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Specific predictions of the macromolecular crowding theory were examined on the volume of internally perfused barnacle muscle cells to analyze the mechanisms by which cells sense volume changes. The predictions tested were that isotonic replacement of large macromolecules by smaller ones should induce volume decreases proportional to the initial macromolecular concentration and size as well as to the magnitude of the concentration reduction. Results showed that isotonic replacement of proteins or polymers with sucrose induced volume reductions which agreed with the prediction.

A study of the involvement of adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) in cell volume regulation in skeletal muscle in intact and internally perfused barnacle muscle cells reveals that in intact cells dibutyryl cAMP and forskolin enhance regulatory volume decrease (RVD) when exposed to Cao-free solutions while dibutyryl cGMP prevents RVD in cells exposed to Cao-containing solutions. cAMP induces a volume loss that is inhibited by the presence of cGMP in perfused cells with buffered intracellular free Ca2+ concentration.

The use of voltage clamp technique on barnacle muscle cells helps study the effects of isosmotic removal of Ca+ and membrane potential on cell volume. Cell volume varies differently with membrane depolarization induced by the substitution of Ca+ with Mg+ in Cai-loaded and Cai-depleted cells. Cai-loaded cells exhibit a decrease in cell volume while Cai-depleted cells exhibit an increase in cell volume, which is sensitive to verapamil. Cell volume increase with Ca+ removal, under constant membrane potentials, is independent of Cai levels.

The use of isolated voltage-clamped barnacle muscle cells helps study the effect of isosmotic removal of extracellular Na+ on cell volume and membrane potential. Membrane depolarization is not essential for the cell volume decrease due to Nao replacement by Triso or Lio, in the presence of Cao. Nao replacement also leads to a membrane depolarization due to activated nonselective cation channels. Similar replacement in the absence of Cao does not alter cell volume or membrane potential.

Influence of extracellular Ca2+ (Cao) on regulatory volume decrease (RVD), extracellular space (ECS) and on sarcolemmal hydraulic water permeability (L'p), was examined. L'p was not affected by the presence or absence of Cao. Cells subjected to anisosmotic media acted as osmometers in 0 and 11 mM Cao, indicating identical istopes and intercepts in van't Hoff plots. Mean speed of RVD was not connected to the level of swelling in reaction to hyposmotic challenges, but PCS decreased in the presence of Cao and hypotonicity.

Hypertonic saline (HS) inhalation therapy benefits cystic fibrosis (CF) patients [Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006; Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, Xuan W, Bye PT; the National Hypertonic Saline in Cystic Fibrosis (NHSCF) Study Group. N Engl J Med 354: 229-240, 2006]. Surprisingly, these benefits are long-lasting and are diminished by the epithelial Na(+) channel blocker amiloride (Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006). Our aim was to explain these effects. Human bronchial epithelial (hBE) cells from CF lungs were grown in inserts and were used in three experimental approaches: 1) Ussing chambers to measure amiloride-sensitive short-circuit currents (INa); 2) continuous perfusion Ussing chambers; and 3) near "thin-film" conditions in which the airway surface of the inserts was exposed to a small volume (30 μl) of isosmotic or HS solution as the inserts were kept in their incubation tray and were subsequently used to measure INa under isosmotic conditions (near thin-film experiments; Tarran R, Boucher RC. Methods Mol Med 70: 479-492, 2002). HS solutions (660 mosmol/kgH2O) were prepared by adding additional NaCl to the isosmotic buffer. The transepithelial short-circuit current (ISC), conductance (GT), and capacitance (CT) were measured by transepithelial impedance analysis (Danahay H, Atherton HC, Jackson AD, Kreindler JL, Poll CT, Bridges RJ. Am J Physiol Lung Cell Mol Physiol 290: L558-L569, 2006; Singh AK, Singh S, Devor DC, Frizzell RA, van Driessche W, Bridges RJ. Methods Mol Med 70: 129-142, 2002). Exposure to apical HS inhibited INa, GT, and CT. The INa inhibition required 60 min of reexposure to the isosmotic solution to recover 75%. The time of exposure to HS required to inhibit INa was <2.5 min. Under near thin-film conditions, apical exposure to HS inhibited INa, but as osmotically driven water moved to the apical surface, the aqueous apical volume increased, leading to an amiloride-insensitive decrease in its osmolality and to recovery of INa that lagged behind the osmotic recovery. Amiloride significantly accelerated the recovery of INa following exposure to HS. Our conclusions are that exposure to HS inhibits hBE INa and that amiloride diminishes this effect.

