Chick Embryo Heart Cells Research Articles

Role of direct RhoA-phospholipase D1 interaction in mediating adenosine-induced protection from cardiac ischemia.

Activation of adenosine A1 or A3 receptors protects heart cells from ischemia-induced injury. The A3 receptor signals via RhoA and phospholipase D (PLD) to induce cardioprotection. The objective of the study was to investigate how RhoA activates PLD to achieve the anti-ischemic effect of adenosine A3 receptors. In an established cardiac myocyte model of preconditioning using the cultured chick embryo heart cells, overexpression of the RhoA-noninteracting PLD1 mutant I870R selectively blocked the A3 agonist (Cl-IBMECA, 10 nM)-induced cardioprotection. I870R caused a significantly higher percentage of cardiac cells killed in A3 agonist-treated than in A1 agonist (CCPA, 10 nM)-treated myocytes (ANOVA and posttest comparison, P<0.01). Consistent with its inhibitory effect on the PLD activity, I870R attenuated the Cl-IBMECA-mediated PLD activation. Cl-IBMECA caused a 41 +/- 15% increase in PLD activity in mock-transfected myocytes (P<0.01, paired t test) while having only a slight stimulatory effect on the PLD activity in I870R-transfected cells. To further test the anti-ischemic role of a direct RhoA-PLD1 interaction, atrial cardiac myocytes were rendered null for native adenosine receptors by treatment with irreversible A1 antagonist m-DITC-XAC and were selectively transfected with the human adenosine A1 or A3 receptor cDNA individually or they were cotransfected with cDNAs encoding either receptor plus I870R. I870R preferentially inhibited the human A3 receptor-mediated protection from ischemia. The RhoA-noninteracting PLD1 mutant caused a significantly higher percentage of cardiac cells killed in myocytes cotransfected with the human A3 receptor than in those cells expressing the human A1 receptor (ANOVA and posttest comparison, P<0.01). The present data provided the first demonstration of a novel physiological role for the direct RhoA-PLD1 interaction, that of potent protection from cardiac ischemia. The study further supported the concept that a divergent signaling mechanism mediates the anti-ischemic effect of adenosine A1 and A3 receptors.

Caspase activation in a cardiac cell-free model of apoptosis

The release of mitochondrial anapoptogenic factors, like cytochrome c, into the cytosol is a fundamental step for caspase activation and induction of apoptosis. In this report it is shown that the polyamine spermine causes the exit of cytochrome c from heart mitochondria. Polyamines are ubiquitous compounds necessary for growth processes, whose excessive accumulation can trigger apoptosis. The release of cytochrome c caused by spermine is a selective process that is independent of mitochondria damage. The cytochrome c-releasing power of spermine is not affected by cyclosporin A, differently from the effect of permeability transition inducers. Addition of cytochrome c to cytosol extracts from chick embryo heart cells (CEHC) or H9C2 ventricular cardiomyoblasts triggers the onset of caspase activity. In a cardiac cell-free model of apoptosis, the latent caspase activity of cytosolic extracts from CEHC can be activated by cytochrome c released from spermine-treated heart mitochondria. The activated caspase activity is inhibited by nitric oxide donor molecules. These data suggest that prolonged and sustained elevation of polyamines, characteristic of heart hypertrophy, could be involved in the development of apoptosis.

Nitric oxide can function as either a killer molecule or an antiapoptotic effector in cardiomyocytes

Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso- N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.

Calcium dynamics in cultured heart cells exposed to defibrillator-type electric shocks

Spatial and temporal changes in intracellular calcium ion concentration and transmembrane voltage were recorded optically from single-isolated cultured chick-embryo heart cells exposed to high-voltage, defibrillator-type shocks. Fluorescence changes were measured during 5 msec electric shocks of field strengths up to 56 volts cm in single myocytes stained with a Ca ++-sensitive or voltage-sensitive dye. Shocks caused a reversible period of depolarization, elevated cytosolic Ca ++, and refractoriness. Intracellular Ca ++ elevation had two temporal phases: first, a Ca ++ spike with morphology independent of shock intensity; and second, a prolonged Ca ++ elevation with a shock-intensity-dependent magnitude and duration, and with greatest Ca ++ elevation at the poles of the cell adjacent to the electrodes. The prolonged elevation (second phase) was initiated earlier at the anode-facing pole of the cell than at the cathode-facing pole. These results suggest that postshock Ca ++ entry consists of two parts: early normal entry through excitation channels plus a prolonged elevation which may be related to cellular damage.

The production of a monoclonal T3-antiidiotypic antibody (T3-MAAB) that mimics the effects of T3 on 2-deoxy-D-glucose uptake in chick embryo heart cells.

