Regulation of protein synthesis in lactating rat mammary tissue by cell volume

Biochimica et Biophysica Acta 1475 (2000) 39^46 www.elsevier.com/locate/bba Regulation of protein synthesis in lactating rat mammary tissue by cell volume A.C.G. Grant, I.F. Gow, V.A. Zammit, D.B. Shennan * Hannah Research Institute, Ayr KA6 5HL, Scotland, UK Received 4 January 2000 ; received in revised form 6 March 2000; accepted 15 March 2000 Abstract The effect of changing cell volume on rat mammary protein synthesis has been examined. Cell swelling, induced by a hyposmotic challenge, markedly increased the incorporation of radiolabelled amino acids (leucine and methionine) into trichloroacetic acid (TCA)precipitable material: reducing the osmolality by 47% increased leucine and methionine incorporation into mammary protein by 147 and 126% respectively. Conversely, cell shrinking, induced by a hyperosmotic shock, almost abolished the incorporation of radiolabelled amino acids into mammary protein : increasing the osmolality by 70% reduced leucine and methionine incorporation into mammary protein by 86 and 93% respectively. The effects of cell swelling and shrinking were fully reversible. Volume-sensitive mammary tissue protein synthesis was dependent upon the extent of the osmotic challenge. Isosmotic swelling of mammary tissue, using a buffer containing urea (160 mM), increased the incorporation of radiolabelled leucine into TCA-precipitable material by 106%. Swelling-induced mammary protein synthesis was dependent upon calcium : removing extracellular calcium together with the addition of EGTA markedly reduced volume-activated protein synthesis. Cell swelling-induced protein synthesis was inhibited by the Ca2‡ ATPase blocker thapsigargin suggesting that volumesensitive protein synthesis is dependent upon luminal calcium. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Mammary ; Protein synthesis ; Cell volume 1. Introduction Cell volume, otherwise termed the cellular hydration state, can change as a consequence of substrate accumulation, aniso-osmolality and/or changes to the rate of oxidative metabolism [1]. It is apparent that cell volume is an important regulator of cellular metabolism [2]. For example, cell swelling increases protein synthesis [3], lipogenesis [4] and glycogen synthesis [5] in hepatocytes. On the other hand, cell shrinking decreases protein synthesis in hepatocytes [3]. It appears, therefore, that cell swelling is an anabolic signal whereas a reduction in the cellular hydration state favours catabolism [2]. Preliminary results from our laboratory suggest that cell volume may be an important regulator of mammary cell metabolism : we have recently shown that rat mammary protein synthesis is regulated by the cellular hydration state [6]. Thus, cell swelling and shrinking, induced by * Corresponding author. Fax: +44-1292-674003; E-mail : shennand@hri.sari.ac.uk osmotic perturbations, increased and decreased, respectively, the incorporation of radiolabelled leucine into trichloroacetic acid-precipitable material. Consistent with these ¢ndings was the observation that casein synthesis was increased and decreased by cell swelling and shrinking, respectively [6]. Interestingly, cell volume had no e¡ect on casein mRNA levels suggesting that the e¡ect of cell volume changes was occurring at the level of translation. Taken together, the results suggest that the cellular hydration state may be an important regulator of mammary protein synthesis in general and casein production in particular. The purpose of the present study was to con¢rm and extend the ¢nding that changes to mammary cell volume have marked e¡ects on protein synthesis. Thus, we have investigated the e¡ect of cell volume changes on the incorporation of radiolabelled amino acids into TCA-precipitable material. In particular we wanted to examine (a) the reversibility of the e¡ects of cell swelling and shrinking on protein synthesis, (b) the e¡ect of isosmotic swelling on protein synthesis, (c) the sensitivity of mammary protein synthesis to osmotic perturbations, and (d) the regulation of volume-sensitive protein synthesis by Ca2‡ . 