Spontaneous recovery of the anoxic diminution in the action potential plateau of the late embryonic and neonatal chicken hearts

The action potential plateau of the embryonic chick hearts at the latest stage of development (19- to 20-day-old) was depressed initially by anoxia but recovered spontaneously under the sustained anoxic condition. A similar spontaneous recovery of the action potential plateau was observed in hearts from neonatal chicks. Propranolol (3 × 10^?6M) did not affect the spontaneous recovery of the action potential plateau, excluding the possible involvement of the release of endogenous catecholamines. Since tissue adenosine triphosphate content continued to decrease during anoxia in these hearts, it is unlikely that the anoxia-resistant metabolic pathway appeared or was enhanced to supply the energy for the rebuilding of the plateau. In a Ca²⁺-free solution the action potential plateau rapidly disappeared and did not recover spontaneously during anoxia; at this time, however, the addition of Ca²⁺ (2 mM) prolonged the plateau. An inward calcium current may play an important role in spontaneous recovery of the action potential plateau during anoxia. The duration of the action potential decreased after hatching. From these results it is suggested that the mechanism underlying the action potential plateau may somehow differ in the hearts at the stages around the time of hatching.     

The ionic basis of electrical activity in embryonic cardiac muscle

The intracellular sodium concentration reported for young, embryonic chick hearts is extremely high and decreases progressively throughout the embryonic period, reaching a value of 43 mM immediately before hatching. This observation suggested that the ionic basis for excitation in embryonic chick heart may differ from that responsible for electrical activity of the adult organ. This hypothesis was tested by recording transmembrane resting and action potentials on hearts isolated from 6-day and 19-day chick embryos and varying the extracellular sodium and potassium concentrations. The results show that for both young and old embryonic cardiac cells the resting potential depends primarily on the extracellular potassium concentration and the amplitude and rate of rise of the action potential depend primarily on the extracellular sodium concentration.     

Influence of Glucose on the Transmembrane Action Potential of Anoxic Papillary Muscle

The response of the cat papillary muscle to anoxia has been found to alter depending on the glucose concentration in the medium. At a glucose concentration of 5 mM anoxia caused a marked reduction in force of contraction and action potential duration within 20 minutes. At a glucose concentration of 50 mM anoxia induced similar changes in the force of contraction but little or no change in action potential duration. Elevation of glucose concentration during an anoxic interval reversed the anoxia-induced changes in action potential but had little effect on force of contraction. This effect of glucose could be partially duplicated by xylose and 2-deoxyglucose and in addition, 2-deoxyglucose has been found to prevent the effect of subsequently added glucose. These sugars appear to be transported by a system responsible for glucose transport but are not metabolized to any extent. It would appear therefore that transport of glucose is in some way related to transport of potassium as increased potassium permeability is thought by many to be responsible for anoxia-induced changes in action potential duration.     

Changes in membrane properties of chick embryonic hearts during development

The electrophysiological properties of embryonic chick hearts (ventricles) change during development; the largest changes occur between days 2 and 8. Resting potential (E_m) and peak overshoot potential (+E _max) increase, respectively, from -35 mv and +11 mv at day 2 to -70 mv and +28 mv at days 12–21. Action potential duration does not change significantly. Maximum rate of rise of the action potential (+V _max) increases from about 20 v/sec at days 2–3 to 150 v/sec at days 18–21; + V _max of young cells is not greatly increased by applied hyperpolarizing current pulses. In resting E_m vs. log [K⁺]_o curves, the slope at high K⁺ is lower in young hearts (e.g. 30 mv/decade) than the 50–60 mv/decade obtained in old hearts, but the extrapolated [K⁺]_i values (125–140 mM) are almost as high. Input resistance is much higher in young hearts (13 MΩ at day 2 vs. 4.5 MΩ at days 8–21), suggesting that the membrane resistivity (R_m) is higher. The ratio of permeabilities, P _Na/P _K, is high (about 0.2) in young hearts, due to a low P _K, and decreases during ontogeny (to about 0.05). The low K⁺ conductance (g _K) in young hearts accounts for the greater incidence of hyperpolarizing afterpotentials and pacemaker potentials, the lower sensitivity (with respect to loss of excitability) to elevation of [K⁺]_o, and the higher chronaxie. Acetylcholine does not increase g _K of young or old ventricular cells. The increase in (Na⁺, K⁺)-adenosine triphosphatase (ATPase) activity during development tends to compensate for the increase in g _K. +E _max and + V _max are dependent on [Na⁺]_o in both young and old hearts. However, the Na⁺ channels in young hearts (2–4 days) are slow, tetrodotoxin (TTX)-insensitive, and activated-inactivated at lower E_m. In contrast, the Na⁺ channels of cells in older hearts (> 8 days) are fast and TTX-sensitive, but they revert back to slow channels when placed in culture.     

