Effectiveness of an intervention to reduce sedentary behaviour as a personalised secondary prevention strategy for patients with coronary artery disease: main outcomes of the SIT LESS randomised clinical trial.

BACKGROUND: A high sedentary time is associated with increased mortality risk. Previous studies indicate that replacement of sedentary time with light- and moderate-to-vigorous physical activity attenuates the risk for adverse outcomes and improves cardiovascular risk factors. Patients with cardiovascular disease are more sedentary compared to the general population, while daily time spent sedentary remains high following contemporary cardiac rehabilitation programmes. This clinical trial investigated the effectiveness of a sedentary behaviour intervention as a personalised secondary prevention strategy (SIT LESS) on changes in sedentary time among patients with coronary artery disease participating in cardiac rehabilitation. METHODS: Patients were randomised to usual care (n = 104) or SIT LESS (n = 108). Both groups received a comprehensive 12-week centre-based cardiac rehabilitation programme with face-to-face consultations and supervised exercise sessions, whereas SIT LESS participants additionally received a 12-week, nurse-delivered, hybrid behaviour change intervention in combination with a pocket-worn activity tracker connected to a smartphone application to continuously monitor sedentary time. Primary outcome was the change in device-based sedentary time between pre- to post-rehabilitation. Changes in sedentary time characteristics (prevalence of prolonged sedentary bouts and proportion of patients with sedentary time ≥ 9.5 h/day); time spent in light-intensity and moderate-to-vigorous physical activity; step count; quality of life; competencies for self-management; and cardiovascular risk score were assessed as secondary outcomes. RESULTS: Patients (77% male) were 63 ± 10 years and primarily diagnosed with myocardial infarction (78%). Sedentary time decreased in SIT LESS (- 1.6 [- 2.1 to - 1.1] hours/day) and controls (- 1.2 [ ─1.7 to - 0.8]), but between group differences did not reach statistical significance (─0.4 [─1.0 to 0.3]) hours/day). The post-rehabilitation proportion of patients with a sedentary time above the upper limit of normal (≥ 9.5 h/day) was significantly lower in SIT LESS versus controls (48% versus 72%, baseline-adjusted odds-ratio 0.4 (0.2-0.8)). No differences were observed in the other predefined secondary outcomes. CONCLUSIONS: Among patients with coronary artery disease participating in cardiac rehabilitation, SIT LESS did not induce significantly greater reductions in sedentary time compared to controls, but delivery was feasible and a reduced odds of a sedentary time ≥ 9.5 h/day was observed. TRIAL REGISTRATION: Netherlands Trial Register: NL9263. Outcomes of the SIT LESS trial: changes in device-based sedentary time from pre-to post-cardiac rehabilitation (control group) and cardiac rehabilitation + SIT LESS (intervention group). SIT LESS reduced the odds of patients having a sedentary time >9.5 hours/day (upper limit of normal), although the absolute decrease in sedentary time did not significantly differ from controls. SIT LESS appears to be feasible, acceptable and potentially beneficial, but a larger cluster randomised trial is warranted to provide a more accurate estimate of its effects on sedentary time and clinical outcomes. CR: cardiac rehabilitation.

Sedentary Behaviour Intervention as a Personalised Secondary Prevention Strategy (SIT LESS) for patients with coronary artery disease participating in cardiac rehabilitation: rationale and design of the SIT LESS randomised clinical trial.

Patients with coronary artery disease (CAD) are more sedentary compared with the general population, but contemporary cardiac rehabilitation (CR) programmes do not specifically target sedentary behaviour (SB). We developed a 12-week, hybrid (centre-based+home-based) Sedentary behaviour IntervenTion as a personaLisEd Secondary prevention Strategy (SIT LESS). The SIT LESS programme is tailored to the needs of patients with CAD, using evidence-based behavioural change methods and an activity tracker connected to an online dashboard to enable self-monitoring and remote coaching. Following the intervention mapping principles, we first identified determinants of SB from literature to adapt theory-based methods and practical applications to target SB and then evaluated the intervention in advisory board meetings with patients and nurse specialists. This resulted in four core components of SIT LESS: (1) patient education, (2) goal setting, (3) motivational interviewing with coping planning, and (4) (tele)monitoring using a pocket-worn activity tracker connected to a smartphone application and providing vibrotactile feedback after prolonged sedentary bouts. We hypothesise that adding SIT LESS to contemporary CR will reduce SB in patients with CAD to a greater extent compared with usual care. Therefore, 212 patients with CAD will be recruited from two Dutch hospitals and randomised to CR (control) or CR+SIT LESS (intervention). Patients will be assessed prior to, immediately after and 3 months after CR. The primary comparison relates to the pre-CR versus post-CR difference in SB (objectively assessed in min/day) between the control and intervention groups. Secondary outcomes include between-group differences in SB characteristics (eg, number of sedentary bouts); change in SB 3 months after CR; changes in light-intensity and moderate-to-vigorous-intensity physical activity; quality of life; and patients' competencies for self-management. Outcomes of the SIT LESS randomised clinical trial will provide novel insight into the effectiveness of a structured, hybrid and personalised behaviour change intervention to attenuate SB in patients with CAD participating in CR. Trial registration number NL9263.

