, completing the carboxy end of the molecule. The truncated protein contains the same key sites as the full length protein: the FITC binding site, phosphorylation sites, and the ouabain binding site. Furthermore, polymerase chain reaction analysis of the same tissue fails to detect mRNA for the a2 or a3 isoforms, although small amounts of al mRNA are present [4]. In the absence of a detectable, ‘classical’ a subunit, these results would suggest that alT may
*Correspondingauthor. Fax: (1) (713) 790 0681.
function as a transport protein in vascular smooth muscle. There are a variety of approaches available to determine if this alT can function as a pump protein. We have chosen to utilize existing models of VSM membranes and intact cultured cells to assess this possible role. Specifically we determined the isoform content of these two well known preparations each of which contains what is accepted as Na pump mediated functions: (i) the Na’,K+-ATPase activities of SL membrane fractions [5], and (ii) ouabain inhibited *‘jRb uptake of confluent canine VSM cells [6,7]. If the ollT isoform is functional in VSM, it should be readily demonstrable in these two model systems.
2. Methods and materials 2. I. Microsomal preparations 2.1.1. Vascular smooth muscle (carotid artery or saphenous vein). Standard procedures were used. In brief, tissues were cleaned in the cold room (0_4”C), minced with a McIlwain Chopper, and dispersed in a Polytron, 3 times for 15-20 s in approximately 15 ml 0.25 M Sucrose, 10 mM Tris, pH 7.4. The supematant from a 2,000 xg 15 min spin was centrifuged at 100,OOOxg for 1 h. The resulting pellet was suspended in about 500~1 of homogenizing solution and protein concentration determined by the method of Bradford [9]. 2.1.2. Kidney medulla and cortex. Crude microsomes were prepared in a manner similar to the VSM microsomes. Instead of a McIlwain Chopper the tissue was minced with scissors. The initial spin was at 1,000 x g 20 min followed by a spin of 100,000 x g 60 min.
2.2. SDS treatment The microsomes were incubated for 45 min at room temperature with
0014-5793/94/$7.00 0 1994 Federation of European Biochemical Societies. All rights reserved. SSDI 0014-5793(93)El443-P
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982
287
J. C Allen et al. I FEBS Letters 337 (1994) 285-288
100 kDa65 kDa -
Fig. 2. Immunoblot of vascular microsomes, using anti-HSL antibody. Vascular microsomes were isolated and treated identically to the microsomes as outlined in Fig. 1.
assessed its content with ‘anti-HSL’, an antibody that reveals only that isoform, and not al full length. Fig. 2 shows an immunoblot of control and SDS-treated VSM microsomes using ‘anti-HSL’. No 100 kDa (al full length) band is present, and SDS treatment does not eliminate the band corresponding to alT. Thus detergent treatment of VSM eliminates cxl protein, while retaining ollT. The opposite is true for kidney microsomes which retain ~1 and eliminate alT upon detergent treatment. 3.2. VSA4 cell culture Since VSM microsomes retained the ollT and Na+,K+ATPase activity after SDS treatment, we wished to determine the major isoform expressed in cells that clearly demonstrate Na pump function. We have shown that [3H]ouabain binding and ouabain inhibited 86Rb uptake are present in a variety of cell cultures of canine VSM [6,7] and presume these to be representative of the Na pump. Fig. 3 shows an immunoblot of total protein from saphenous vein smooth muscle cells grown for 1, 4 and 5 days probed with ‘anti-LEAVE’ using canine kidney medulla for comparison. It can be seen that while both al and alT were present in kidney microsomes (see Fig. 1). only alT was detectable in proliferating VSM cells.
4. Discussion The data contained in this paper strongly suggest but do not prove that the 66 kDa, cllT protein may serve a transport function in VSM. We showed that SDS treatment of VSM microsomes enhanced the Na’,K’-ATPase activity of the preparation, eliminated the ccl protein, and retained alT protein. This is in sharp contrast to similar SDS treatment of kidney microsomes, which resulted in elimination of alT and retention of the al and Na’,K’-ATPase. Thus SDS treated VSM microsomes show Na’,K+-ATPase activity, ouabain binding, no detectable full-length al protein, and alT protein.
It appears that despite the presence of both al and a 1T isoforms in crude microsomal fractions from kidney and VSM, the proteins have differing membrane-associative characteristics. This is not surprising in view of their large structural differences and may also reflect different lipid profiles of the membranes of different tissues as well. Regardless of the reasons for such tissuespecific differences, we suggest that selective retention in the cell membrane of either of the 01 subunits reflects interaction with the ,f?subunit necessary to form a functional pump site. Since no detectable full-length al subunit is retained in VSM fractions this argues that the cllT subunit functions as a pump protein in this tissue and that al does not associate with /3. Furthermore, these data suggest that this truncated protein, lacking 40% of the -COOH terminal sequence, can be targeted to the membrane and fulfil1 a transport function. Some workers have suggested that glutamic acid residues 955 and 956 are important in the ion transport function of the full length protein [ 16,171. Since alT lacks this region, this would seem to preclude a transport role for this protein. However, more recent evidence utilizing site-directed mutagenesis of these predicted cation binding sites argues against the contribution of these sites and the carboxy terminus [18]. Thus specific designation of ion transport residues remains unclear and cllT may yet prove to retain the necessary motifs for Na’ and K’ transport. Since intact tissues (or at least microsomes) demonstrate both al and alT proteins, suggestions that alT serves a transport function cannot be made without cytochemical localization studies, or the determination of the actual molecular nature of the pump complex (i.e. (al)@?) or (ollT)(/$). For these reasons, we assessed the protein components contained in confluent cultures of canine vascular smooth muscle cells, which we know express functional Na pump sites [6,7]. These cells show a large component of alT and no detectable al. Clearly since microsomes show an al full-length protein before SDS treatment, the lack of detectable al in confluent
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Fig. 3. Immunoblot of saphenous vein smooth muscle cells, and canine kidney medullary microsomes. Saphenous vein cells were grown for indicated time on 100 mm plastic dishes, lysed with NP 40, and Western analysis performed as in Fig. 1 and Section 2. Canine kidney microsomes were assessed as described above.