0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 4 5 - 3 40 A.C.G. Grant et al. / Biochimica et Biophysica Acta 1475 (2000) 39^46 2. Materials and methods 2.1. Animals and isolation of mammary tissue explants Lactating Wistar rats, 10^15 days post partum, which were suckling between 8 and 10 pups were used in this study. Animals were maintained on a 12 h light:dark cycle and were allowed free access to water and chow. Mammary tissue explants were isolated from the abdominal mammary glands as previously described [7]. Care was taken to remove as much connective tissue as possible. 2.2. Measurement of mammary protein synthesis Protein synthesis by mammary tissue explants (each weighing 0.5^2.0 mg wet weight) was measured at 37³C using a method similar to that described by Millar et al. [6]. The incorporation of [3 H]leucine (1 WCi/ml) or [35 S]methionine (2.5 WCi/ml) into trichloroacetic acid (TCA)-precipitable material was taken as a measure of protein synthesis. These amino acids were chosen to measure protein synthesis because (a) the transport of leucine and methionine appears not to be rate limiting and (b) the intracellular concentration of both amino acids is low [6,8]. All bu¡ers contained amino acids at concentrations similar to that found in the plasma of lactating rats [9^11] (see Table 1). Furthermore, the experiments were conducted under conditions where the extracellular Na‡ concentration remained unchanged: this was to minimise any change to the transmembrane Na‡ gradient which is the driving force for the uptake of a number of amino acids by the mammary gland [12]. The isosmotic bu¡er contained (mM) 55 NaCl, 80 choline^Cl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4. The hyposmotic bu¡ers were similar in composition except that they contained less, or even no, choline^Cl. The hyperosmotic bu¡er was similar in composition to the isosmotic solution except that sucrose (200 mM) was added to increase the osmolality. All bu¡ers were continuously gassed with 100% O2 . Tissue explants were pre-incubated in the bu¡ers for 20 min prior to the addition of the radiolabelled amino acids. At pre-determined times after the addition of radiolabelled amino acids, explants were removed from the incubation bu¡ers and placed in preweighed vials containing 1 ml of a 10% solution of TCA. After the tissue weight was determined the vials were allowed to stand for at least 60 min. The tubes were then centrifuged at 13 000Ug and the resulting pellet was washed thrice by centrifugation and resuspension in 10% TCA. After this, the pellet was incubated in 100 Wl of formic acid (90%) for at least 16 h. Distilled H2 O (900 Wl) was added to each vial and the contents of the vials were mixed with 20 ml of UltimaGold liquid scintillation cocktail. The speci¢c activity of radiolabelled amino acids in each incubation vial was measured by counting the radioactivity in 100 Wl samples. The total tissue water, and thus dry matter, was determined in parallel incubations for each of the conditions studied by drying explants to constant weight at 110³C. 2.3. Materials [3 H]leucine and [35 S]methionine were purchased from Amersham International (UK). All other chemicals were obtained from Sigma (UK). 2.4. Statistics Di¡erences were assessed by Student's paired or unpaired t-test as appropriate and were considered signi¢cant when P 6 0.05. 