Hepatocyte growth factor/scatter factor stimulates Ca2+-activated membrane K+ current and migration of MDCK II cells.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates migration of various cells and has been linked via Met tyrosine kinase-signaling to transformation and the metastatic phenotype. Migration of transformed MDCK-F cells depends on activation of a charybdotoxin-sensitive, volume-activated membrane K+ current. Thus, we used patch-clamp electrophysiology and transwell migration assays to determine whether HGF/SF stimulation of MDCK II cell migration depends on the activation of membrane K+ currents. HGF/SF activated a membrane K+ current that increased over 24 hr, and which could be modulated by increasing intracellular calcium concentration, [Ca2+]i. Charybdotoxin (ChTX, 50 nM), iberiotoxin (IbTX, 100 nM), stichodactyla toxin (Stk, 100 nM) and clotrimazole (CLT, 1 mM) all inhibited this current. HGF/SF (100 scatter units/ml) significantly increased MDCK II cell migration over 8 hr compared to control cells. Addition of ChTX (50 nM), IbTX (100 nM), Stk (100 nM) or CLT (1 microM) inhibited the HGF/SF-stimulated MDCK II cell migration. We conclude that the activation of membrane Ca2+-activated K+current is necessary for HGF/SF stimulation of MDCK II cell.

Blocking swelling-activated chloride current inhibits mouse liver cell proliferation.

A non-transformed mouse liver cell line (AML12) was used to show that blocking swelling-activated membrane Cl- current inhibits hepatocyte proliferation. Two morphologically distinguishable cell populations exhibited distinctly different responses to hypotonic stress. Hypotonic stress (from 280 to 221 mosmol kg(-1)) to rounded, dividing cells activated an ATP-dependent, outwardly rectifying, whole-cell Cl- current, which took 10 min to reach maximum conductance. A similar anionic current was present spontaneously in 20 % of the dividing cells. Hypotonic stress to flattened, non-dividing cells activated no additional current. The Eisenman halide permeability sequence of swelling-activated anionic current in the dividing cells was SCN(-) > I(-) > Br(-) > Cl(-) > gluconate. Addition of either 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), tamoxifen or mibefradil inhibited swelling-activated anionic current. Hyperosmolarity by added sucrose inhibited the spontaneous anionic current in dividing cells. Added Cl- channel blockers NPPB (IC50 = 40 microM), DIDS (IC50 = 31 microM), tamoxifen (IC50 = 1.3 microM) and mibefradil (IC50 = 7 microM) inhibited proliferative growth of AML12 as determined by cell counts over 4 days or by protein accumulation over 2 days. Only the inhibitory effects of NPPB and mibefradil reversed with the drug washout. Hyperosmolarity by added sucrose (50 and 100 mM) also inhibited cell proliferation. Of the hydrophobic inhibitors neither NPPB at 40 microM nor tamoxifen at 1.3 microM, added for 48 h, reduced cellular ATP; however, DIDS at 31 microM significantly reduced cellular ATP with an equivalent increase in cellular ADP. We conclude that those membrane Cl- currents that can be activated by hypotonic stress are involved in mechanisms controlling liver cell growth, and that NPPB, tamoxifen and mibefradil at their IC50 for growth do not suppress the metabolism of mouse hepatocytes.

Membrane potassium channels and human bladder tumor cells. I. Electrical properties.

These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca(2+)-dependent outward K+ current, 0.57 +/- 0.13 nS/pF at -70 mV holding potential and 3.10 +/- 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca(2+)-activated, voltage-dependent K+ channel (KCa) of 214 +/- 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 +/- 1 pS at positive transmembrane potential (Vm) and 145 +/- 13 pS at negative Vm. Glibenclamide (50 microM), Ba2+ (1 mM) and quinine (100 microM) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50 = 46 microM. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mM) and by its activation with diazoxide (100 microM). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP.