Luminal and abluminal plasma membranes were isolated from bovine brain microvessels and used to identify and characterize Na(+)-dependent and facilitative taurine transport. The calculated transmembrane potential was -59 mV at time 0; external Na(+) (or choline under putative zero-trans conditions) was 126 mM (T=25 °C). The apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24 μM to 11.4 μM. Abluminal membranes had both Na(+)-dependent taurine transport as well as facilitative transport while luminal membranes only had facilitative transport. The apparent K(m) for facilitative and Na(+)-dependent taurine transport were 0.06±0.02 μM and 0.7±0.1 μM, respectively. The Na(+)-dependent transport of taurine was voltage dependent over the range of voltages studied (-25 to -101 mV). The transport was over 5 times greater at -101 mV compared to when V(m) was -25 mV. The sensitivity to external osmolality of Na(+)-dependent transport was studied over a range of osmolalities (229 to 398 mOsm/kg H(2)O) using mannitol as the osmotic agent to adjust the osmolality. For these experiments the concentration of Na(+) was maintained constant at 50mM, and the calculated transmembrane potential was -59 mV. The Na(+)-dependent transport system was sensitive to osmolality with the greatest rate observed at 229 mOsm/kg H(2)O., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The aims of this work were to determine: 1) whether Ca2+ exit via the plasmalemmal Ca2+ ATPase (PMCA) is coupled to H+ entry via a Ca2+/H+ exchange; 2) whether operation of PMCA has an absolute requirement on external H+ (Ho); and 3) the stoichiometry and voltage-dependence of the Ca2+/H+ exchange. Barnacle muscle cells were used because of the ease with which they can be internally-perfused (e.g., with 45Ca), voltage-clamped and impaled with a pH electrode. Thus, the simultaneous measurement of plasmalemmal Ca2+ and H+ fluxes can be measured. The effects of Ho, intracellular ATP, PMCA blockers, and membrane potential (VM) were studied on PMCA-mediated Ca2+/H+ exchange. The results indicate that: i) Ca2+ efflux is promoted by external acidification, is accompanied by a membrane depolarization, and by an intracellular acidification greater than the one resulting from Ho "leak" and PMCA-mediated ATP hydrolysis; ii) Ho-dependent Ca2+ efflux is inhibited by PMCA blockers and by ATP depletion and is accelerated by membrane depolarization (~3 fold by 20 mV depolarization); iii) the coupling ratio of the Ca2+/H+ exchange depends on Ho: at an extracellular pH (pHo)=6.5, the ratio is 1Ca2+:~3H+; at pHo=8.2, Ca2+ efflux rate is 3 times slower and the ratio is 1Ca2+: <1H+.

The goal of this study was to assess the effect of extracellular glucose and K+ ((K)o) on the intracellular osmolyte content and cell volume maintenance and regulation in a human embryonic kidney cell line (tsA201a). Cell volume maintenance was studied by isotonic (313 +/- 5 mOsm) replacement of culture media by a glucose-free Ringer solution containing (in mM) 0, 3, 6, or 10 K+. Cell volume regulation was studied by exposing cells to hypotonic (250 +/- 5 mOsm) glucose-free Ringer solution containing the various (K)o. The results showed that: 1) intracellular osomlyte content (i.e. Na+, Cl-, Urea and free amino acids (FAA)) and cell volume increased when culture media was replaced with isotonic Ringer at all (K)o; 2) osmolyte content decreased with continuous exposure to isotonic Ringer at all (K)o but cell volume changes depended on (K)o. Volume recovery occurred at 6 and 10 mM K+; 3) exposure to hypotonic Ringer induced swelling at all (K)o followed by a reduction in measured intracellular osmolytes. Regulatory volume decrease occurred in 6 or 10 mM K+ but swelling continued in 0 or 3 mM K+; and 4) addition of ouabain produced swelling without recovery under iso- and hypotonic conditions. These results indicate that the removal of extracellular glucose produced a transient inhibition of the Na+/K+ ATPase resulting in a transient increase in the intracellular content of Urea, FAA and cell volume and (K)o regulated an as yet unidentified intracellular osmolyte.

In numerous animal cells, experimental manipulations that increase the intracellular free Ca2+ concentration induce cell volume reduction. This may occur under isosmotic conditions, e.g. when external Ca2+ (Ca(o)) is replaced by Mg2+ (42) or during exposure to hypoosmotic conditions (i.e. regulatory volume decrease, RVD) in the presence of Ca(o). We determined the osmolytes responsible for volume reduction under isosmotic and hypoosmotic conditions in barnacle muscle cells. Organic osmolytes (i.e. free amino acids and methylamines) and inorganic ions accounted for approximately 78% and 22% of the intracellular isosmotic activity, respectively. Isosmotic Ca(o) removal induced a net loss of KCI (with a ratio of 1K:1Cl) and free amino acids (FAA, mainly glycine and taurine). During RVD. the same ions (but in a proportion of 2K:1Cl) and FAA were lost. Since RVD was accompanied by extracellular alkalinization, the 2K:1Cl loss may be explained by the presence of a K+/H+ exchanger (or K+-OH- co-transporter) or Cl-/OH- exchanger. The lack of RVD in the absence of Ca(o) cannot be attributed to the loss of intracellular osmolytes during isosmotic Ca(o) removal because addition of Ca(o) during cell swelling promoted RVD.