3. Results 3.1. The e¡ect of an osmotic shock on mammary protein synthesis The ¢rst step in the investigation was to con¢rm that cell volume perturbations a¡ected the rate of protein synthesis in tissue explants isolated from the mammary glands of rats during peak lactation. Therefore, we examined the e¡ect of changing the external osmolality on the incorporation of radiolabelled leucine and methionine into trichloroacetic acid-precipitable material. It is evident from Fig. 1 that the rate of protein synthesis was linear under all the conditions used. Consistent with previous results from this laboratory [6], Fig. 1A shows that incubating mammary tissue in a hyposmotic medium increased (P 6 0.01) leucine incorporation into rat mammary protein compared to that found under isosmotic conditions. On the other hand a hyperosmotic shock (sucrose addition) decreased (P 6 0.01) leucine incorporation compared to that measured under isosmotic conditions. The rate of leucine incorporation into mammary tissue protein under isosmotic (290 þ 4 mosmol/kg), hyposmotic (153 þ 1 mosmol/kg) and hyperosmotic (492 þ 14 mosmol/kg) conditions was respectively 3.16 þ 0.64, 7.83 þ 1.16 and 0.43 þ 0.09 Wmol/h/g dry tissue weight (n = 6, þ S.E.M.). Therefore, leucine incorporation into TCA-precipitable material increased by 147% as a result of cell swelling and was inhibited by 86% as a consequence of cell shrink- Table 1 Concentrations (WM) of amino acids in incubation bu¡ers Ala 680 Arg 130 Asp 50 Asn 45 Cys 160 Gln 680 Glu 100 Gly 350 His 245 Ile 145 Leu 225 Lys 245 Met 115 Phe 115 Pro 250 Ser 445 Thr 390 Trp 86 Tyr 150 Val 240 A.C.G. Grant et al. / Biochimica et Biophysica Acta 1475 (2000) 39^46 41 and decreased methionine incorporation by 126 and 93%, respectively. The ratio of leucine/methionine incorporation into TCA-precipitable material under isosmotic and hyposmotic conditions was 7.52 and 8.24, respectively. Cell shrinking is known to increase proteolysis in hepatocytes [2]. Thus, there is the possibility that the reduction in mammary protein synthesis following hyperosmotic exposure shown in Fig. 1A is due to a decrease in the speci¢c activity of radiolabelled leucine following proteolysis. Therefore, we examined the e¡ect of changing the osmolality of the incubation medium on [3 H]leucine incorporation into protein under conditions where the concentration of unlabelled leucine was increased from 225 WM to 4 mM. Under these conditions it was predicted that unlabelled leucine derived from proteolysis should have less e¡ect on label dilution. Fig. 2 shows that when leucine was used at a concentration of 4 mM a hyperosmotic shock decreased (P 6 0.02) mammary protein synthesis compared to that found under isosmotic conditions. It is evident that a hyposmotic challenge also increased mammary protein synthesis (P 6 0.002) under these experimental conditions. Furthermore, the rate of protein synthesis under each condition was linear over the entire time course. The rate of leucine incorporation under isosmotic, hyposmotic and hyperosmotic conditions was respectively 6.64 þ 1.07, 11.43 þ 1.48 and 1.17 þ 0.03 Wmol/h/g dry tissue weight (n = 4, þ S.E.M.). Thus, cell swelling and shrinking increased and decreased protein synthesis by 72 and 82%, respectively. 3.2. Reversibility of volume-sensitive protein synthesis Fig. 1. (A) The time course of [3 H]leucine incorporation into mammary protein under (b) isosmotic (290 þ 4 mosmol/kg), (F) hyposmotic (153 þ 1 mosmol/kg) and (R) hyperosmotic conditions (492 þ 14 mosmol/kg). All bu¡ers contained amino acids at concentrations shown in Table 1. The isosmotic bu¡er contained (mM) 55 NaCl, 80 choline^Cl, 4 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 (+1 WCi/ml [3 H]leucine). The hyposmotic bu¡er was similar in composition except that it did not contain any choline^Cl. The hyperosmotic bu¡er was similar in composition to the isosmotic bu¡er except that it also contained 200 mM sucrose. Each point is the mean þ S.E.M. of six experiments using tissue from separate animals. (B) The time course of [35 S]methionine incorporation onto mammary protein under (b) isosmotic (292 þ 3 mosmol/kg), (F) hyposmotic (154 þ 2 mosmol/kg) and (R) hyperosmotic conditions (504 þ 4 mosmol/kg). The composition of the bu¡ers