Development of slow Ca2+-Na+ channels during organ culture of young embryonic chick hearts

Young (3-days-old) embryonic chick hearts have slowly-rising spontaneous action potentials, dependent on tetrodotoxin-insensitive slow Na+ channels. When the hearts were placed into organ culture for 5-11 days, action potential duration was markedly increased by 260-370%, and a notch appeared between the initial spike phase and the plateau phase in some hearts. The spike amplitude was mainly dependent on [Na]0, whereas the plateau amplitude was dependent on [Ca]0. Thus, the young embryonic hearts develop slow Ca2+-Na+ channels (while retaining the slow Na+ channels) during organ culture, and the spike phase and the plateau phase of the slow action potentials are mainly dependent on currents through slow Na+ channels and through slow Ca2+-Na+ channels, respectively. The effects of Mn2+ (a specific blocker of slow Ca2+-Na+ channels) and verapamil (a blocker of slow Na+ channels as well as of slow Ca2+-Na+ channels) on the spike phase and the plateau phase were examined. Mn2+ (0.5 mM) and verapamil (5 microM) depressed the plateau duration and overshoot. Verapamil did not decrease the maximum rate of rise (Vmax), but Mn++ produced a small, but significant, decrease. High concentrations (10/30 microM) of verapamil depressed the action potential amplitude and Vmax, and abolished the spontaneous action potentials. These results indicate that slow Ca2+-Na+ channels appear de novo during organ culture of young embryonic hearts.     

Calcium-dependent slow potassium conductance in rat skeletal myotubes

A prolonged hyperpolarizing afterpotential (amplitude 5–20 mV, half decay time about 400 msec at 25°C) follows the action potential in myotubes and myosacs cultured from rat skeletal muscle. This slow hyperpolarizing afterpotential (hap) is mediated by an increase in membrane K conductance, because its reversal potential follows the Nernst potential for K and is not affected by other ions. The conductance increase measured during the hap (up to four times the resting input conductance) correctly predicts the time course of the slow hap. The slow hap is Ca dependent. Its amplitude decreases when bath [Ca] is lowered, and both amplitude and duration increase when bath [Ca] is raised. The slow hap is blocked by intracellular injection of the calcium chelator, EGTA. It is inhibited by solutions containing 2–4 mM manganese or 1–5 mM barium, but is not blocked by 5–20 mM tetraethylammonium. Myotubes bathed in zero [Na], high [Ca] solutions show calcium action potentials, which are inhibited by 2–10 mM manganese, nickel or cobalt. Myotubes bathed in isotonic Ca salts (or in 2 mM Ca plus 5 mM caffeine) show long-lasting (up to 10 sec) spontaneous hyperpolarizations accompanied by prolonged contractions. These hyperpolarizations are associated with a large increase in input conductance, and they reverse in sign near the K equilibrium potential. They appear to reflect activation of the Ca-sensitive K conductance by Ca released from intracellular stores. The observation that spontaneous hyperpolarizations usually occur with no prior depolarization argues that at least a portion of the slow, Ca-sensitive K conductance system can be activated by internal Ca alone, with no requirement for plasma membrane depolarization. Cultured myotubes also have a faster K conductance system, which is inhibited by 5–20 mM tetraethylammonium or 1–5 mM barium, and is not dependent on Ca for its activation.     