Membrane potassium channels and human bladder tumor cells: II. Growth properties.

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 microM for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 microM (IC50 = 73 microM) when growth was assayed in the absence of added serum. This microM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane KATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10(-5) M bromodeoxyuridine. Glibenclamide (100 microM) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane KATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane KATP channel are involved in mechanisms controlling HTB-9 cell growth. However, KATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.

Ethanol increases hepatocyte water volume.

Mouse hepatocytes respond to osmotic stress with adaptive changes in transmembrane potential, Vm, such that hypotonic stress hyperpolarizes cells and hypertonic stress depolarizes them. These changes in Vm provide electromotive force for redistribution of ions such as Cl-, and this comprises part of the mechanism of hepatocyte volume regulation. We conducted the present study to determine whether ethanol administered in vitro to mouse liver slices increases hepatocyte water volume, and whether this swelling triggers adaptive changes in the Vm. Cells in mouse liver slices were loaded with tetramethylammonium ion (TMA). Changes in hepatocyte water volume were computed from measurements with ion sensitive microelectrodes of changes in intracellular activity of TMA (a1TMA) that resulted from water fluxes. Ethanol (70 mM) increased hepatocyte water volume immediately, and this peaked at 17% by 7 to 8 min, by which time a plateau was reached. Liver slices also were obtained from mice treated 12 hr prior with 4-methylpyrazole (4 mM). The effect of ethanol on their hepatocyte water volume was identical to that from untreated mice, except that the onset and peak were delayed 2 min. Hepatocyte Vm showed no differences between control or ethanol-treated cells during the course of volume changes. In contrast, hyposmotic stress, created by dropping external osmolality 50 mosm, increased Vm from -30 mV to -46 mV. Ethanol did not inhibit this osmotic stress-induced hyperpolarization, except partially at high concentrations of 257 mM or greater. We infer that ethanol-induced swelling of hepatocytes differs from that resulting from hyposmotic stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Redistribution of hepatocyte chloride during L-alanine uptake.

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (Vm), intracellular K+, Cl-, and Na+ activities (aiK, aiCl and aiNa), and water volume during L-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 mM L-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mM tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during L-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control Vm was -33 +/- 1 mV. L-alanine uptake first depolarized Vm by 2 +/- 0.2 mV and then hyperpolarized Vm by 5 mV to -38 +/- 1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, aiNa increased by 30% from 19 +/- 2 to 25 +/- 3 mM (P < 0.01), and aiK did not change significantly from 83 +/- 3 mM. However, with added ouabain (1 mM) L-alanine caused only a 2-mV increase in Vm, but now aiK decreased from 61 +/- 3 to 54 +/- 5 mM (P < 0.05). Hyperpolarization of Vm by L-alanine uptake also resulted in a 38% decrease of aiCl from 20 +/- 2 to 12 +/- 3 mM (P < 0.001). Changes in Vm and VCl-Vm voltage traces were parallel during the time of L-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl- with the Vm in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Contributions of K+, Na+, and Cl- to the membrane potential of intact hamster vascular endothelial cells.

The transmembrane potential (Vm) of vascular endothelial cells (EC) is an important property that may be involved in intra- and intercellular signal transduction for various vascular functions. In this study, Vm of intact aortic and vena caval EC from hamsters were measured using conventional microelectrodes. Vascular strips with the luminal surface upwards were suffused in a tissue chamber with Krebs solution in physiological conditions. The resting Vm of aortic and vena caval EC was found to be -40 +/- 1 mV (n = 55) and -43 +/- 1 mV (n = 15), respectively. The Vm recordings were confirmed to have originated from EC by scanning and transmission electron microscopy combined with the comparison of electrical recordings between normal and endothelium-denuded aortic strips. The input resistance varied from 10-240 M omega, which implied the presence of electrical coupling between vascular EC. Elevating the K+ level in the suffusate from 4.7 mM to 50 and 100 mM depolarized aortic EC by 19% and 29% and vena caval EC by 18% and 29%, respectively. These low percentages indicated a relatively small contribution of [K+] to the resting Vm of vascular EC. A positive correlation (r > 0.69) between the resting Vm and the magnitude of depolarization by the high [K+]o may be related to the involvement of voltage-dependent K+ channels. The hyperpolarization caused by lowering both [Na+]o and [Cl-]o suggested the disengagement of some electrogenic transport systems in the membrane, such as a Na(+)-K(+)-Cl- cotransporter. The transference number (t(ion)), as an index of membrane conductance for specific ions, was calculated for K+ (15-20%), Na+ (16%), and Cl- (9-15%), demonstrating that both Na+ and Cl- as well as K+ contribute to the overall resting Vm. Our study documented some basic electrophysiology of the vascular EC when both structural and functional properties of the cell were maintained, thus furthering the understanding of the essential role of endothelial cells in mediating vascular functions.

