J Physiol Volume 512, Number 2, 481-485, October 15, 1998
The Journal of Physiology (1998), 512.2, pp. 481-485
© Copyright 1998 The Physiological Society
Integrin and cytoskeletal involvement in signalling cell volume changes to glutamine transport in rat skeletal muscle
Sylvia Y. Low and Peter M. Taylor
Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, UK
Received 24 June 1998; accepted after revision 13 August 1998.
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ABSTRACT |
- Muscle glutamine transport is modulated in response to changes in cell volume by a mechanism dependent on active phosphatidylinositol 3-kinase. We investigated the possibility that this mechanism requires interactions between the extracellular matrix (ECM), integrins and the cytoskeleton as components of a mechanochemical transduction system.
- Using skeletal muscle cells, we studied effects of (a) inactivating integrin-substratum interactions by using integrin-binding peptide GRGDTP with inactive peptide GRGESP as control, and (b) disrupting the cytoskeleton using colchicine or cytochalasin D, on glutamine transport after brief exposure to hypo-osmotic, isosmotic or hyperosmotic medium (170, 300 and 430 mosmol kg-1, respectively).
- Neither GRGDTP nor GRGESP significantly affected basal glutamine uptake (0·05 mM; 338 ± 58 pmol min-1 (mg protein)-1) but GRGDTP specifically prevented the increase (71 %) and decrease (39 %) in glutamine uptake in response to hypo- and hyperosmotic exposure, respectively.
- Colchicine and cytochalasin D prevented the increase and decrease in glutamine uptake in response to changes in external osmolality. They also increased basal glutamine uptake by 59 ± 19 and 85 ± 16 %, respectively, in a wortmannin-sensitive manner.
- These results indicate involvement of ECM-integrin-mediated cell adhesion and the cytoskeleton in mechanochemical transduction of cell volume changes to chemical signals modulating glutamine transport in skeletal muscle. Phosphatidylinositol 3-kinase may function to maintain the mechanotransducer in an active state.
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INTRODUCTION |
The amino acid transporter system Nm is quantitatively the most important glutamine transporter in skeletal muscle and is rapidly activated in response to increases in cell volume such as are induced by various nutrient and endocrine stimuli of physiological importance (Low et al. 1996b, 1997b). Cell swelling may act as a general anabolic signal in skeletal muscle, stimulating synthesis of glycogen and, possibly, protein (Low et al. 1996a). We have shown in skeletal muscle that rapid (half-time for response, t½ < 1 min) stimulation of glutamine transport by cell swelling requires active phosphatidylinositol 3-kinase, and subsequent (t½ 
30 min) stimulation of glycogen synthesis additionally involves activation of the p70S6 kinase signalling pathway (Low et al. 1996a, 1997a). Similar metabolic signalling mechanisms operate in liver, where amino acids, insulin and hypo-osmotic cell swelling also stimulate anabolic processes (Baquet et al. 1990; Häussinger, 1996; Krause et al. 1996). Indeed, hepatic system N has functional similarities to system Nm, which include rapid activation in response to swelling (Bode & Kilberg, 1991; Häussinger, 1996).
We are currently investigating the sensor and signalling mechanisms involved in the transduction of cell swelling (and other forms of mechanical strain) to altered transport activities in skeletal muscle. These mechanisms may have important functions in the regulation of metabolism and cell volume. Phosphatidylinositol 3-kinase is associated with integrins at the focal adhesion complex (Parsons, 1996) and integrins have been implicated as a component of a mechanochemical transduction system through their interactions with the cytoskeleton and extracellular matrix (Chen & Grinnell, 1995; Ingber, 1997). We have recently demonstrated that blockade of integrin binding to the extracellular matrix or disruption of the cytoskeleton prevents swelling-induced increases in muscle glycogen synthesis (Low et al. 1997a). It has also previously been shown in liver cells that disruption of cytoskeletal elements (e.g. microfilaments and microtubules) prevents modulation of metabolism by cell volume changes (Theodoropoulos et al. 1992). Thus, the purpose of the present study was to investigate whether an intact cytoskeleton and integrins are components of a mechanochemical sensor-transducer system with the capability to modulate rapidly glutamine transport in response to cell volume changes in skeletal muscle.