The calcium channel agonist, Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues

Effects of a novel slow channel activator, Bay K-8644 (Bay K), were studied on slow action potential (APs) in young and old embryonic chick hearts, and on its antagonism of the effects of diacetyl monoxime (DAM). The slow APs of young hearts are mediated by slow Na+ channels, whereas those of old hearts are mediated by slow Ca2+ channels. In slow APs of old (13-18 days old) embryonic chick hearts superfused with a high (22 mM) K+ solution, Bay K (10-6 M) gradually increased the amplitude, maximum rate of rise (Vmax), and duration of the slow APs. The actions of Bay K persisted for a long time (greater than 30 min) after washout of the drug. DAM (10 mM) depressed the Vmax, duration and amplitude of the slow APs. Some of the changes in slow AP parameters produced by DAM, e.g., Vmax decrease, were antagonized by the addition of Bay K (10(-6) M). In 3-day-old embryonic chick hearts. Bay K potentiated the slow APs and DAM depressed them; Bay K antagonized these effects of DAM. Thus, the actions of Bay K and DAM are likely to be produced, respectively, via the activation and depression of slow Ca2+ channels in old embryonic chick hearts. In addition, the drugs seem to influence slow Na+ channels found in young embryonic chick hearts.     

A prolonged,voltage-dependent calcium permeability revealed by tetraethylammonium in the soma and axon of Aplysia giant neuron

The soma but not the axon of the giant neuron, R2, of Aplysia can generate an all-or-none Ca spike in Na-free or TTX-containing medium (Junge and Miller, 1974). Extracellular axonal recordings made at several distances from the soma provide evidence that the transition in ability to fire a spike in Na-free medium occurs within the first 250 μm of the axon. Application of 25 mM TEA-Br to the bathing medium causes a more than tenfold increase in the duration of the somatic action potential. The duration of the axonal action potential in TEA decreases with distance from the soma. At distances greater than 3 mm from the soma this concentration of TEA causes little or no increase in the duration of the axon spike. The effect of 25 mM TEA on both the soma and proximal axon is blocked reversibly by 30 mM CoCl₂ or 1 mM CdCl₂. The duration of the somatic action potential in TEA increases with an increase in Ca concentration of the bath. At a constant concentration of Na, the voltage level of the somatic plateau increases with Ca concentration in the manner predicted for a Ca electrode. In the presence of 11 mM Ca²⁺ the potential of the plateau is relatively insensitive to Na concentration. The TEA plateau in R2 reveals a prolonged voltage-dependent permeability to Ca. The duration of the plateau may indicate the degree of Ca activation during a spike.     

Electrical activity of cultured cells of the guinea pig heart. Comparison with the rat]

For the first time, action potentials were recorded with the use of microelectrodes from myocardial cells beating spontaneously which were obtained by trypsination of guinea-pig embryonic hearts. 4-5 weeks old embryos have been found most suitable for such experiments. The development of cultures and the recordings obtained were compared to those observed in rat hearts under the same experimental conditions. These results showed that inspite of the known physiological differences between adult rat hearts and adult guinea-pig hearts, the cardiomyoblasts cultured under the same conditions possess common features relating to the repolarization phase of action potential, specially a lack of the plateau.     

Slow inward and outward currents of rat ventricular fibers under anoxia

Voltage and current clamp experiments were performed on rat ventricular strips under anoxia. 1. Under the influence of anoxia the membrane depolarized by 5 to 10 mV and the action potential amplitude decreased by 15 mV. The plateau disappeared and the duration of the action potential was shortened. 2. The slow inward current was reduced by 50 to 80% and its reversal potential became more negative by about 31 mV. The conductance of the slow inward channel decreased by 26%. 3. The net outward current was slightly depressed.     