was the same as that described above except that the media contained 2.5 WCi/ml [35 S]methionine. Each point is the mean þ S.E.M. of four experiments using tissue from separate animals. ing. Cell swelling and shrinking also increased (P 6 0.01) and decreased (P 6 0.01), respectively, the incorporation of radiolabelled methionine into rat mammary protein (Fig. 1B). Methionine incorporation into mammary tissue protein under isosmotic (292 þ 3 mosmol/kg), hyposmotic (154 þ 2 mosmol/kg) and hyperosmotic (504 þ 4 mosmol/ kg) conditions was respectively 0.42 þ 0.02, 0.95 þ 0.08 and 0.03 þ 0.01 Wmol/h/g dry tissue weight (n = 4, þ S.E.M.). Therefore, cell swelling and shrinking increased We designed experiments to test whether or not the e¡ect of a hyperosmotic shock on rat mammary protein Fig. 2. The time course of [3 H]leucine incorporation into mammary protein under (b) isosmotic (294 þ 13 mosmol/kg), (F) hyposmotic (159 þ 9 mosmol/kg) and (R) hyperosmotic (500 þ 10 mosmol/kg) conditions when the extracellular concentration of leucine was 4 mM. The composition of the incubation bu¡ers was the same as that described in the legend to Fig. 1A except that the concentration of unlabelled leucine was 4 mM. Each point is the mean þ S.E.M. of four experiments using tissue from separate animals. 42 A.C.G. Grant et al. / Biochimica et Biophysica Acta 1475 (2000) 39^46 Fig. 3. The reversibility of the e¡ect of a hyperosmotic challenge on [3 H]leucine incorporation into mammary protein. Mammary explants were initially incubated in a medium containing (mM) 160 sucrose, 55 NaCl, 80 choline^Cl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine. (+amino acids ^ see Table 1) (osmolality = 461 þ 7 mosmol/kg). At t = 22 min the medium was made isosmotic by adding an equal volume of bu¡er containing (mM) 55 NaCl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1) (¢nal osmolality = 307 þ 5 mosmol/kg). Each point is the mean þ S.E.M. of four experiments using tissue from separate animals. synthesis is reversible (Fig. 3). In this set of experiments, the incubation bu¡er was initially hyperosmotic (461 þ 7 mosmol/kg) but at t = 22 min the bu¡er was made isosmotic (307 þ 5 mosmol/kg) without changing the speci¢c activity of [3 H]leucine. The rate of leucine incorporation Fig. 4. The reversibility of the e¡ect of a hyposmotic shock on [3 H]leucine incorporation into mammary protein. Mammary tissue explants were initially incubated in a medium containing (mM) 55 NaCl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1) (osmolality = 152 þ 1 mosmol/kg). At t = 22 min the medium was made isosmotic by adding an equal volume of a bu¡er containing (mM) 160 choline^Cl, 55 NaCl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1) (¢nal osmolality = 283 þ 3 mosmol/kg). Each point is the mean þ S.E.M. of four experiments using tissue from separate animals. under hyperosmotic conditions was 0.73 þ 0.13 Wmol/h/g dry tissue weight (n = 4, þ S.E.M.). When the bu¡er was made isosmotic leucine incorporation increased to 3.16 þ 0.29 Wmol/h/g dry tissue weight (n = 4, þ S.E.M., P 6 0.01). It is apparent, therefore, that the e¡ect of a hyperosmotic challenge is reversible. The stimulation of mammary protein synthesis by a hyposmotic shock was also found to be a reversible process (Fig. 4). In these experiments the incubation bu¡er was initially hyposmotic but at t = 22 min the bu¡er was rendered isosmotic again without changing the speci¢c activity of [3 H]leucine. Thus, changing the osmolality of the incubation bu¡er from 152 þ 1 mosmol/kg to 283 þ 3 mosmol/kg decreased the rate of leucine incorporation into TCA-precipitable material from 8.70 þ 1.27 to 4.44 þ 0.61 Wmol/h/g dry tissue weight (n = 4, þ S.E.M., P 6 0.02). 