The Ig fraction of rabbit anti-T3 antibody was injected into the spleens of BALB/c mice. Four days later, the lymphocytes were recovered from their spleens and were fused with cells of the 653 myeloma cell line. Screening of the hybrid colonies was carried out in a T3 RIA system. Positive colonies were those whose supernatant displaced 125I-labeled T3 from its antibody. The positive cultures were recloned and one was injected ip into mice. The crude IgG fraction of the ascites fluid was affinity purified on an affigel-10 column containing a covalently bound rabbit anti-T3-IgG. In order to eliminate possible endogenous T3 contamination during the affinity purification, the column was stripped with a 40% solution of acetonitrile in 0.2 M acetic acid, neutralized, and then the purification proceeded as described. The affinity purified antibody was an IgG2a isotype. This monoclonal antibody (T3-MAAB) displaced labeled T3 from its antibody in an RIA system. It also mimicked T3 in the stimulation of [3H]2-deoxy-D-glucose (2-DOG) uptake in cultured chick embryo heart cells. After 6 h exposure, the dose-response curve of 2-DOG uptake to T3-MAAB was shifted to the left by at least one order of magnitude when compared to the dose-response curve obtained with T3. After 24 h exposure, T3 had the expected additional stimulatory effect that was dependent on neosynthesis of proteins, while T3-MAAB did not. Also at 24 h exposure, T3-MAAB did not stimulate the incorporation of labeled leucine and uridine into the heart cells while T3 at an equivalent concentration did. The MAAB activity could be abolished by boiling, while boiling did not affect the activity of an equivalent concentration of T3, thus excluding a T3 contamination-mediated effect. We conclude, therefore, that (a) a monoclonal hybridoma producing an antibody that mimics T3 was established; (b) this antibody competed with labeled T3 for anti-T3 antibody and, like T3, stimulated sugar uptake into cultured chick embryo heart cells; and (c) this antibody, unlike T3, did not stimulate the neosynthesis of proteins.

Extent of Increase in Intracellular Calcium Concentration in Electrically Stimulated Chick Embryo Heart Cells Does Not Depend on Resting Intracellular Calcium Concentration

Using Fura-2 digital imaging microscopy, we found two cell populations in chick embryo heart with different resting intracellular calcium concentration ([Ca2+]i). Electrical stimulation of the cells led to the same increase in [Ca2+]i in both cell types, e.g. from 118 +/- 24 nM up to a peak of 243 +/- 67 nM (SD, n = 18) and from 44 +/- 15 nM up to a peak of 135 +/- 59 nM (SD, n = 13) in cells with high and low resting [Ca2+]i, respectively. All the cells showed the same rate of decline in [Ca2+]i. Due to synchronic stimulation and contraction, this method is suitable to measure [Ca2+]i transients at a high time resolution in individual cells.

Purification and properties of endogenous ouabain-like substances from hemofiltrate and adrenal glands.

Chick embryo heart cells in culture were found to contain two cell types differing in their [Ca2+]i. Elevation of [Ca2+]i in cells with 99.4 +/- 7.1 nM [Ca2+]i was induced half-maximally at 2 x 10(-9) M ouabain and of cells with 27.9 +/- 4.4 nM [Ca2+]i at 4 x 10(-8) M ouabain. The localization of a sodium pump with differing sensitivity for ouabain in different cell types of the heart raised the question of the existence of endogenously formed ouabain. From 6,000 I hemofiltrate of uremic patients, a substance of 582 Da cross-reacting with ouabain antibodies was purified 42,000-fold. The substance was slightly more hydrophobic on a C-18 reversed-phase high-performance liquid chromatography (HPLC) than ouabain was. It inhibited 86Rb+ uptake into red blood cells and raised like ouabain [Ca2+]i in both types of chick heart cells. From pig adrenal glands, a substance inhibiting 86Rb+ uptake into erythrocytes was purified by affinity chromatography on a column containing antibodies against ouabain. A major part of the eluted substances chromatographed on a C-18-HPLC column as ouabain did, but a minor part was somewhat more hydrophobic. It is presumed that more than a single endogenous ouabain-like factor exists in mammals.

