Figure 1.
Schematic representation of known and putative ionic regulatory volume increase (RVI) mechanisms in mammalian skeletal muscle.
The sodium, potassium, chloride cotransporter (NKCC) facilitates the inward flux of these three ions into cells. The NKCC can be inhibited to a large extent by 1 mM bumetanide. The two main monocarboxylate transporters (MCTs) in muscle are MCT1 and MCT4. Phloretin (1 mM) inhibits all lactate- flux through MCT1 and about 90% of flux through MCT4. pCMBS inhibits all lactate- flux through MCT4 and about 90% of flux through MCT1. Data presented in the present paper favour a preferential influx of lactate- through MCT1 and a preferential efflux of lactate- through MCT4.
Figure 2.
Cell volume loss with subsequent volume recovery in response to increased NaCl or sucrose.
Time course of volume changes in single fibres where extracellular osmolarity was raised 49/L by addition of NaCl (•, n = 6 fibres from 4 muscles from 2 mice) or sucrose (▪, n = 8 fibres from 4 muscles from 2 mice), or by raising osmolarity by 94 mosmol/L using sucrose (▾ n = 11 fibres from 6 muscles from 3 mice). * The initial rate of volume loss and RVI were greater when NaCl was used to raise osmolarity compared to sucrose.
Figure 3.
Osmolarity dependence of volume loss and attenuated volume loss with elevated [NaLac] compared to NaCl.
Time course of volume changes in single fibres where extracellular osmolarity was raised by 14 (▴), 34 (•) and 49 mosmol/L (▪) by raising [NaCl] or [NaLac]. NaCl + 49 mosmol/L: n = 6 fibres from 4 muscles from 2 mice; NaLac + 49 mosmol/L: n = 30 fibres from 16 muscles from 8 mice). NaCl + 34 mosmol/L: n = 14 fibres from 7 muscles from 3 mice; NaLac + 34 mosmol/L: n = 14 fibres from 7 muscles from 3 mice or 7.5 mM NaCl. NaCl + 14 mosmol/L: n = 20 fibres from 6 muscles from 3 mice; NaLac + 14 mosmol/L: n = 12 fibres from 6 muscles from 3 mice.
Figure 4.
NKCC inhibition does not affect muscle volume responses when osmolarity is raised using NaLac.
Time course of volume changes in single fibres to raised osmolarity (+49 mosmol/L) using NaCl or NaLac, in the absence or presence of NKCC activity inhibition using 1 mM bumetanide. NaLac controls (▪, n = 39 fibres from 21 muscles from 6 mice) compared to NKCC inhibition (⋄; n = 30 fibres from 16 muscles from 8 mice). NaCl controls (▴; n = 13 fibres from 6 muscles from 4 mice) compared to NKCC inhibition (○; n = 16 fibres from 6 muscles from 3 mice). The top panel expands the first 400 s. * significantly lower than NaCl control.
Figure 5.
MCT inhibition increases the magnitude of volume loss and influences the RVI response.
Time course of volume changes in single fibres where MCT activity was inhibited using either phloretin (▾, n = 7 fibres from 4 muscles from 2 mice) or pCMBS (•, n = 11 fibres from 6 muscles from 3 mice), or where NKCC activity was inhibited using bumetanide (⋄, n = 39 fibres from 21 muscles from 6 mice) compared to control (▪; n = 30 fibres from 16 muscles from 8 mice). Fibres were incubated in the presence of inhibitor for 30 minutes prior to addition of NaLac to raise extracellular osmolarity by 49 mosmol/kg. pCMBS resulted in a greater volume loss and more rapid volume recovery than seen in controls or in bumetanide treated fibres. Phloretin resulted in a greater volume loss than seen in controls, bumetanide treated fibres and pCMBS treated fibers. The rates of volume recovery in pCMBS and phloretin treated fibres were similar.
Figure 6.
Combined inhibition of MCT and NKCC minimizes volume loss and associated RVI.
Time course of volume changes in single fibres where NKCC and monocarboxylate transport activity was inhibited using bumetanide with phloretin (▪ n = 19 fibres from 8 muscles from 4 mice) or bumetanide with pCMBS (<$>\raster(130%)="rg1"<$> n = 13 fibres from 7 muscles from 4 mice) compared to NaLac control (• n = 30 fibres from 16 muscles from 8 mice) and NaLac after NKCC inhibition (▴ n = 39 fibres from 21 muscles from 6 mice). Fibres were incubated in the presence of inhibitors for 30 minutes prior to addition of NaLac to raise extracellular osmolarity by 57 mosmol/kg. The top panel expands the first 400 s.
Figure 7.
Time course of volume changes in single fibres treated with DMSO.
Cell volume did not change after addition of DMSO to final concentration of 2 to 3% (<$>\raster(130%)="rg1"<$>) for 2700 s (45 minutes; no change after the 1500 s shown in the figure). At 45 minutes these DMSO-treated fibres experienced a 57 mosmol/L increase in extracellular osmolarity using NaCl (▪; time was re-set to 0 s). The response of fibres not treated with DMSO to increased osmolarity (▾ 57 mosmol/L using NaCl; data from Fig. 1) is shown for comparison. DMSO treatment: n = 16 fibres from 8 muscles from 3 mice. NaCl treatment: n = 12 fibres from 6 muscles from 3 mice.