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.

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.

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.

Serum somatomedin stimulation in thyroxine-treated hypophysectomized rats.

Somatomedin activity in rat serum was measured by sulfate incorporation into embryonic chick cartilage. Hypophysectomized male rats were treated daily for 21 days with 20 microgram/kg L-thyroxine (T4) and/or 25 microgram bovine (b) GH. Serum from rats given T4 alone or with bGH had somatomedin potencies of 0.88 and 0.83, respectively, comparable to normal serum (1.00) and significantly greater than serum from bGH-treated hypophysectomized rats (0.62). Hypophysectomized rats had no measurable somatomedin activity. T4 treatment resulted in a return to normal of the basal metabolic rate and serum T4 and T3 concentrations. Addition of T4 or T3 to serum from normal rats to produce concentrations comparable to those found in serum of hypophysectomized rats after T4 and/or bGH treatment did not alter the somatomedin activity. Higher concentrations of T4 (10(-7) M) or T3 (10(-8) or 10(-7) M) did, however, enhance the sulfation potency of normal serum. T4 (10(-9) M to 10(-6) M) or T3 (10(-10) M to 10(-7) M) added to serum-free medium provided minimal sulfation activity that represented only a fraction of the activity of serum. T4 may stimulate production of somatomedin in the absence of adequate bGH or may be metabolized to an active sulfation factor.

Transmembrane potential of rat hepatocytes in primary monolayer culture.

Transmembrane potentials of rat hepatocytes in primary monolayer culture on collagen gels were measured with glass microelectrodes. Potentials for cells in culture for 23-30 h comprised two populations. The mean +/- SD for a population of stable low potentials was -9.7 +/- 2.0 mV (n = 93). This was compared with -23.6 +/- 9.4 mV (n = 42), the mean value for stable potentials that followed spontaneous increases in the low potentials, 0.5-2.0 min after the impalement. The estimated input resistance increased during these spontaneous hyperpolarizations. In some cells, after 48 h in culture, the transmembrane potential oscillated rhythmically, with an amplitude of 25 mV and a period of 7 min. Suffusing the cells with 120 mM potassium chloride decreased the potential and eliminated the oscillations. The stable high potentials were considered more accurate estimates of the hepatocyte transmembrane potential, based on comparison with values for intact liver. Low potentials may have resulted from current leaking through an electrode shunt resistance, followed by an increase in potential as the membrane "sealed" the shunt pathway. However, these events may also reflect cells capable of two stable transmembrane potentials.

Insulin depolarization of rat hepatocytes in primary monolayer culture.

Rat hepatocytes in primary monolayer culture demonstrated two stable states of transmembrane potential (Em). Low potentials ranging from -10 to -15 mV followed impalements with glass microelectrodes, and in some cells low potentials increased spontaneously or in response to a train of intermittent current (5 nA) to stable potentials of -50 mV, which were comparable to hepatocyte Em in vivo. Adding insulin at 20 mU/ml depolarized the stable higher Em 22.4 +/- 2.6 mV (n = 6) over an 11-min interval, and input resistance increased 21.4 +/- 4.7 M omega (n = 6) during the depolarization. The insulin effect was dose dependent, because adding insulin at 0.2 mU/ml depolarized the stable high Em 5.0 +/- 1.5 mV (n = 3) and increased input resistance 6.3 +/- 1.8 M omega (n = 3). In one experiment the Em repolarized 32 min after insulin was washed out. Adding metabolic inhibitors KCN (1 mM) and 2,4-dinitrophenol (10 and 1 mM) and increasing external potassium (60 mM, with external sodium reduced equivalently) also depolarized Em, but they did not increase input resistance. Thus insulin depolarized hepatocytes from a stable high Em, which is equivalent to the Em of liver in vivo, to a stable low Em, which occurs in hepatocytes in primary monolayer culture. This hormone action is consistent with changes in membrane ion conductance, and it further demonstrates that these cells can sustain two stable states of Em.

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)

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)

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)

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)

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)

Transmembrane potential and amino acid transport in rat hepatocytes in primary monolayer culture.