Barnacle muscle cells regulate their volume when exposed to anisotonic conditions. Due to their large size, these cells can be internally perfused. Interestingly, perfused cells maintain their volume regulatory properties (17,21). Thus, the osmotic properties of barnacle muscle cells can be studied under conditions in which the intracellular and extracellular osmolalities, the membrane potential (V(M)), the cell volume and the intracellular pressure can all be measured simultaneously. In this manuscript we report the effect that various rates of isosmotic (1000 mOsm x kg H2O(-1)) intracellular perfusion have on cell volume, intracellular pressure, intracellular osmolality, V(M), and the apparent sarcolemmal hydraulic water permeability (L'p). Replacement of the cytosol with the perfusate at a perfusion rate of 0.83 microl x min(-1) took 120 min. During this transition period, the cell volume increased from 45.1+/-6.9 microl to 73.7+/-5.8 microl, the intracellular osmolality decreased from 1406+/-133 to 1188+/-64 mOsm x kg H2O(-1), and the intracellular pressure underwent a transient drop of 2.8 cm H2O. After 2.5 hr of continuous perfusion at 0.83 microl min(-1), the above mentioned parameters reached steady values: the L'p was 1.35 x 10(-5) cm x sec(-1) x Osm(-1) x kg H2O(-1); cell volume was 67.2+/-6 microl; the intracellular osmolality was 1052+/-10 mOsm x kg H2O(-1); the intracellular pressure was 5.6+/-0.4 cm H2O; V(M) depolarized slowly at a rate of 0.03 mV x min(-1). Stepwise increases in the rate of perfusion (from 0.83 to 3.18 microl min(-1)) produced reversible increases in the intracellular pressure, L'p and cell volume and decreases in intracellular osmolality. We conclude that intracellular perfusion: i/ produces a transient removal of intracellular osmotically active components; ii/ promotes sarcolemmal water filtration; iii/ induces a laminar flow of perfusate at the center of the cell, and iv/ enables calculations of sarcolemmal L'p values under isosmotic conditions.

In excitable cells, the concentration of intracellular free Mg2+ ([Mg2+]i) is several hundred times lower than expected if Mg2+ ions were at electrochemical equilibrium. Since Mg2+ is a permeant ion across the plasmalemma, it must be constantly extruded. An ATP-dependent Na/Mg exchanger has been proposed as the sole mechanism responsible for Mg2+ extrusion. However, this hypothesis fails to explain numerous observations including the fact that K+ and Cl- appear to be involved in Mg2+ transport. Until now three main limitations have hampered the studies of plasmalemmal Mg2+ transport: i) 28Mg, the only useful radioactive isotope of Mg2+, has a short half-life and is difficult to obtain; ii) squid giant axons, the ideal preparation to carry out transport studies under "zero-trans" conditions, are only available during the summer months; and iii) the ionic fluxes mediated by the Mg2+ transporter are very small and difficult to measure. The purpose of this manuscript is to review how these limitations have been recently overcame and to propose a novel hypothesis for the plasmalemmal Mg2+ transporter in squid axons and barnacle muscle cells. Overcoming the limitations for studying the plasmalemmal Mg2+ transporter has been possible as a result of the following findings: i) the Mg2+ exchanger can operate in "reverse", thus extracellular Mg2+-dependent ionic fluxes (e.g., Na+ efflux) can be utilized to measure its activity; ii) internally perfused, voltage-clamped barnacle muscle cells which are available all year long can be used in addition to squid axons; and iii) phosphoinositides (e.g., PIP2) produce an 8-fold increase in the ionic fluxes mediated by the Mg2+ exchanger. The hypothesis that we postulate is that, in squid giant axons and barnacle muscle cells, a 2Na+2K+2Cl:1Mg exchanger is responsible for transporting Mg2+ across the plasmalemma and for maintaining [Mg2+]i under steady-state conditions.

Deficient myocardial cyclic AMP concentrations contribute to abnormal Ca2+ handling and systolic and diastolic dysfunction in chronic heart failure (CHF). We tested the hypothesis that decreased cyclic AMP in skeletal muscle of animals with failure may contribute to the weakness and easy fatiguability also common in patients with CHF. We compared intracellular Ca2+ signaling and contractility in skeletal muscle preparations from rats 6 weeks after myocardial infarction-induced CHF versus sham-operated controls. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control and CHF rats. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches. Treatment with 2mM dibutyryl cyclic AMP returned tension and Ca2+ towards normal levels. There was no evidence of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling that could be reversed with cyclic AMP supplementation as previously reported for the heart. This suggests that deficient cyclic AMP levels may contribute to both cardiac and skeletal muscle dysfunction in CHF.