Hepatocyte water volume and potassium activity during hypotonic stress.

Hepatocytes exhibit a regulatory volume decrease (RVD) during hypotonic shock, which comprises loss of intracellular K+ and Cl- accompanied by hyperpolarization of transmembrane potential (Vm) due to an increase in membrane K+ conductance, (GK). To examine hepatocyte K+ homeostasis during RVD, double-barrel, K+-selective microelectrodes were used to measure changes in steady-state intracellular K+ activity (aKi) and Vm during hyposmotic stress. Cell water volume change was evaluated by measuring changes in intracellular tetramethylammonium (TMA+). Liver slices were superfused with modified Krebs physiological salt solution. Hyposmolality (0.8 x 300 mosm) was created by a 50 mM step-decrease of external sucrose concentration. Hepatocyte Vm hyperpolarized by 19 mV from -27 +/- 1 to -46 +/- 1 mV and aiK decreased by 14% from 91 +/- 4 to 78 +/- 4 mM when slices were exposed to hyposmotic stress for 4-5 min. Both Vm and aKi returned to control level after restoring isosmotic solution. In paired measurements, hypotonic stress induced similar changes in Vm and aKi in both control and added ouabain (1 mM) conditions, and these values returned to their control level after the osmotic stress. In another paired measurement, hypotonic shock first induced an 18-mV increase in Vm and a 15% decrease in aKi in control condition. After loading hepatocytes with TMA+, the same hypotonic shock induced a 14-mV increase in Vm and a 14% decrease in aTMAi. This accounted for a 17% increase of intracellular water volume, which was identical to the cell water volume change obtained when aKi was used as the marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse hepatocyte membrane potential and chloride activity during osmotic stress.

Hepatocyte transmembrane potential (Vm) during osmotic stress responds as an osmometer, in part because of changes in membrane K+ conductance. This may contribute to the electromotive force that drives transmembrane Cl- fluxes. To test this, double-barreled ion-sensitive microelectrodes were used to measure changes in steady-state intracellular Cl- activity (aiCl) during osmotic stress applied to mouse liver slices. Hyperosmotic and hyposmotic conditions were created by rapidly switching to a solution in which sucrose concentrations were increased or reduced, respectively. Hyperosmotic stress [1.4 x control osmolality (280 mosmol/kgH2O)] decreased hepatocyte Vm 46% from -39 +/- 1 to -21 +/- 1 mV (SE; n = 16 animals). Corresponding aiCl increased twofold from 19 +/- 2 to 38 +/- 3 mM. This shifted the Cl- equilibrium potential (ECl) 19 mV, from -38 +/- 0.3 to -19 +/- 2 mV. Hyposmotic stress [0.71 x control osmolality (290 mosmol/kgH2O)] increased hepatocyte Vm 64% from -28 +/- 1 to -46 +/- 1 mV (SE; n = 13 animals). Corresponding aiCl decreased 0.53-fold from 17 +/- 1 to 8 +/- 1 mM. This shifted the ECl 20 mV from -26 +/- 2 to -46 +/- 3 mV. Thus hepatocyte aiCl is in electrochemical equilibrium with Vm. The paired measurements above were repeated after addition of K(+)-channel blockers quinine or Ba2+. Ba2+ (2 mM) had no effect on either Vm or aiCl during hyperosmotic stress; however, Ba2+ significantly inhibited changes in Vm and aiCl during hyposmotic stress. Effects of quinine (0.5 mM) on Vm and aiCl during both hyperosmotic stress and hyposmotic stress were similar to those of Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hyperosmotic medium on hepatocyte volume, transmembrane potential and intracellular K+ activity.