Transmembrane potential (Em) and alpha-aminoisobutyric acid (AIB) transport were measured in primary monolayer cultures of rat hepatocytes obtained from unoperated control rats and from rats 12 hr following partial hepatectomy. Measurements were performed 20-24 hr after plating the cells. The capacity of both kinds of cells to concentrate AIB depended upon extracellular sodium: however, the steady-state accumulation in regenerating cells was twice that of control cells. Transmembrane potentials, recorded with glass microelectrodes, were -13 +/- 0.6 mV and -27 +/- 1.6 mV in control and regenerating cells, respectively. Ouabain (1 mM) depolarized regenerating cell to -18 +/- 1.0 mV, but it had no effect on control cells. The initial rates of 1 mM AIB transport into control and regenerating cells were 1.2 +/- 0.1 and 3.1 +/- 0.1 nanomoles/mg protein x 4 min, respectively. Ouabain (1 mM) reduced the initial rate of AIB transport into regenerating cells to 2.7 +/- 0.1 nanomoles/mg protein x 4 min, but it had no effect on AIB transport into control cells. Glucagon (10(-7) M) added to control cells 12 hr before measurements hyperpolarized Em to -31 +/- 1.3 mV and increased AIB transport rate to 3.1 nanomoles/mg protein x 4 min. The results suggest a relationship between increases in Em and increases in AIB transport in rat hepatocytes. An electrogenic Na-K pump may be involved in both of these events.

Membrane potential measurements during rat liver regeneration.

Membrane potential was measured in perfused rat liver and was shown to increase from -33 +/- 1.0 mV in livers from normal rats to -50 +/- 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na-K pump.

Effect of temperature on transmembrane potential of mouse liver cells.

Mouse liver transmembrane potential (Vm), measured under steady-state conditions with conventional microelectrodes, was -40 +/- 0.6 mV, and intracellular Na+ and K+ activities, measured with liquid ion-exchanger ion-sensitive microelectrodes, were 17 +/- 2 and 104 +/- 4 mM, respectively. The corresponding K+ and Na+ equilibrium potentials (EK and ENa) were -88 and 48 mV. Vm also varied as a linear function of temperature. In the range of 37-27 degrees C, the temperature coefficient (Q10) of 1.61 was greater than the Q10 of 1.033 predicted for a direct proportion of absolute temperature. A decrease in hepatocyte EK accounted for only a small portion of the total decrease in Vm resulting due to cooling from 37 to 25 degrees C. In contrast, slopes of the linear portion of Vm versus log10 external K+ activity were -24 and -14 mV/10-fold change in external K+ activity at 37 and 25 degrees C, respectively. This is consistent with an increase of membrane Na+- to -K+ permeability ratio (PNa/PK) with cooling. Ba2+ and quinine, which block membrane K+ channels, reversibly inhibited increases in hepatocyte Vm resulting due to heating from 37 to 40 degrees C. This suggests that membrane PK varies directly with temperature. We postulate that effects of temperature on liver Vm result from temperature effects on membrane K+ channel conductance and on the Na+-K+ pump. The results also are consistent with temperature effects on kinetic parameters for opening and closing of membrane K+ channels.

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.

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.

Transmembrane potential and intracellular potassium ion activity in fetal and maternal liver.

We have compared transmembrane potentials (Em) of maternal liver with Em of fetal liver, and as an initial step to account for differences in Em, we have measured intracellular potassium ion activities (aiK) in both tissues. Paired segments of maternal and fetal (day 17) mouse liver were suffused (15 ml/min) with Krebs' physiologic salt solution equilibrated with 95% 02-5% CO2 (pH 7.3-7.4) at 37 degrees C. To measure Em, cells were impaled with open-tip microelectrodes filled with 0.5 M KCl. Intracellular voltage recordings that were stable +/- 2 mV for at least 10 s were considered valid impalements. Maternal liver mean Em = -41 +/- 1 (SEM) mV, n = V 10 animals. In contrast, fetal liver mean Em = -23 +/- 1 (SEM) mV, n = 10 animals. In the same segments we measured aiK with potassium-selective liquid ion-exchanger microelectrodes. Maternal liver mean aik = 95 +/- 7 (SEM) mM and fetal liver mean aiK = 62 +/- 4 (SEM) mM. in addition, Em and aiK of fetal liver increased to values comparable to those of maternal liver during the first 8 days of neonatal life. The differences of Em and aik between fetal and maternal liver, and the changes in these values that occur in the neonate, may result from activity of a membrane Na-K exchange pump that increases with tissue development.