Glucose uptake by chicken embryo hearts at various stages of development

d-glucose, but not l-glucose, was found to readily enter the cells of 5- to 6-day chick embryo heart. This suggests the operation of a specific transport system for glucose. The rate of glucose uptake was found to decrease as development proceeds from 5 to 15 days of development, but no further decrease was found between 15 and 20 days. Uptake of glucose is a saturable process, from 5–6 days of embryonic life on. The large decrease in glucose uptake between 5 and 10 days of development is found to be associated with a fourfold increase in the apparent K_m of the uptake process. From 10 days of development onward, the apparent K_m remains about 40 mM. The rate of 2-deoxyglucose uptake also decreased from 5 to 15 days of embryonic life with no further decrease from 15 to 20 days. Glucose competitively inhibits the uptake of 2-deoxyglucose with a K_i close to the K_m for glucose uptake. The uptake of 2-deoxyglucose is stimulated by physiological levels of insulin as early as 5–6 days, although the extent to which insulin enhances uptake is not quite as great as at 15 days of development.     

Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics

Cultured cerebellar granule cells were co-loaded with Ca²⁺-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (????_m). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca²⁺]ⁱ. This sMD was insensitive to a removal of external Ca²⁺ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca²⁺ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6?C8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]_c) and ????_m changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]_c and ????_m. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ????_m despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca²⁺ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.     

An atomic absorption method for cation measurements in Kjeldahl digests of biological materials

Sulfate ion produced little or no interference in absorption by sodium, potassium, and magnesium, but produced a large depression in calcium absorbance in the atomic absorption spectrophotometric measurement of these cations in an acetylene-air flame. Nearly maximal depression of calcium absorbance by 2 mM sulfate was followed by a plateau region of only slight depression from 2 mM to 1 M sulfate concentration. Presence of 25 mM lanthanum in the samples resulted in no depression of calcium absorbance up to 2 mM sulfate, a sharp decrease to about 30 mM sulfate and a plateau from 30 mM up to 1 M sulfate. From these observations, it was determined that the addition of H₂SO₄ to provide approximately 40 mM added sulfate in standards and samples permitted accurate measurement of calcium even though the original sample contained relatively high and variable sulfate.     

Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600

During Days 4 to 7 in ovo, beating of embryonic chick hearts becomes progressively more sensitive to inhibition by tetrodotoxin, an inhibitor of fast Na⁺ channels, and progressively less sensitive to inhibition by D600, an inhibitor of slow Ca2+/Na+ channels. The developmental change in tetrodotoxin sensitivity is not retained in heart cells cultured in monolayer. In contrast, the developmental change in D600 sensitivity is retained. Veratridine-stimulated ²²Na⁺ influx mediated by fast Na⁺ channels is inhibited by tetrodotoxin (K_i = 1.6 nM) in cells prepared from either 3-day or 12-day embryos. These results suggest that young embryonic hearts contain physiologically inactive Na⁺ channels. ²²Na⁺ influx mediated by slow Ca2+/Na+ channels is inhibited by D600 with a K_i of 40 nM for cells from 3-day hearts and 8 μM for cells from 12-day hearts. Beating of heart cells in aggregate cultures is also inhibited by D600. Aggregates which have reactivated after inhibition by tetrodotoxin are 10-fold more sensitive to inhibition by D600 than untreated controls. The results suggest that the primary developmental event is a change in slow Ca2+/Na+ channels which reduces their sensitivity to D600 and diminishes their ability to support beating without the activity of the fast Na⁺ channel.     