3.3. Isosmotic swelling increases mammary protein synthesis The e¡ect of a hyposmotic shock on mammary protein synthesis depicted in Fig. 1 could be due to cell swelling or a reduction in the osmolality of the incubation medium per se. Therefore, to distinguish between these two possibilities we tested the e¡ect of an isosmotic bu¡er which contained a relatively high concentration of urea on mammary protein synthesis. The rationale behind this experimental approach is that mammary cells will swell on account of the high permeability of urea. Fig. 5 shows that isosmotic swelling, induced by urea, markedly increased the rate of leucine incorporation into TCA-precipitable material. Leucine incorporation was increased by 106% (n = 6, P 6 0.001) by the inclusion of urea in the incubation bu¡er. Fig. 5. The e¡ect of isosmotic cell swelling on [3 H]leucine incorporation into mammary protein. The control isosmotic bu¡er contained (mM) 55 NaCl, 80 choline^Cl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1 (osmolality = 294 þ 1 mosmol/kg). The bu¡er containing urea was similar in composition except that it contained urea (160 mM) and no choline^Cl (osmolality = 306 þ 3 mosmol/kg). Data shown are the means þ S.E.M. of six experiments using tissue from separate animals. A.C.G. Grant et al. / Biochimica et Biophysica Acta 1475 (2000) 39^46 43 Fig. 6. Sensitivity of [3 H]leucine incorporation into mammary protein to a hyposmotic shock. All bu¡ers contained (mM) 55 NaCl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1). Osmolality was varied by adding choline^Cl to the bu¡er (0^160 mM). Each point is the mean þ S.E.M. of six experiments using tissue from separate animals. Fig. 7. Sensitivity of [3 H]leucine incorporation into mammary protein to a hyperosmotic challenge. All bu¡ers contained (mM) 55 NaCl, 80 choline^Cl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1). The bu¡ers were made hyperosmotic by adding sucrose. Each point is the mean þ S.E.M. of six experiments using tissue from separate animals. 3.4. Sensitivity of mammary protein synthesis to osmotic perturbations thesis was markedly reduced under isosmotic conditions when the incubation medium was Ca-free (+EGTA): leucine incorporation decreased from 2.81 þ 0.27 to 1.29 þ 0.09 Wmol/h/g dry tissue weight (n = 4, þ S.E.M., P 6 0.02). It is also evident from Fig. 8 that removing extracellular calcium markedly inhibited the e¡ect of a hyposmotic challenge on leucine incorporation into mammary tissue protein. Thus, the volume-sensitive increase in leucine incorporation in the presence and absence of extracellular calcium (+EGTA) was 2.81 þ 0.40 and The sensitivity of mammary protein synthesis to a reduction in the osmolality of the incubation medium was examined. Fig. 6 shows the e¡ect of decreasing the osmolality over the range 295 to 156 mosmol/kg. The osmolality of the incubation bu¡er was changed by reducing the choline^Cl concentration. It is apparent that the protein synthesis increased linearly between 295 and 226 mosmol/ kg and was maximal by 188 mosmol/kg. Fig. 7 shows the e¡ect of increasing the external osmolality over the range 298 to 500 mosmol/kg on mammary protein synthesis. The osmolality of the incubation bu¡er was increased by the addition of sucrose. It is evident that the decrease in mammary protein synthesis is dependent upon the extent of the hyperosmotic challenge. The relationship between protein synthesis and the osmolality of the incubation medium shown in Fig. 7 could adequately be described by a monoexponential equation: Pr ˆ ae3kr 1† where Pr is protein synthesis (%) at a given osmolality r (mosmol/kg), a is a constant (4924.5) and k is the decay constant (0.013 (mosmol/kg)31 ). 