Hepatocyte transmembrane potential (Vm) behaves as an osmometer and varies with changes in extracellular osmotic pressure created by altering the NaCl concentration in the external medium (Howard, L.D. and Wondergem, R. (1987) J. Membr. Biol. 100, 53). We now have demonstrated similar effects on Vm by increasing external osmolality with added sucrose and not altering ionic strength. We also have demonstrated that hyperosmotic stress-induced depolarization of Vm results from changes in membrane K+ conductance, gK, rather than from changes in the K+ equilibrium potential. Vm and aKi of hepatocytes in liver slices were measured by conventional and ion-sensitive microelectrodes, respectively. Cell water vols. were estimated by differences in wet and dry weights of liver slices after 10-min incubations. Effect of hyperosmotic medium on membrane transference number for K+, tK, was measured by effects on Vm of step-changes in external [K+]. Hepatocyte Vm decreased 34, 52 and 54% when tissue was superfused with medium made hyperosmotic with added sucrose (50, 100 and 150 mM). Correspondingly, aKi increased 10, 18 and 29% with this hyperosmotic stress of added sucrose. Tissue water of 2.92 +/- 0.10 kg H2O/kg dry weight in control solution decreased to 2.60 +/- 0.05, 2.25 +/- 0.06 and 2.22 +/- 0.05 kg H2O/kg dry weight with additions to medium of 50, 100 and 150 mM sucrose, respectively. Adding 50 mM sucrose to medium decreased tK from 0.20 +/- 0.01 to 0.05 +/- 0.01. Depolarization by 50% with hyperosmotic stress (100 mM sucrose) also occurred in Cl-free medium where Cl- was substituted with gluconate. We conclude that hepatocytes shrink during hyperosmotic stress, and the aKi increases. The accompanying decrease in Vm is opposite to that expected by an increase in aKi, and at least in part results from a concomitant decrease in gK. Changes in membrane Cl- conductance most likely do not contribute to osmotic stress-induced depolarization, since equivalent decreases in Vm occurred with added sucrose in cells depleted of Cl- by superfusing tissue with Cl-free medium.

Involvement of cell calcium and transmembrane potential in control of hepatocyte volume.

This study examined the role of hepatocyte calcium and cytoskeleton in activation of hyposmotic stress-induced increases in hepatocyte transmembrane potential and control of cell volume. Hepatocyte transmembrane potential was measured by glass microelectrodes in mouse liver slices before and after exposure to hyposmotic medium. Hepatocytes were loaded with tetramethylammonium by briefly exposing liver slices to nystatin, a cation poreforming antibiotic. Changes in hepatocyte steady-state water volume were determined by changes in intracellular tetramethylammonium activity measured with tetramethylammonium-sensitive, double-barrel micro-electrodes 4 min after exposure to hyposmotic medium. Hyposmotic stress of 74% of the control osmolality (approximately 280 mOsm) hyperpolarized hepatocyte transmembrane potential by 1.83 times the control hepatocyte transmembrane potential, and cell water volume increased by a factor of 1.19. The Ca2+ channel blocker verapamil (100 mumol/L) completely inhibited hyposmotic stress-induced hyperpolarization of hepatocyte transmembrane potential. This inhibitory effect diminished at doses of 37.5 or 50 mumol/L, but even these hyperpolarizations were decreased significantly compared with control. Hyposmotic stress during added verapamil dosage (50 mumol/L) also resulted in 23% greater cell swelling compared with control. Ca(2+)-free medium plus ethylene glycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid (5 mmol/L) inhibited hyposmotic stress-induced increases in hepatocyte transmembrane potential and resulted in 16% greater cell swelling compared with control. Calmodulin inhibitors trifluoperazine (100 mumol/L) and promethazine (100 mumol/L) inhibited the hyperpolarization of hepatocyte transmembrane potential caused by hyposmolality, as did 3,4,5-trimethoxybenzoate 8-(N,N-diethylamino)octyl ester) (50 mumol/L), which inhibits mobilization of Ca2+ from intracellular stores. Cytochalasin B (50 mumol/L), which disrupts microfilaments, also inhibited hyperpolarization of hepatocyte transmembrane potential with osmotic stress.(ABSTRACT TRUNCATED AT 250 WORDS)

An electrophysiological technique to measure change in hepatocyte water volume.

We have applied an electrophysiologic technique (Reuss, L. (1985) Proc. Natl. Acad. Sci. USA 82, 6014) to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA+) during transient exposure of cells to nystatin. Intracellular TMA+ activity (alpha 1TMA) was measured with TMA(+)-sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions alpha iTMA remained constant over 4 min in a single impalement. Hyperosmotic solutions (50, 100 and 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 and 100 mM) increased and decreased alpha iTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(alpha iTMA)0/(alpha iTMA)4min] -1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less predicted for a perfect osmometer. Corresponding measurements of Vm showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba2+ (2 mM) was present during hyposmotic stress of 0.66 X 286 mosmol (control), cell water volume increased by a factor of 1.44 +/- 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 +/- 0.02, P less than 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 +/- 0.04 to 1.24 +/- 0.09, P less than 0.01. We conclude that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of Vm in the presence of K(+)-channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.

