Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

Multitrack system for superfusing isolated cardiac myocytes

A new system for studying mechanical activity of freshly isolated cardiac myocytes from up to four experimental groups simultaneously is described. Suspensions of cardiac myocytes isolated from adult rat hearts were drawn into microhematocrit capillary tubes, which were then mounted in parallel fashion between two four-channel tubing manifolds placed on the movable stage of an inverted microscope. Within a few minutes, cells settled and attached to the bottom of the tubes and then could be superfused with various test solutions. The system allowed for electrical field stimulation, rapid changes in bathing solutions, control of temperature, and simulation of ischemia and reperfusion with measurements of the effects of such interventions on both populations of cells (low power survey) and individual myocytes (high power). Myocyte responses to these various interventions are described. The primary advantage of this system is the ability to conduct experiments on cardiac myocytes isolated concurrently from multiple experimental groups at the same time and under identical conditions.     

Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches.

Strophanthidin inhibits KATP channels in 2,4-dinitrophenol-poisoned heart cells (). The current study shows that the Na/K pump interacts with KATP current (IK-ATP) via submembrane ATP depletion in isolated giant membrane patches and in nonpoisoned guinea pig cardiac cells in whole-cell configuration. IK-ATP was inhibited by ATP, glibenclamide, or intracellular Cs+. Na/K pump inactivation by substitution of cytoplasmic Na+ for Li+ or N-methylglucamine decreased both IK-ATP by 1/3 (1 mM ATP, zero calcium), and IC50 of ATP for IK-ATP (0.3 +/- 0.1 mM) by 2/5. The Na+/Li+ replacement had no effect on IK-ATP at low pump activity ([ATP] </= 0.1 mM or 100 microM ouabain) or when IK-ATP was completely inhibited by 10 mM ATP. In whole-cell configuration, ouabain inhibited up to 60% of inwardly rectifying IK-ATP at 1 mM ATP in the pipette but not at 10 mM ATP and 10 mM phosphocreatine when IK-ATP was always blocked. However, mathematical simulation of giant-patch experiments revealed that only 20% of ATP depletion may be attributed to the ATP concentration gradient in the bulk solution, and the remaining 80% probably occurs in the submembrane space.     

Oxygen pressure gradients in isolated cardiac myocytes

Intracellular oxygen pressure within intact isolated cardiac myocytes is studied as a function of steady state extracellular oxygen pressure. The fractional saturation of myoglobin with oxygen is used to report sarcoplasmic oxygen pressure. The fractional oxidation of cytochrome oxidase, the fractional oxidation of cytochrome c, the rate of respiratory oxygen uptake, and lactate accumulation are used to reflect the availability of oxygen at the inner mitochondrial membrane. These probes of mitochondrial function show no large change with decreasing extracellular oxygen pressure until that pressure is less than 2 torr and intracellular myoglobin is largely deoxygenated. Sarcoplasmic oxygen pressure in resting cells is nearly the same as extracellular oxygen pressure and is about 2 torr less in cells whose respiration has been increased 3.5-fold by mitochondrial uncoupling. Oxygen pressure at the mitochondrial inner membrane differs from sarcoplasmic oxygen pressure by no more than 0.2 torr and from extracellular oxygen pressure by no more than 2 torr. We conclude that differences of oxygen pressure within the cardiac myocyte are very small. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. We conclude also that mitochondria of the cardiac myocyte become oxygen limited only when sarcoplasmic myoglobin is almost entirely deoxygenated.     

Background K+ current in isolated canine cardiac Purkinje myocytes.

The current-voltage (I-V) relation of the background current, IK1, was studied in isolated canine cardiac Purkinje myocytes using the whole-cell, patch-clamp technique. Since Ba2+ and Cs+ block IK1, these cations were used to separate the I-V relation of IK1 from that of the whole cell. The I-V relation of IK1 was measured as the difference between the I-V relations of the cell in normal Tyrode (control solution) and in the presence of either Ba2+ (1 mM) or Cs+ (10 mM). Our results indicate that IK1 is an inwardly rectifying K+ current whose conductance depends on extracellular potassium concentration. In different [K+]0's the I-V relations of IK1 exhibit crossover. In addition the I-V relation of IK1 contains a region of negative slope (even when that of the whole cell does not). We also examined the relationship between the resting potential of the myocyte, Vm, and [K+]0 and found that it exhibits the characteristic anomalous behavior first reported in Purkinje strands (Weidmann, S., 1956, Elektrophysiologie der Herzmuskelfaser, Med. Verlag H. Huber), where lowering [K+]0 below 4 mM results in a depolarization.     