J.C. Allen et al. IFEBS
288
cells may reflect the up-regulation of the alT, and downregulation of al. Such modulation is currently under investigation. Freshly dispersed cells do show a small amount of al, which seems to disappear during proliferation (data not shown). Thus strong circumstantial evidence suggests that alT may serve a transport function in VSM. We have shown that in the absence of detectable al, VSM microsomes show a Na’,K’-ATPase activity and [3H]ouabain binding. In addition, in VSM confluent cells, containing apparently normal Na pump function, al was undetectable and alT was clearly demonstrable. We have shown earlier that canine VSM contains both pl mRNA [4] and protein [15], but have not yet determined its associative characteristics with the alT in the sarcolemma. It does, however, remain attached to the sarcolemma after extensive purification attempts with detergents. Furthermore, the SDS data in this paper reveal nothing about the nature of the association of alT with the different membranes, or the reasons for such different associative characteristics. More extensive characterization with additional detergents is necessary to more clearly define these phenomena. Despite these limitations we can conclude that both al and alT exist in both canine kidney and VSM canine tissues but their membrane associative characteristics vary greatly. The al is resistant to SDS treatment in the kidney membrane, and the alT is more tightly retained in VSM. Since there is no detectable al in confluent cultured VSM, it is possible that alT serves as a pump subunit in VSM. In the absence of al in VSM, a.lT may complex with an appropriate /3, insert in the membrane and may form functional pump sites in the SL of this tissue. This would account for the Na’,K’-ATPase activity in VSM SL preparations. The manifestations of alT not associated with a p subunit, and not inserted in the membrane could be significantly different from alT complexed with B, and bound to a membrane. Acknowledgements: Supported by NIH Grants: HL-24585 (JCA) and
HL-39846 (TAP) and by AHA 90-1336 (RMM). TAP is the recipient of an RCDA (HL-002305). RMM is an Established Investtgator of the AHA.
Letters 337 (1994) 285-288
References
111Kaplan, J.H. and De Weer, P. (1991) The Sodium Pump: Structure, Mechanisms and Regulation, Rockefeller University Press, New York. 121Kaplan, J.H. and De Weer, P. (1991) The Sodium Pump: Recent Developments, Rockefeller University Press, New York. [31 Medford, R.M., Hyman, A.M., Allen, J.C., Pressley, T.A., Allen, P.D. and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 183088 18312. [41 Allen, J.C.. Medford, R.M.. Zhao, X. and Pressley. T.A. (1991) Circ. Res. 69. 3944. PI Allen, J.C.. Kahn, A.M., Navran, S.S. (1986) Am. J. Physiol. 250. C536C540. 161Allen, J.C., Navran, S.S., Seidel, C.L., Dennison, D.K., Amann, J.M. and Jemelka. S.J. (1989) Am. J. Physiol. 256, C786792. [71 Feltes, T.F., Seidel, C.L., Dennison, D.K., Amick, S. and Allen. J.C. (1993) Am. J. Physiol. 264, C169-C174. PI Jorgensen, P.L. (1974) in: Methods in Enzymology Volume XXX11 Biomembranes, Part B, pp. 2777290, Academic Press, New York. [91 Bradford, M.M. (1976) Anal. Biochem. 72, 248-259. UOI Pressley, T.A. (1992) Am. J. Phystol. 262, C743-C751. 1111Eggermont, J.A., Vrolix, M.. Raemaekers, L., Wuytack. F. and Casteels, R. (1986) Circ. Res. 62, 26627812. WI Allen, J.C., Smith, D.J. and Gerthoffer, W.T. (1981) Life SCI. 28. 945-950. u31 Navran, S.S., Adair, S.E., Jemelka. S.K., Setdel. C.L. and Allen, J. (1988) Pharm. Exp. and Ther. 245, 6088613. [I41 Jorgenson, P.L. and Anderson, J.P. (1988) J. Membr. Biol. 103. 955120. USI Allen, J.C., Navran, S.S. and Jemelka, S.K. (1988) The functional role of Na pump modulation in vascular smooth muscle, in, Vascular Neuroeffector Mechanisms (Bevan, J.A., Majewskt, H., Maxwell, R.A. and Story, D.F.. Eds.) Vol. 10, pp. 217-224, ICSU Press. U61 Argello, J.M. and Kaplan, J.H. (1991) J. Btol. Chem. 266. 146277 14636. 1171Goldshleger, R.. Tal, D.M., Stem, W.D. and Karlish. S.J.D. ( 1992) Proc. Natl. Acad. Sci. USA 89, 6911-6915. P81 Van Huysse, J.W., Jewell, E.A. and Lingrel, J.B. (1993) Btochemistry 32, 819-826.