Effects of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea.

Isolated hepatocytes from the elasmobranch Raja erinacea were examined for their regulatory responses to a solute load following electrogenic uptake of L-alanine. The transmembrane potential (Vm) was measured with glass microelectrodes filled with 0.5 M KCl (75 to 208 M omega in elasmobranch Ringer's solution) and averaged -61 +/- 16 mV (S.D.; n = 68). L-Alanine decreased (depolarized) Vm by 7 +/- 3 and 18 +/- 2 mV at concentrations of 1 and 10 mM, respectively. Vm did not repolarize to control values during the 5-10 min impalements, unless the amino acid was washed away from the hepatocytes. The depolarizing effect of L-alanine was dependent on external Na+, and was specific for the L-isomer of alanine, as D- and beta-alanine had no effect. Hepatocyte Vm also depolarized on addition of KCN or ouabain, or when external K+ was increased. Rates of 86Rb+ uptake and efflux were measured to assess the effects of L-alanine on Na+/K+-ATPase activity and K+ permeability, respectively. Greater than 80% of the 86Rb+ uptake was inhibited by 2 mM ouabain, or by substitution of choline+ for Na+ in the incubation media. L-Alanine (10 mM) increased 86Rb+ uptake by 18-49%, consistent with an increase in Na+/K+ pump activity, but had no effect on rubidium efflux. L-Alanine, at concentrations up to 20 mM, also had no measurable effect on cell volume as determined by 3H2O and [14C]inulin distribution. These results indicate that Na+-coupled uptake of L-alanine by skate hepatocytes is rheogenic, as previously observed in other cell systems. However, in contrast to mammalian hepatocytes, Vm does not repolarize for at least 10 min after the administration of L-alanine, and changes in cell volume and potassium permeability are also not observed.

Quinine decreases hepatocyte transmembrane potential and inhibits amino acid transport.

Effects of L-alanine, 2-(methylamino)isobutyric acid (MeAIB), and quinine on mouse hepatocyte transmembrane potential (Vm) are compared with effects of quinine on MeAIB transport into isolated mouse hepatocytes in primary monolayer culture. In liver slices, L-alanine (10 mM) decreased Vm 6 +/- 0.4 mV from control Vm (-37 +/- 0.2 mV). With L-alanine still present, Vm repolarized and stabilized at Vm of -2 +/- 0.5 mV greater than control Vm. Quinine (1 mM) decreased Vm reversibly by 7 +/- 0.9 mV. Depolarization was 11 +/- 1.5 mV when L-alanine and quinine were added together, but now Vm did not repolarize. Transient depolarization also resulted from addition of either L-alanine or MeAIB to isolated hepatocytes in primary culture. Moreover, quinine (1 mM) inhibited steady-state MeAIB uptake by 91%. Quinine decreased Vmax for MeAIB transport from 9.0 +/- 1.0 to 4.8 +/- 1.9 nmol MeAIB.mg protein-1.4 min-1, but it did not change Km of 0.60 mM. Quinine inhibition of MeAIB transport was reversible. Quinine also increased hepatocyte steady-state volume from 3.2 +/- 0.8 to 4.9 +/- 1.2 microliter/mg protein. Thus quinine may inhibit Na+-amino acid cotransport by blocking conductive K+ channels, thereby decreasing Vm and the transmembrane electrochemical Na+ gradient, and it may deplete the intracellular amino acid pool by disrupting hepatocyte volume regulation.

Effects of anisosmotic medium on cell volume, transmembrane potential and intracellular K+ activity in mouse hepatocytes.

Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37 degrees C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm) ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4 degrees C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (Vm) and intracellular K+ activity (aik) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration. Vm increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and in [10(experimental Vm/control Vm)] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8-1.5. The aik did not change when medium osmolality was decreased 40-70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60 mM K+ or 1 mM quinine HCl or at 27 degrees C resulted in no change in Vm compared with a 20-mV increase in Vm without the added agents or at 37 degrees C. We conclude that mouse hepatocytes maintain their volume and aik in response to anisosmotic medium; however, Vm behaves as an osmometer under these conditions. Also, increases in Vm by hypoosmotic stress were abolished by conditions or agents that inhibit K+ conductance.