A microscopic model for the current-voltage behaviour of the Na,K-pump

The current voltage characteristic of the Na, K pump is described on the basis of a modified Post-Albers cycle. The voltage dependence of the rate constants is derived from the elementary chargetranslocations associated with the single reaction steps. Charge displacements result from movements of the sodium- or potassium-loaded binding sites, as well as from motions of polar groups in the pump molecule. If part of the transmembrane voltage drops between the alkali-ion binding sites and the aqueous solution, the binding constants become voltage-dependent. Depending on the values of the microscopic parameters, the current-voltage characteristic may assume a variety of different shapes. Saturating behaviour results when one or more voltage-independent reaction steps become rate limiting. Non-monotonic current-voltage curves exhibiting regions of negative pump conductance are predicted when, at least in one of the transitions, charge is moved against the direction of overall charge-translocation. The theoretical predictions are compared with recent experimental studies of voltage-dependent pump currents.     

G-protein-mediated regulation of the insulin-responsive glucose transporter in isolated cardiac myocytes.

Isolated muscle cells from adult rat heart were used to study the involvement of G-proteins in the regulation of the glucose transporter by insulin and isoprenaline. Efficient modification of G-protein functions was established by measuring isoprenaline-stimulated cyclic AMP production, viability and ATP content after treating the cells with cholera toxin and pertussis toxin for 2 h. Under these conditions cholera toxin decreased the stimulatory action of insulin on 3-O-methylglucose transport by 56%, but pertussis toxin had no effect. Basal transport was not affected by toxin treatment. Isoprenaline increased 3-O-methylglucose transport by 63%. This effect was not mimicked by dibutyryl cyclic AMP, but was completely blocked by cholera toxin. Streptozotocin-diabetes abolished isoprenaline action and decreased stimulation of transport by 64%. Concomitantly, cholera-toxin sensitivity of glucose transport was lost in cells from diabetic animals. This was paralleled by a large decrease (87 +/- 4%) in mRNA expression of the insulin-regulatable glucose transporter, as shown by Northern-blot analysis of RNA isolated from cardiomyocytes of diabetic rats. These data suggest a functional association between the insulin-responsive glucose transporter and a cholera-toxin-sensitive G-protein mediating stimulation by insulin and isoprenaline.     

Characterization of catecholamine uptake2 in isolated cardiac myocytes

Extraneuronal catecholamine uptake was investigated in isolated quiescent rat myocardial cells. By administration of (³H-)(–)noradrenaline concentration of 22 nmol/l up to 1000 mol/l the following data were obtained: (1) The K_M of the uptake process amounted to 260 mol/l, the V_max to 4.24 nmol/(10 min × mg Protein) corresponding to 179 nmol/(min × g_WWt)(WWT = Wet Weight). (2) The uptake was largely inhibited by the uptake₂-inhibitors corticosterone (100 mol/l), isoprenaline (IC so = 30.6 mol/l), and O-methylisoprenaline (IC₅₀ = 2.1 pmol/l), but not by the uptake₁-inhibitors cocaine (100 mol/l) and desipramine (10 mol/l). (3) The affinity-values K_M and IC₅₀ closely agreed with those already known, but the V_max-value was higher than those obtained in whole rat hearts by a factor of at least 1.79. This is caused presumably by the voltage dependence of the uptake mechanism and the resulting inhibition of uptake 2 during the periods of depolarisation in beating hearts of other studies.     

Superoxide-dependent and superoxide-independent pathways for reduction of nitroblue tetrazolium in isolated rat cardiac myocytes.

Spectroscopic studies indicated that nitroblue tetrazolium (NBT) could be reduced to blue formazan by several distinct reactions in suspensions of isolated rat cardiac myocytes. Both NADPH- and NADH-linked pathways for reduction of NBT were observed. NADPH-linked NBT reduction showed little activity in the absence of digitonin, but could be stimulated an average of 9.5-fold by digitonin permeabilization of the plasma membrane. NADH-linked NBT reduction occurred in the absence of digitonin, and could be increased an average of 3.5-fold by digitonin treatment. Analysis of the effects of cell viability on the extent of digitonin stimulation with these substrates suggested that the NADPH-linked reaction involved a cytosolic component, while the NADH-linked reaction involved an intracellular membrane enzyme system. With either NADPH or NADH, NBT reduction was completely inhibited by dicoumarol (100 microM). Dicoumarol-insensitive NBT reduction could subsequently be observed following the addition of 2 mM cyanide, a level of cyanide known to inhibit cytosolic superoxide dismutase. Cyanide-stimulated, dicoumarol-insensitive NBT reduction was augmented by the presence of either antimycin or doxorubicin, two agents which enhance superoxide formation by different mechanisms. The results indicate the existence of multiple pathways for both superoxide-independent and superoxide-dependent reduction of NBT. Dicoumarol-insensitive, cyanide-stimulated NBT reduction may be useful as a spectroscopic probe for intracellular superoxide formation.     

Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.     

Involvement of Na,K-pump in SEPYLRFamide-mediated reduction of cholinosensitivity in Helix neurons

SEPYLRFamide acts as an inhibitory modulator of acetylcholine (ACh) receptors in Helix lucorum neurones. Ouabain, a specific inhibitor of Na,K-pump, (0.1 mM, bath application) decreased the ACh-induced inward current (ACh-current) and increased the leak current. Ouabain decreased the modulatory SEPYLRFamide effect on the ACh-current. There was a correlation between the effects of ouabain on the amplitude of the ACh-current and on the modulatory peptide effect. Ouabain and SEPYLRFamide inhibited the activity of Helix aspersa brain Na,K-ATPase. Activation of Na,K-pump by intracellular injection of 3 M Na acetate or 3 M NaCl reduced the modulatory peptide effect on the ACh-current. An inhibitor of Na/Ca-exchange, benzamil (25 muM, bath application), and an inhibitor of Ca(2+)-pump in the endoplasmic reticulum, thapsigargin (TG, applied intracellularly), both prevented the effect of ouabain on SEPYLRFamide-mediated modulatory effect. Another inhibitor of Ca(2+)-pump in the endoplasmic reticulum, cyclopiazonic acid (applied intracellularly), did not prevent the effect of ouabain on SEPYLRFamide-mediated modulatory effect. These results indicate that Na,K-pump is responsible for the SEPYLRFamide-mediated inhibition of ACh receptors in Helix neurons. Na/Ca-exchange and intracellular Ca(2+) released from internal pools containing TG-sensitive Ca(2+)-pump are involved in the Na,K-pump pathway for the SEPYLRFamide-mediated inhibition of ACh receptors.     

Genetic manipulation of calcium-handling proteins in cardiac myocytes. I. Experimental studies

Two genetic experimental approaches, de novo expression of parvalbumin (Parv) and overexpression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), have been shown to increase relaxation rates in myocardial tissue. However, the relative effect of Parv and SERCA2a on systolic function and on beta-adrenergic responsiveness at varied pacing rates is unknown. We used gene transfer in isolated rat adult cardiac myocytes to gain a fuller understanding of Parv/SERCA2a function. As demonstrated previously, when Parv is expressed in elevated concentration (>0.1 mM), the transduced myocytes showed a reduction in sarcomere-shortening amplitude: 129 +/- 17, 81 +/- 8, and 149 +/- 14 nm for control, Parv, and SERCA2a, respectively. At physiological temperature, shortening amplitude responses of Parv and SERCA2a myocytes to the beta-adrenergic agonist isoproterenol (Iso) were not statistically different from that of control myocytes. However, in SERCA2a myocytes, in which baseline was slightly elevated and the Iso-stimulated value was slightly lower, the increase in shortening was slightly less than in Parv or control myocytes: 108 +/- 14, 169 +/- 39, and 34 +/- 12% for control, Parv, and SERCA2a, respectively. In another test set, Parv myocytes had the strongest early postrest potentiation among all groups studied (rest time = 2-10 s), and SERCA2a myocytes were the least sensitive to variations in stimulation rhythm. To replicate the deficient Ca2+ removal observed in heart failure, we used 150 nM thapsigargin. Under these conditions, control myocytes exhibited slowed relaxation, whereas Parv myocytes retained their rapid kinetics, showing that Parv is still able to control relaxation, even when SERCA2a function is impaired.     

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.     

Two functionally different Na/K pumps in cardiac ventricular myocytes

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half- saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half- maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.     

Involvement of hormone processing in insulin-activated glucose transport by isolated cardiac myocytes.