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METHODS |
Skeletal muscle cells were harvested from thigh muscles of 1-day-old neonatal rats (killed by cervical dislocation) and cultured as described previously (Low et al. 1996b). All experiments were performed on 10-day-old, confluent, multinucleated myotubes. Radiotracers were obtained from NEN Research Products (Stevenage, UK) except L-[3H]glutamine which was obtained from Amersham International (Aylesbury, UK). The peptide GRGESP was custom made by GENOSYS (Cambridge, UK). The peptide GRGDTP and all other chemicals were obtained from Sigma.
Uptake of L-[3H]glutamine (0·05 mM) was measured as described previously (Low et al. 1996b). The basic experimental medium contained (mM): 60 NaCl, 4·9 KCl, 2·5 MgSO4, 20 Tris-HCl and 1 CaCl2; osmolality was adjusted using sucrose as required by the experimental designs and checked using an osmometer. In all experiments involving altered extracellular osmolality, myotubes were exposed for 2 min to hypo-osmotic (170 mosmol kg-1) or hyperosmotic (430 mosmol kg-1) medium and measurements of 0·05 mM L-[3H]glutamine uptake were made over the final minute of this period as described previously (Low et al. 1996b).
To assess the effects of cytoskeletal disruption or integrin-binding blockade, cells were incubated with cytochalasin D (0·5 µM; which prevents the formation of contractile microfilaments), colchicine (3 µM; which disrupts microtubules), integrin-binding peptide GRGDTP (25 µg ml-1; active) or inactive control peptide GRGESP (25 µg ml-1) for 60 min in isosmotic medium, and, where necessary, the switch to hypo- or hyperosmotic conditions was for the final 2 min of the incubation, with glutamine (0·05 mM) uptake measured (as described earlier) over the final minute. The RGD-containing peptide disrupts integrin-extracellular matrix interactions by competing with the extracellular matrix for binding sites on the extracellular domain of integrins. In certain experiments, wortmannin (0·1 µM; an inhibitor of phosphatidylinositol 3-kinase) was added after 30 min (i.e. half-way through the pre-incubation period), or rapamycin (0·1 µM; an immunosuppressant which prevents the activation of p70S6 kinase) was added for 60 min as indicated, i.e. drugs were added prior to the time the hypo- or hyperosmotic exposure commenced.
Data are presented as the mean values ± S.E.M. for n muscle cell preparations; each experimental measurement in an individual preparation was performed in triplicate using three separate wells in a culture plate. Statistical significance was assessed using the paired t test; differences were considered significant where P < 0·05.
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RESULTS |
Glutamine (0·05 mM) uptake was increased by 71 ± 15 % and decreased by 39 ± 14 % from control values (338 ± 58 pmol min-1 (mg protein)-1; n = 12 preparations) after exposure to hypo- and hyperosmotic media, respectively (Fig. 1), confirming previous results (Low et al. 1996b, 1997b). These osmotically induced changes in glutamine uptake were blocked by pre-incubation of the cells with the active integrin-binding peptide GRGDTP (Fig. 1), whereas the inactive peptide GRGESP had no significant effect (Fig. 1). The increase (
35 %) in basal glutamine uptake produced by the active peptide did not achieve statistical significance.
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Figure 1. Effect of 2 min hypo- or hyperosmotic exposure on glutamine uptake in muscle cells in NaCl medium in the presence of GRGDTP or GRGESP peptide
[3H]-Labelled glutamine (0·05 mM) uptake was measured over the final 1 min of a 2 min exposure of muscle cells to medium of 170 or 430 mosmol kg-1. GRGDTP (25 µg ml-1, active) or GRGESP (25 µg ml-1, control) was added for 60 min prior to the time the hypo- or hyperosmotic exposure commenced. NaCl concentration was 60 mM in all experiments; osmolality was adjusted using sucrose. Each value represents the mean ± S.E.M. for 12 preparations. Statistical significance from the control value at 300 mosmol kg-1 was measured using the paired t test; * P < 0·05.