3.5. Regulation of volume-sensitive protein synthesis by Ca2‡ Several studies have shown that mammary protein synthesis under isosmotic conditions is dependent upon calcium [13,14]. In view of this we tested the e¡ect of calcium on volume-activated protein synthesis. In accordance with previous results, Fig. 8 shows that mammary protein syn- Fig. 8. The e¡ect of Ca2‡ on swelling-induced [3 H]leucine incorporation into mammary protein. The isosmotic bu¡er contained (mM) 55 NaCl, 80 choline^Cl, þ 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1) (osmolality = 303 þ 9 mosmol/kg). The hyposmotic bu¡er was similar in composition except that it did not contain choline^Cl (osmolality = 159 þ 5 mosmol/kg). Tris^EGTA (0.5 mM) was also added to the Ca‡ -free bu¡ers. Data shown are the means þ S.E.M. of four experiments using tissue from separate animals. 44 A.C.G. Grant et al. / Biochimica et Biophysica Acta 1475 (2000) 39^46 Fig. 9. The e¡ect of thapsigargin on swelling-induced [3 H]leucine incorporation into mammary protein. The isosmotic bu¡er contained (mM) 55 NaCl, 80 choline^Cl, 5 KCl, 2 CaCl2 , 1 MgSO4 , 10 glucose and 10 Tris^MOPS, pH 7.4 plus 1 WCi/ml [3 H]leucine (+amino acids ^ see Table 1) (osmolality = 305 þ 1 mosmol/kg). The hyposmotic bu¡er was similar in composition except that it contained no choline^Cl (osmolality = 165 þ 1 mosmol/kg). When required, thapsigargin was used at a ¢nal concentration of 1 WM. DMSO was present at 0.1% (v/v) in all bu¡ers. Data shown are the means þ S.E.M. of six experiments using tissue from separate animals. 0.91 þ 0.14 Wmol/h/g dry tissue weight, respectively (n = 4, þ S.E.M., P 6 0.02). The results shown in Fig. 8 constitute prima facie evidence that protein synthesis under isosmotic and hyposmotic conditions is dependent upon external calcium. However, the e¡ect of removing external calcium (+EGTA) on protein synthesis could be due to decreasing intraorganelle Ca2‡ . To determine whether intraorganelle (i.e. luminal) Ca2‡ plays a role in determining protein synthesis we examined the e¡ect of thapsigargin, an endoplasmic reticulum (ER) Ca2‡ -ATPase inhibitor [15], on mammary protein synthesis under isosmotic and hyposmotic conditions. The results of these experiments are shown in Fig. 9. The inclusion of thapsigargin in the incubation medium at a concentration of 1 WM inhibited mammary protein synthesis by 35% under isosmotic conditions. Thus, leucine incorporation in the absence and presence of thapsigargin under isosmotic conditions was 2.11 þ 0.32 and 1.36 þ 0.23 Wmol/h/g dry tissue weight, respectively (n = 6, þ S.E.M., P 6 0.01). Thapsigargin also reduced volume-activated protein synthesis. Thus, volume-sensitive leucine incorporation in the absence and presence of thapsigargin was 2.87 þ 0.49 and 1.75 þ 0.35 Wmol/h/g dry tissue weight, respectively (n = 6, þ S.E.M., P 6 0.01). 4. Discussion We have examined protein synthesis by rat mammary tissue explants by measuring the rate of incorporation of radiolabelled amino acids into TCA-precipitable material. Under control conditions (i.e. isosmotic) we found that the ratio of leucine to methionine incorporation into mammary protein was 7.52. Even though we were measuring total protein synthesis this ratio is comparable to the ratio of leucine/methionine in rat milk proteins (5.51) found by Davis et al. [16] suggesting that our technique for measuring protein synthesis by mammary tissue is adequate. Furthermore, the ¢nding that protein synthesis was linear under all the conditions studied suggests that the secretion of protein during the time course of our experiments was negligible. In this connection it has been shown that secretion of [35 S]methionine-labelled protein into the medium from mouse mammary acini was only detectable after a lag period of 45^60 min [17,18]. However, the overall rates of protein synthesis reported here are larger than that found in a previous study from this laboratory [6] : this may re£ect that there was a di¡erence between the way the TCA-precipitates were treated. In our previous study the TCA-precipitates were stored at 320³C until they were processed. 