Isolated muscle cells from adult rat heart were used to study the relationship between myocardial insulin processing and insulin action on 3-O-methylglucose transport at 37 degrees C. Internalization of the hormone as measured by determination of the non-dissociable fraction of cell-bound insulin increased linearly up to 10 min, reaching a plateau by 30-60 min at 3 nM-insulin. At this hormone concentration the onset of insulin action was found to be biphasic, with a rapid phase up to 8 min, followed by a much slower phase, reaching maximal insulin action by 30-60 min. Insulin internalization was totally blocked by phenylarsine oxide, whereas dansylcadaverine had no effect on this process. Initial insulin action (5 min) on glucose transport was not affected by chloroquine and dansylcadaverine, but was completely abolished by treatment of cardiocytes with phenylarsine oxide. This drug effect was partly prevented by the presence of 2,3-dimercaptopropanol. Under steady-state conditions (60 min), the stimulatory action of insulin was decreased by about 60% by both chloroquine and dansylcadaverine. This study, demonstrates that insulin action on cardiac glucose transport is mediated by processing of the hormone. The data suggest dual pathways of insulin action involving initial processing of hormone-receptor complexes and lysosomal degradation.     

Reverse mode of the sarcoplasmic reticulum calcium pump and load-dependent cytosolic calcium decline in voltage-clamped cardiac ventricular myocytes

We have characterized [Ca](i) decline in voltage-clamped rabbit ventricular myocytes with progressive increases in sarcoplasmic reticulum (SR) calcium load. "Backflux" through the SR calcium pump is a critical feature which allows realistically small values for SR calcium leak fluxes to be used. Total cytosolic calcium was calculated from the latter part of [Ca](i) decline using rate constants for cellular calcium buffers. Intra-SR calcium buffering characteristics were also deduced. We found that the net SR calcium pump flux and rate of [Ca](i) decline decreased as the SR free [Ca] rose, with pump parameters held constant. We have therefore characterized for the first time in intact myocytes both forward and reverse SR calcium pump kinetics as well as intra-SR calcium buffering and SR calcium leak. We conclude that the reverse flux through the SR calcium pump is an important factor in comprehensive understanding of dynamic SR calcium fluxes.     

Monoamine oxidase, an intracellular probe of oxygen pressure in isolated cardiac myocytes

The activity of monamine oxidase, an enzyme located almost exclusively at the outer mitochondrial membrane, toward the substrate phenylethylamine is used to report the oxygen pressure at the outer mitochondrial membrane of intact cardiac myocytes isolated from hearts of adult rats. The rate of substrate oxidation, under the conditions used, follows the Michaelis-Menten relation, and accordingly can be used as a measure of the local chemical activity of dissolved oxygen. The oxygen pressure at the outer mitochondrial membrane of myocytes, at rest and after 2- to 3-fold stimulation of respiratory oxygen consumption, differs from the extracellular oxygen pressure by at most 2 torr. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. At physiologically relevant concentrations of the substrates phenylethylamine and norepinephrine, monoamine oxidase activity is relatively insensitive to extracellular oxygen pressure in the range 155 to 8 torr, suggesting a limited role for regulation of biogenic amine oxidation by oxygen availability.     

The uptake and distribution of radiolabelled thyroxine by isolated cardiac myocytes.

The uptake and distribution of ¹⁴C and ¹²⁵I-labelled thyroxine was studied in ventricular myocytes, isolated from the hearts of male Sprague-Dawley rats. Equilibrium was established between the radioactivity of the incubation medium and the cells within 15 minutes. At equilibrium the concentration of ¹⁴C-thyroxine in the cells was approximately 50 times the concentration in the incubation medium. Fractionation of the cells revealed that the equilibrium had been attained for all fractions except the nuclear. The radioactivity of the nuclear fraction showed an increase for at least 60 minutes of incubation. At equilibrium the distribution of radioactivity was: Soluble fraction 51.3%, Mitochondria 33.6%, Microsomal 7.0% and Nuclear 7.0%. When the values for these fractions were corrected for mitochondrial contamination the specific activity (CPM/MG protein) of the mitochondrial fraction was by far the highest, exceeding the next highest fraction (the supernatant) by an order of magnitude. The presence of equimolar amounts of triiodothyronine produced little change in the pattern of uptake of the label by any of the cell fractions. The uptake of labelled thyroxine was profoundly affected by the presence of calcium in the media. The uptake of ¹⁴C-thyroxine by cells incubated in media containing 1.25mM calcium was less after 60 minutes than in cells incubated in calcium free buffer. Fractionation of the cells revealed that the amount of label bound to the mitochondria of cells in calcium containing medium was significantly increased while the radioactivity bound to the other cellular fractions was decreased. The data indicate that the cell fraction with the highest specific activity was the mitochondria. The relation of these findings to some of the current theories of thyroid hormone action is discussed.