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Osmotically induced alterations in glutamine uptake were also absent in cells treated with colchicine or cytochalasin D, although basal glutamine uptake was increased 59 ± 20 and 85 ± 16 %, respectively, by these drug treatments (Fig. 2). Note that the inability of these cells to respond to swelling is not simply a result of cytoskeletal disruption already producing the maximum achievable short term increase in muscle glutamine transport, because cells treated with rapamycin show an equivalent increase in basal glutamine uptake but still exhibit a further increase in transport after cell swelling (Low et al. 1997b).
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Figure 2. Effect of 2 min hypo- or hyperosmotic exposure on glutamine uptake in muscle cells in NaCl medium in the presence of cytochalasin D, colchicine or rapamycin
[3H]-Labelled glutamine (0·05 mM) uptake was measured over the final 1 min of a 2 min exposure of muscle cells to medium of 170 or 430 mosmol kg-1. Cytochalasin D (0·5 µM, Cyt D), colchicine (3 µM, Col) or rapamycin (100 nM, Rap) was added for 60 min prior to the time the hypo- or hyperosmotic exposure commenced. NaCl concentration was 60 mM in all experiments; osmolality was adjusted using sucrose. Values are means ± S.E.M. of 12 preparations. Statistical significance from the control value at 300 mosmol kg-1 was measured using the paired t test; * P < 0·05, ** P < 0·01.
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Wortmannin increased basal glutamine uptake by 55 ± 18 % (Table 1) and blocked the osmotically induced changes in glutamine uptake (data not shown; see Low et al. 1997b). Wortmannin did not influence the inhibition of osmotically induced changes in glutamine uptake by GRGDTP, but it abolished increases in basal glutamine uptake observed after cytoskeletal disruption using colchicine or cytochalasin D (Table 1).
Table 1. Effect of wortmannin on glutamine uptake in muscle cells
| 0·05 mM glutamine uptake (pmol min-1 (mg protein)-1) |
| Medium composition | - Wortmannin | + Wortmannin |
| Control | 312 ± 63 | 483 ± 85 ** |
| + GRGDTP | 396 ± 78 | 488 ± 99 |
| + GRGESP | 332 ± 65 | 463 ± 56 * |
| + Cytochalasin D | 589 ± 91  | 428 ± 76 * |
| + Colchicine | 575 ± 82 | 439 ± 89 * |
[3H]-Labelled glutamine (0·05 mM) uptake was measured over the final 1 min of a 60 min exposure of muscle cells to medium containing 25 µg ml-1 GRGDTP/GRGESP, 0·5 µM cytochalasin D or 3 µM colchicine in the presence or absence of 100 nM wortmannin. Values represent the mean measurements ± S.E.M. for 6 preparations, and statistical significance of differences between values was assessed using the paired t test: * P < 0·05 and ** P < 0·01 between values with and without wortmannin, P < 0·05 from control in the absence of wortmannin.
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DISCUSSION |
There is accumulating evidence that changes in cell volume associated with altered cell glutamine concentration have both physiological and pathophysiological roles in the regulation of membrane transport and metabolism in skeletal muscle (Low et al. 1996a, 1997b; Neu et al. 1996; Rennie et al. 1996). Mechanical strain produced at the surface of cells by physical processes such as swelling, shrinking, stretch or movement is now recognized to be an important signal for modulation of cellular metabolism, growth and development (Chen & Grinnell, 1995; Plopper et al. 1995; Clarke & Feeback, 1996; Häussinger, 1996; Ingber, 1997). One mechanism by which cells may transduce mechanical stimuli into chemical signals evoking metabolic responses involves transmembrane integrins (Parsons, 1996; Ingber, 1997). Integrins make physical attachments to specific proteins of the extracellular matrix (ECM) and help 'anchor' the microtubular struts and tensile microfilaments of the intracellular cytoskeleton to the substratum at focal adhesion complexes. These perform a mechanochemical signalling function highlighted in the recently proposed tensegrity model of cytoskeletal mechanics (Ingber, 1997). The present results demonstrate that experimental disruption of the ECM-integrin-cytoskeleton axis suppresses osmotically induced changes in glutamine uptake. These disruptions included (a) inhibition of ECM-integrin interactions by the use of a soluble peptide (GRGDTP) that competes with ECM proteins for binding sites on the integrin 'receptor', and (b) destruction of either microtubular or microfilamentous elements of the cytoskeleton using colchicine and cytochalasin D, respectively. We infer from these results that an integrin-dependent mechanochemical signalling mechanism is involved in transducing changes in cell volume to an effect on the system Nm glutamine transport mechanism. The exact nature of this effect is not clear as yet, but the fact that membrane transport mechanisms for other solutes (e.g. glucose, glutamate, methylaminoisobutyrate) in muscle are affected differently (Low et al. 1996a, b) argues against a generalized effect on membrane surface area (e.g. increase after cell swelling) or on transmembrane solute gradients. The effect of GRGDTP peptide demonstrates that integrin-dependent adhesion to the ECM (rather than integrin receptor occupation per se) is required for mechanochemical transduction. A role for integrins in skeletal muscle mechanotransduction has previously been suggested by Chen & Grinnell (1995), who showed that muscle stretch enhances neurotransmitter release from motor nerve terminals by a mechanism that is suppressed by integrin-binding peptides and also by certain integrin antibodies. Mechanical strain generated in muscle cells by swelling or stretch may therefore be transduced to biochemical signals at least partly by the same mechanism.