Cultured chick-embryo heart cells respond differently to ouabain as measured by the increase in their intracellular Na + concentration

Using digital imaging microscopy with the sodium-sensitive fluorescent indicator sodium-binding benzofuran isopptalate (SBFI), we examined the cytosolic free sodium ion concentration ([Na +] i) in single chick-embryo heart cells. The distribution of the [Na +] i was homogeneous within one cell, but we found a wide cell to cell variation in the range of 3 to 18 mM [Na +] i. In contrast to former experiments showing a heterogeneity of chick-embryo heart cells with respect to their [Ca 2+] i (Ahlemeyer et al. (1992) Eur. J. Biochem. 205. 269–275), we could not distinguish cell populations with different [Na +] i. We found a lognormal distribution of the resting [Na +] i with a median value of 8.8 mM with a standard deviation of 4.5 mM ( n=90). After the addition of varying concentrations of ouabain, we found a biphasic dose-response curve as measured by the increase in [Na +] i. Ouabain showed its half-maximal effect on the [Na +] i between 10 −9 M and 10 −8 M and at 4.3·10 −6 M under steady-state conditions. The finding of a heterogeneity of chick-embryo heart cells with respect to their ouabain-induced increase in [Na +] i is consistent with our previous observations of cells differing in their [Ca 2+] i and in the sensitivity of their sodium pumps to cardiac glycosides.

Chick embryo heart cells with high and low intracellular calcium concentrations respond differently to ouabain

In cell cultures of 10-day-old chick embryo hearts, we found two cell populations, one with high intracellular calcium concentration ([Ca 2+] i) of 116 ± 34 nM (S.E., high [Ca 2+] i cells, n = 154) and another one with low [Ca 2+] i of 46 ± 14 nM [Ca 2+] i (S.E., low [Ca 2+] i cells, n = 171), as revealed by fura-2 digital imaging fluorescence microscopy. The proportion of the high [Ca 2+] i cells varied as a function of the cell density from 10–60% of all cells. Histochemical staining of the cells showed that cells with high and low [Ca 2+] i did not represent differences between muscle and non-muscle cells. When the cells were exposed to different concentrations of ouabain, the high [Ca 2+] i cells showed a half maximal effect at 2 · 10 −9 M ouabain, but only a small increase in [Ca 2+] i of 30%. The low [Ca 2+] i cells reached their half maximal increase in [Ca 2+] i at 4 · 10 −8 M ouabain. A second increase in [Ca 2+] i in this cell type was observed between 10 −6 and 10 −5 M ouabain. Toxic concentrations of ouabain produced an excessive increase in [Ca 2+] i in low [Ca 2+] i cells, whereas high [Ca 2+] i cells showed morphological degeneration due to their higher sensitivity to ouabain. In conclusion, we demonstrate that chick embryo heart contains cells with high and low [Ca 2+] i which show differences in the sensitivity of their sodium pumps to cardiac glycosides.

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na + concentration ([Na +] i) in individual chick embryo heart cells. Inhibition of the Na +-H + exchanger using Na +-free (Li + substituted) medium and inhibition of the Na +-efflux through the Na +-Ca 2+ exchanger using Ca 2+-free medium didn't change the [Na +] i. The opening of voltage-dependent Na + channels with veratridine (150 μg/ml) and inhibition of the Na +-K +-Cl −-cotransporter with bumetanide (10 μM) led to an increase in [Na +] i by 107% and 86%, respectively, suggesting that the Na + channels and the Na +-K +-Cl − cotransporter predominantly regulate the [Na +] i in cultured chick embryo heart cells.

Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative.

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.

Mechanisms of reoxygenation-induced calcium overload in cultured chick embryo heart cells

We examined mechanisms by which Ca enters cultured myocardial cells during posthypoxic reoxygenation. Monolayer cultures of chick embryo ventricular cells were prepared from hearts 10 days in ovo. Cells were exposed to hypoxic conditions (PO2 less than 1.5 Torr), and 45Ca uptake during subsequent reoxygenation was then examined in the absence and presence of modulators of Ca channel-dependent Ca entry and Na-Ca exchange. Modulation of Ca entry by free radical-scavenging enzymes was also examined. Hypoxia for 120 min followed by reoxygenation increased Ca content from 1.9 to 6.1 nmol/mg protein (P less than 0.05) at 30 min. Verapamil (10(-5) M) added before reoxygenation reduced Ca overload to 3.1 +/- 0.2 nmol/mg protein (P less than 0.05), but both verapamil and BAY K 8644 were without effect on modulating Ca entry if added 30 min after reoxygenation. 24Na content of cells increased from 70 nmol/mg protein in control cells to 157 nmol/mg protein (P less than 0.05) after hypoxia and reoxygenation, favoring Ca entry via Na-Ca exchange. Dichlorobenzamil significantly ameliorated reoxygenation-induced Ca overload, as did catalase and superoxide dismutase. We conclude that reoxygenation-induced Ca overload is unlikely to occur via the Ca channel. It occurs in part via Na-Ca exchange and is substantially ameliorated by enzymatic O2 free radical scavengers.