4.1. The e¡ect of cell volume changes on mammary protein synthesis The results con¢rm and extend the preliminary observation that a change to mammary cell volume markedly a¡ects the rate of protein synthesis. Thus, cell swelling, induced by a hyposmotic shock, markedly increased the rate of mammary protein synthesis whereas cell shrinking, induced by a hyperosmotic challenge inhibited protein synthesis. Importantly, the e¡ect of a hyperosmotic shock was reversible suggesting that the e¡ect of cell shrinking on mammary protein synthesis cannot be attributed to a loss of cell viability. It is interesting to note that mammary protein synthesis was very sensitive to changes in the osmolality of the incubation medium in the region 260^325 mosmol/kg. This suggests that a small increase or decrease in cell volume in vivo could give rise to relatively large changes in mammary protein synthesis. We also found that isosmotic cell swelling increased mammary protein synthesis suggesting that the increase in synthesis following a hyposmotic shock is not simply due to a reduction in the osmolality of the incubation bu¡er per se but rather as a consequence of changing the intracellular osmotic pressure. However, it should be borne in mind that mammary cells, in common with most other cell types, probably regulate their volume following osmotic perturbations. Indeed, it has been shown that mammary tissue expresses a volume-sensitive amino acid transport system [19]. If mammary cells are regulating their volume during the time course of our experiments then it is possible that cell volume per se is not the controlling factor [2]. Other mechanisms, directly or indirectly associated with cell volume regulation (i.e. intracellular tonicity, macromolecular crowding) may be candidates for the regulation of protein synthesis described in this report. A.C.G. Grant et al. / Biochimica et Biophysica Acta 1475 (2000) 39^46 Our experiments indicate that cell volume is an important factor in the regulation of mammary protein synthesis. This is in accordance with the e¡ect of cell volume perturbations on protein synthesis in hepatocytes. It may transpire that the control of protein synthesis by the cellular hydration state is a widespread phenomenon. However, it is interesting to note that there is one major di¡erence between the e¡ect of cell volume changes found in mammary tissue and hepatocytes. We have shown that a hyposmotic challenge increased mammary protein synthesis above and beyond that found under isosmotic conditions. In contrast, protein synthesis in hepatocytes, in the presence of external amino acids, proceeds at similar rates under hyposmotic and isosmotic conditions [3,20]. 4.2. Regulation of volume-activated protein synthesis by Ca2 + The results clearly show, in accordance with previous studies [13,14,21], that mammary tissue protein synthesis, under isosmotic conditions, is dependent upon calcium. Thus, removing extracellular calcium in the presence of EGTA markedly reduced the incorporation of radiolabelled leucine into mammary protein. We now show that protein synthesis activated by cell swelling is also dependent upon calcium. At ¢rst sight, this may be taken to suggest that protein synthesis is dependent upon the presence of extracellular Ca2‡ . Indeed, cell swelling has been shown to transiently increase cytosolic calcium in both mouse and rat mammary cells in a fashion which requires external Ca2‡ [22,23]. However, removing extracellular Ca2‡ in the presence of EGTA will probably reduce intraorganelle Ca2‡ which in turn could reduce protein synthesis. In this connection, we found, in accordance with the results of Duncan and Burgoyne [14], that thapsigargin, an ER Ca2‡ -ATPase inhibitor, inhibited protein synthesis under isosmotic conditions. The ¢nding that thapsigargin also inhibited protein synthesis under hyposmotic conditions suggests that the luminal calcium concentration is also an important factor in the regulation of volumesensitive protein synthesis. It should be borne in mind, however, that the present results do not rule out cytosolic calcium as a potential regulator of mammary protein synthesis. 