The amino acid transport systems Nm and A show opposing but equally rapid responses to swelling and shrinking of muscle cells (Low et al. 1996b). We have hypothesized elsewhere (Rennie et al. 1996) that swelling-induced activation of system Nm acts as a component of a 'positive-feedback' mechanism amplifying the anabolic effects of circulating nutrients after a meal. In contrast, activation of system A in response to cell shrinkage is likely to contribute to cell volume regulation (Low et al. 1996b). The system A transporter appears to be physically associated with integrin 
3/
1 in cell membranes (McCormick & Johnstone, 1995), possibly facilitating the rapid response of amino acid transport to changes in mechanical strain at the muscle membrane via the integrin-dependent mechanochemical signalling mechanism. Integrin activation rapidly stimulates tyrosine phosphorylation of signalling molecules at focal adhesions, and we have evidence (inhibition by genistein; Low et al. 1997b) that tyrosine phosphorylation is necessary for swelling-induced activation of system Nm. Integrin-dependent signalling also involves cytoskeletal rearrangement and subsequent activation of p70S6 kinase (Malik & Parsons, 1996; Parsons, 1996), which appear to be required for 'downstream' stimulation of glycogen synthesis in response to muscle cell swelling (Low et al. 1996a).
Phosphatidylinositol 3-kinase appears to act as a permissive rather than a transducing element in the anabolic signal generated by cell swelling (Low et al. 1997b), conceivably because it maintains integrins in an 'active' conformation promoting cell adhesion (Kovacsovics et al. 1995; Plopper et al. 1995; Shimizu et al. 1995). The primary effect of inhibiting phosphatidylinositol 3-kinase with wortmannin may therefore be to disable an adhesion-dependent 'volume sensor' rather than to block downstream mechanochemical signals for modulation of membrane transport and metabolism. The effects of cytoskeletal disruption, wortmannin and GRGDTP peptide on muscle cells all include stimulation of basal glutamine transport to a greater or lesser extent, consistent with the suggestion (Low et al. 1997b) that inactivation of the 'sensor' helps relieve a normal depressor effect on glutamine transport.
We conclude that the modulation of amino acid transport system Nm in response to altered muscle cell volume involves an integrin-dependent mechanochemical transduction mechanism. The mechanism also requires active phosphatidylinositol 3-kinase, an enzyme common to signalling pathways activated by stimuli including soluble hormones as well as mechanical forces (Plopper et al. 1995; Clarke & Feeback, 1996; Häussinger, 1996; Ingber, 1997). This mechanism for regulating glutamine transport is well suited to effecting a rapid and sensitive element of metabolic control in skeletal muscle (Low et al. 1997b) and may also enable feedback/feedforward interactions between signalling mechanisms responsive to nutrient-induced mechanical and endocrine stimuli (e.g. cell swelling and insulin, respectively). Integrin-dependent mechanochemical transduction may also have a role in initiation and co-ordination of mechanisms for regulating cell volume.
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Acknowledgements
This work was supported by the UK Medical Research Council and the University of Dundee.
Corresponding author
S. Y. Low: Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, UK.
Email: s.y.low{at}dundee.ac.uk
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