Free calcium in isolated chick embryo heart cells measured using quin2 and fura-2.

Cytosolic free calcium (Cai) was measured using quin2 and fura-2 in isolated chick embryo heart cells. Account was taken of extracellular quin2 and fura-2 (which could not be entirely washed away) by adding Mn. Shortly after loading with quin2, Cai was 49 nM (n = 7) but then rose continuously at a rate varying between 13 and 88%/h. By varying the time between cell isolation and quin2 loading, it was ascertained that the loading was causing the rise in Cai. In one set of experiments, Cai was stable in time and the apparent Cai increased steadily from 55 to 179 nM as dye loading (quin2 or fura-2) was decreased from 1 mM to 5 microM. We conclude that although quin2 and fura-2 are useful for comparing Cai levels and determining whether Cai changes as a result of certain maneuvers, they do not provide an absolute measure of Cai in isolated embryonic heart cells.

Enkephalins increase cyclic adenosine monophosphate content, calcium uptake, and contractile state in cultured chick embryo heart cells.

Peripheral vascular effects of opioid peptides are well known, but direct myocardial effects have not been established. We studied the inotropic response of spontaneously beating cultured chick embryo ventricular cells to the enkephalin analogue [D-Ala2]-enkephalin. Amplitude of cell motion increased in a concentration-dependent manner with 0.53 microM [D-Ala2]-enkephalin producing half-maximal response. The mechanism of this positive inotropic effect was investigated by examining alterations in 45Ca influx, cyclic AMP accumulation and adenylate cyclase activity in response to [D-Ala2]-enkephalin. At maximally inotropic concentrations, the 45Ca influx rate increased 39%, adenylate cyclase was stimulated by 30%, and cyclic AMP content rose more than twofold. Thus, in contrast to neural tissue, receptors for enkephalin in cultured heart cells are coupled to adenylate cyclase in a stimulatory manner. Occupancy of these receptors produces an increase in cyclic AMP levels and exerts a positive inotropic effect via a verapamil-sensitive enhancement of Ca influx.

The Stimulation of Sugar Transport in Heart Cells Grown in a Serum-Free Medium by Picomolar Concentrations of Thyroid Hormones: The Effects of Insulin and Hydrocortisone*

Chick embryo heart cells were propagated in a defined serum-free medium. They formed a confluent, synchronously contracting monolayer that is not different from myocytes grown in serum containing media. The uptake of 2-deoxy-D-[1-3H]glucose in these cells was stimulated by exposure to physiological concentrations of T3 (1 pM) and T4 (10 pM). Actinomycin-D and puromycin did not block the stimulation of 2-deoxy-D-[1-3H]glucose uptake when given with T3 throughout a 6-h incubation period. Cells grown in the absence of both insulin and hydrocortisone were unresponsive to T3. Insulin at 200 nM restored the sensitivity of the cells to 0.1 pM T3. Addition of 10 nM hydrocortisone to the growth medium enhanced the effects of T3 synergistically. The T3-stimulated sugar uptake was completely blocked by 5 X 10(-6) M cytochalasin B, suggesting that T3 acts, like insulin, by the translocation of glucose transporters to the plasma membrane.

Effects of calcium flux inhibitors on contracture and calcium content during inhibition of high energy phosphate production in cultured heart cells

The effects of inhibition of oxidative phosphorylation by 1 mM cyanide (CN) and of glycolysis by 20 mM 2-deoxyglucose (2DG) on contraction and relaxation of cultured monolayers of chick embryo heart cells were determined. Exposure to these agents first induced a gradual decline in contractility and a transient impairment of relaxation. Spontaneous beating then ceased, associated with increased relaxation, followed by a marked and prolonged contracture. This contracture was completely reversible after washout of metabolic inhibitors. The effects of calcium flux inhibitors on the time course of development of contracture were studied. Verapamil, which inhibits Ca influx via the slow Ca channel, delayed the onset of contracture when used in pretreatment, but had no effect if added to cultures after exposure to CN + 2DG. Lanthanum, which inhibits Ca influx both via the slow Ca channel and via Na-Ca exchange, delayed onset of contracture if added after exposure to CN and 2DG, but accelerated contracture if added prior to treatment with CN + 2DG. Cellular exchangeable calcium content, measured after exposure to CN and 2DG for the same time period that produced contracture, was reduced compared to the control level while unidirectional Ca influx rate was not measurably altered. Exchangeable Ca content was unaffected by pretreatment with verapamil and La, but was reduced if cells were exposed to La after metabolic inhibition. These findings suggest that after metabolic inhibition intracellular storage capacity for Ca+ is reduced in cultured heart cells. Ca influx via the slow Ca channel after metabolic inhibition does not appear to contribute to development of contracture in this model system. However, Ca entry via Na-Ca exchange may accelerate cellular contracture developing after metabolic inhibition of ATP production.