Control of action potential duration by calcium ions in cardiac Purkinje fibers

It is well known that cardiac action potentials are shortened by increasing the external calcium concentration (Cao). The shortening is puzzling since Ca ions are thought to carry inward current during the plateau. We therefore studied the effects of Cao on action potentials and membrane currents in short Purkinje fiber preparations. Two factors favor the earlier repolarization. First, calcium-rich solutions generally raise the plateau voltage; in turn, the higher plateau level accelerates time- and voltage-dependent current changes which trigger repolarization. Increases in plateau height imposed by depolarizing current consistently produced shortening of the action potential. The second factor in the action of Ca ions involves iK1, the background K current (inward rectifier). Raising Cao enhances iK1 and thus favors faster repolarization. The Ca-sensitive current change was identified as an increase in iK1 by virtue of its dependence on membrane potential and Ko. A possible third factor was considered and ruled out: unlike epinephrine, calcium-rich solutions do not enhance slow outward plateau current, ikappa. These results are surprising in showing that calcium ions and epinephrine act quite differently on repolarizing currents, even though they share similar effects on the height and duration of the action potential.     

Muscarinic activation causes biphasic inotropic response and decreases cellular Na+ activity in canine cardiac purkinje fibers

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na⁺ activity (a _Na ⁱ ), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to a _Na ⁱ . In fibers driven at 1 Hz, CCh (10 µM) initially and transiently decreased and then increased the twitch tension by 36±8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD₉₀), from 403±7 to 389±7 ms. The a _Na ⁱ decreased from 7.4±0.4 to 6.7±0.3 mM (n=23, p<0.05). Atropine (1 µM) decreased the twitch tension by 21±6% (n=7, p<0.05) without significant effects on the action potential and a _Na ⁱ , and inhibited the effects of CCh. Cs⁺ (20 mM) increased the plateau height and APD₉₀, enhanced the twitch tension by 66±24%, but decreased a _Na ⁱ from 7.3±0.3 to 6.3±0.4 mM (n=6, p<0.05). In the presence of 20 mM Cs⁺, some fibers generated slow responses. The addition of 10 µM CCh further increased the twitch tension and APD₉₀, and decreased a _Na ⁱ from 6.3±0.4 to 5.3±0.3 mM. Ouabain (0.3 µM) increased the twitch tension and a _Na ⁱ , and inhibited the CCh-induced decrease of a _Na ⁱ . In the presence of ouabain, 20 mM Cs⁺ depolarized the fiber and generated slow responses with a decreased a _Na ⁱ . The addition of 10 µM CCh enhanced the slow action potential, and increased a _Na ⁱ although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na⁺-K⁺ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M₂ receptors, is associated with the activation of K⁺ channels. The stimulatory effect, on the other hand, is probably due to the action on the M₁ receptors, resulting in increases in Ca²⁺ currents.     

The activity and properties of poly(adenosine diphosphate ribose) polymerase in vitro during the embryonic development of the South African clawed toad Xenopus laevis

Characterization of poly(ADPribose) polymerase in isolated nuclei of Xenopus laevis embryos shows that maximum activity in vitro occurs at 25°C in 10 mM Tris-HCl buffer, pH 8.0, containing 20 mM MgCl₂, 1 mM dithiothreitol, and 3 mM NaF. Under these conditions the apparent K_m for NAD⁺ was 0.125 mM. The activity of the polymerase during embryogenesis was measured using both a whole embryo extract and isolated embryonic nuclei. Both of these sources of enzyme demonstrated an increase of approximately eightfold in the activity per cell, between early cleavage (stages 2–4; Nieuwkoop and Faber, 1956, “Normal Table of Xenopus laevis (Daudin),” North-Holland, Amsterdam) and late neurula (stages 23–24). From late neurula to early tadpole (stages 38–39) the activity of the extracted enzyme, calculated per cell, decreased by 64%. During the same period the activity of the enzyme in isolated nuclei increased by 40% to reach its maximum activity in early tail bud (stages 27–28), and thereafter decreased by 23%. These results indicate a possible involvement of poly(ADPribose) polymerase in these embryos in the cell differentiation processes, rather than in cell proliferation.     