4.3. Physiological signi¢cance of volume-sensitive protein synthesis In this study we used aniso-osmotic solutions and ureacontaining bu¡ers as an experimental tool to change mammary cell volume. It must be stressed that under physiological conditions mammary cells will never be exposed to such conditions. However, we predict that mammary cell volume will change in vivo as a consequence of substrate accumulation and/or changes to the rate of oxidative metabolism. In addition, the rate of removal of metabolic 45 products from mammary cells will also in£uence cell volume. Thus, mammary cell volume may change in vivo following a post-prandial increase in plasma nutrients. This could be a way of matching nutrient supply with the synthesis of milk components such as protein. There is good evidence that hormones are able to in£uence the cellular hydration state. For example, insulin increases hepatocyte volume and consequently the metabolic activity by regulating transport mechanisms such as Na‡ /H‡ exchange and Na‡ -K‡ -Cl3 cotransport [24]. In this connection, action of hormones on membrane transport processes (e.g. Na‡ -coupled cotransport mechanisms) could act to increase mammary cell volume. In this manner cell volume could function as a signalling system in the hormonal control of mammary metabolism. The next step in the investigation will be to develop a method to measure mammary cell volume under physiological conditions and to identify the factors (i.e. nutrient uptake, hormones) which modulate it. Acknowledgements The authors are grateful to Mrs J. Thomson for expert technical assistance. This work was funded by the Scottish Executive Rural A¡airs Department. References [1] D. Haussinger, Biochem. J. 313 (1996) 697^710. [2] F. Lang, G.L. Busch, M. Ritter, H. Volkl, S. Waldegger, E. Gulbins, D. Haussinger, Physiol. Rev. 78 (1998) 247^306. [3] B. Stoll, W. Gerok, F. Lang, D. Haussinger, Biochem. J. 287 (1992) 217^222. [4] A. Baquet, A. Lavoinne, L. Hue, Biochem. J. 273 (1991) 57^62. [5] M. Al-Habori, M. Peak, T.H. Thomas, L. Agius, Biochem. J. 282 (1992) 789^796. [6] I.D. Millar, M.C. Barber, M.A. Lomax, M.T. Travers, D.B. Shennan, Biochem. Biophys. Res. Commun. 230 (1997) 351^355. [7] D.B. Shennan, Q. J. Exp. Physiol. 74 (1989) 927^938. [8] D.B. Shennan, I.D. Millar, D.T. Calvert, Proc. Nutr. Soc. 56 (1997) 177^191. [9] J. Vina, I.R. Puertes, J.M. Estrela, J.R. Vina, J.L. Gablis, Biochem. J. 194 (1981) 99^102. [10] J. Vina, I.R. Puertes, T. Saez, J.R. Vina, FEBS Lett. 126 (1981) 250^ 252. [11] G.R. Jansen, M.B. Schilby, M. Masor, D.A. Sampson, J.B. Longenecker, J. Nutr. 116 (1986) 376^387. [12] D.B. Shennan, M. Peaker, Physiol. Rev. (2000) in press. [13] C.J. Wilde, H.R. Hasan, D.A. White, R.J. Mayer, Biochem. Biophys. Res. Commun. 103 (1981) 934^942. [14] J.S. Duncan, R.D. Burgoyne, Biochem. J. 317 (1996) 487^493. [15] O. Thastrup, A.P. Dawson, O. Schar¡, B. Foder, P.J. Cullen, B.K. Drobak, P.J. Bjerrum, S.B. Christensen, M.R. Hanley, Agents Actions 27 (1989) 17^23. [16] T.A. Davis, M.L. Fiorotto, P.J. Reeds, J. Nutr. 123 (1993) 947^956. [17] M.D. Turner, M.E. Rennison, S.E. Handel, C.J. Wilde, R.D. Burgoyne, J. Cell Biol. 117 (1992) 269^278. [18] M.E. Rennison, S.E. Handel, C.J. Wilde, R.D. Burgoyne, J. Cell Sci. 102 (1992) 239^247. 46 A.C.G. Grant et al. / Biochimica et Biophysica Acta 1475 (2000) 39^46 [19] D.B. Shennan, S.A. McNeillie, D.E. Curran, Exp. Physiol. 79 (1994) 797^808. [20] A.J. Meijer, L.A. Gustafson, J.J.F.P. Luiken, P.J.E. Blommaart, H.P. Caro, G.M. Van Woerkom, C. Spronk, L. Boon, Eur. J. Biochem. 215 (1993) 449^454. [21] J.J. Smith, C.S. Park, T.W. Keenan, Int. J. Biochem. 14 (1981) 573^ 576. [22] A.W. Sudlow, R.D. Burgoyne, P£ugers Arch. 433 (1997) 609^616. [23] I.F. Gow, C. Burns, D.B. Shennan, Biochem. Soc. Trans. 26 (1998) S389. [24] L. Agius, M. Peak, G. Beresford, M. Al-Habori, T.H. Thomas, Biochem. Soc. Trans. 22 (1994) 516^522.