Production of nerve growth-stimulating factor(s) from chick embryo heart cells: Use of Cytodex ® 3 microcarriers and serum-free media

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME/F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.

The development of physiologic responsiveness to muscarinic agonists in chick embryo heart cell cultures. Role of high affinity receptors and sensitivity to guanine nucleotides.

Prior to ingrowth of the vagus nerve (4-5 days in ovo), embryonic chick hearts are relatively unresponsive to muscarinic stimulation ( Pappano , A. J. (1977) Pharmacol. Rev. 29, 3-33). We studied the correlation between the development of physiologic responsiveness in the embryonic chick heart and changes in the properties of muscarinic receptors. In cultures from hearts 10 days in ovo, muscarinic agonists decreased beating rate by 15% and increased the rate of K+ efflux by 35%. In cultures of embryonic hearts 3 1/2 days in ovo, muscarinic receptors had no effect on beating rate and mediated only an 11% increase in the rate of K+ efflux. We previously demonstrated that in cells cultured from hearts 10 days in ovo, 26% of receptors bound agonist with a high affinity (RH) and that incubation with guanine nucleotides mediated the conversion of RH to a low affinity form (RL ( Galper , J. B., Dziekan , L. C., O' Hara , D. S., and Smith, T. W. (1982) J. Biol. Chem. 237, 10344-10356]. In cultures of hearts 3 1/2 days in ovo, RH constituted 52% of total [3H] quinuclidinyl benzilate-binding sites, and guanine nucleotides had no effect on the conversion of RH to RL. Growth of cells cultured from hearts 3 1/2 days in ovo in medium supplemented with specific lots of serum resulted in a 3-fold increase (from 11 to 30%) in the ability of muscarinic agonists to increase K+ permeability. This increased sensitivity to muscarinic stimulation was accompanied by a 20% increase in RH and sensitivity of 75% of RH to guanine nucleotides. Thus, enhanced number of RH and development of guanine nucleotide responsiveness are associated with the development of a physiologic response. The relationship of these developmental changes to the appearance of a guanine nucleotide regulatory protein is discussed.

Regulation of guanylate cyclase activity by polyamines in chick embryo heart cell cultures: C.M.Caldarera, B.Tantini, C.Pignatti, A.Marmiroli and C.Clô. Institute of Biological Chemistry, School of Medicine, University of Bologna, Bologna (Italy)

Regulation of guanylate cyclase activity by polyamines in chick embryo heart cell cultures: C.M.Caldarera, B.Tantini, C.Pignatti, A.Marmiroli and C.Clô. Institute of Biological Chemistry, School of Medicine, University of Bologna, Bologna (Italy)

Stimulation of sugar transport in cultured heart cells by triiodothyronine (T3) covalently bound to red blood cells and by T3 in the presence of serum.

T3, covalently bound to red blood cells (RBCs), stimulated the uptake rate of 2-deoxy-D-glucose (2-DOG) in cultured chick embryo heart cells. The response, measured 6 h after exposure, was at least the same than that to free T3. An inhibitor of rhodamine-T3 internalization, bacitracin, did not affect the stimulation of sugar uptake by T3 regardless of whether T3 was covalently bound or free. Pretreatment of RBC-T3 with anti-T3 immunoglobulin G completely blocked the effect of T3, whereas normal rabbit immunoglobulin G failed to do so. Addition of 5% chick serum to the medium stimulated 2-DOG uptake to 144% of the control at 6 h. Adding T3 (10 nM) to the serum-containing medium increased 2-DOG uptake to 171% of the control. The effect of T3 alone or in the presence of serum was not inhibited by cycloheximide, puromycin, or actinomycin D. A T3 dose response curve, in medium containing 10% dehormonized serum, showed enhancement of the T3 effect when compared with the curve obtained in the serum-free medium. The minimal effective concentration of T3 was 10 pM in the presence of serum and 100 pM in its absence. The slope of the linear portion of the dose response curve was greatly increased and the maximal response markedly enhanced by serum. The ED50 was 0.33 nM vs. 0.43 nM in terms of total T3 concentration and 0.16 nM vs. 0.43 nM in terms of free T3 in the presence or absence of serum, respectively. These data suggest that T3, in physiological concentrations, activates sugar transport through an external contact with the cell surface.