Book Review

Two factors were examined to determine their effect on the life history, reproduction and life table parameters in the predacious mite Paraseiulus talbii (Athias-Henriot), cultured in the laboratory at 32°C and 75% RH, using tydeid mite, Orthotydeus californicus (Banks) as food. The factors investigated included age of mating females and food deprivation periods during adult stage. Age of mating females has an influence on fecundity and adult longevity; old females decreased egg production and shortened adult longevity compared with young females under constant conditions of abundant prey. A significant lower fecundity, shorter oviposition period and adult longevity were recorded on females when exposed to different food deprivation programmes. Old mating females (30-days old) as well as females exposed to severe food deprivation (three days starvation/a week) resulted in the lower net reproductive rate (Ro), intrinsic rate of natural increase (r_m) and finite rate of increase (λ) values (Ro = 7.301 and 7.486, r_m = 0.202 and 0.198, λ = 1.224 and 1.219) compared with young females (0-day old) and without food deprivation (Ro = 41.312, r_m = 0.308, λ = 1.361). The influence of the duration of copulation on egg production was also studied. Artificial curtailment of copulation to half or quarter the average duration resulted in a reduction in the percentage of reproducing females, fecundity, oviposition duration and the proportion of females in the progeny.     

Energy and protein sources for development of pig embryos cultured beyond hatching in vitro

The effects of supplementation of synthetic culture media with different energy and protein sources on in vitro development of pig embryos beyond the 4–8-cell stage have been explored.Minimal Essential Medium (MEM) supplemented with glucose (1 mg/ml) proved superior to Krebs-Ringer Bicarbonate (KRB) supplemented with glucose (1 mg/ml) in its capacity to support embryonic development to the expanded blastocyst stage (P < 0.05). Inclusion of pyruvate (0.25 mM) or lactate (25 mM) in either MEM or KRB based media inhibited embryonic development. As pyruvate and lactate are important and readily utilizable energy sources for development of most other mammalian embryos in vitro, it is suggested that the observed inhibitory effects of these substrates reflect comparatively lower critical ranges of concentrations of pyruvate and lactate for optimum development of pig embryos in vitro.As a supplementary protein source to MEM, heat inactivated (HI) human serum (10% υ/υ) was superior (P < 0.05) to HI-pig serum (10% υ/υ), HI-foetal calf serum (10% υ/υ) or bovine serum albumin (5 mg/ml). The proportion of 4–8-cell pig embryos which developed beyond hatching in MEM supplemented with HI-human serum (> 56%) was higher than any other reported for in vitro culture of pig embryos through the same developmental period and this medium is recommended for future studies on in vitro development of pig embryos from the four-cell through the hatched/expanded blastocyst stages.     

Factors influencing glucose flux and the effect of insulin in cultured human cells

Uptake of glucose-³H into cultured HLM cells was measured. Equilibration of intracellular and extracellular pools occurred after 25 min. Glucose influx was determined subsequently by measuring the glucose-³H entering in precisely 1 min. Although saturation kinetics were demonstrated these were not of the simple Michaelis-Menten type. The K_m of the glucose carrier system is probably about 60 mM glucose. Galactose did not compete with glucose. Insulin stimulated glucose flux without increasing the value of V _max. The stimulation was fully demonstrable after 10 min, could be elicited at concentrations of 10^-4 units/ml, and was absent 2–4 hr after removal. Increasing pH had little or no effect in stimulating glucose flux. Increasing osmotic pressure caused a marked increase and reduced the effect of insulin. Glucose influx was unaffected by anoxia. Glucose influx was increased and the effect of insulin abolished in the absence of K⁺. Glucose influx was increased by mercuric chloride, iodoacetate, and fluoride which abolished the effect of insulin. Dinitrophenol decreased the rate of glucose uptake but did not alter the effect of insulin. Phlorizin reduced the rate of glucose uptake and abolished the effect of insulin. ATP and AMP enhanced the rate of glucose uptake. These findings are discussed in relation to the mode of action of insulin.