Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 X 10(-5) M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in Vmax of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50-70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.     

Glucose uptake in human and animal muscle cells in culture

Human muscle cells were grown in culture from satellite cells present in muscle biopsies and fusion-competent clones were identified. Hexose uptake was studied in fused myotubes of human muscle cells in culture and compared with hexose uptake in myotubes of the rat L6 and mouse C2C12 muscle cell lines. Uptake of 2-deoxyglucose was saturable and showed an apparent Km of about 1.5 mM in myotubes of all three cell types. The Vmax of uptake was about 6000 pmol/(min.mg protein) in human cells, 4000 pmol/(min.mg protein) in mouse C2C12 muscle cells, and 500 pmol/(min.mg protein) in L6 cells. Hexose uptake was inhibited approximately 90% by cytochalasin B in human, rat, and mouse muscle cell cultures. Insulin stimulated 2-deoxyglucose uptake in all three cultures. The hormone also stimulated transport of 3-O-methylglucose. The sensitivity to insulin was higher in human and C2C12 mouse myotubes (half-maximal stimulation observed at 3.5 X 10(-9) M) than in rat L6 myotubes (half-maximal stimulation observed at 2.5 X 10(-8) M). However, insulin (10(-6) M) stimulated hexose uptake to a larger extent (2.37-fold) in L6 than in either human (1.58-fold) or mouse (1.39-fold) myotubes. It is concluded that human muscle cells grown in culture display carrier-mediated glucose uptake, with qualitatively similar characteristics to those of other muscle cells, and that insulin stimulates hexose uptake in human cells. These cultures will be instrumental in the study of human insulin resistance and in investigations on the mechanism of action of antidiabetic drugs.     

Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Differential role of insulin receptor autophosphorylation sites 1162 and 1163 in the long-term insulin stimulation of glucose transport, glycogenesis, and protein synthesis.

The long-term regulatory effect of insulin on glucose transport activity and glucose transporter expression was examined in Chinese hamster ovary (CHO) transfectants that overexpress either human insulin receptors of the wild type (CHO-R cells) or human insulin receptors mutated at two major autophosphorylation sites, Tyr1162 and Tyr1163 (CHO-Y2 cells). Previous studies showed that, when acutely stimulated by insulin, CHO-Y2 cells exhibit decreased receptor kinase activity along with decreased signaling of several pathways, including that for glucose transport, as compared with CHO-R cells. We now report the following. (i) When treated for 24 h with insulin (10(-10) to 10(-6) M), CHO-R and CHO-Y2 cells displayed closely similar concentration-dependent increases in 2-deoxyglucose uptake. In both transfectants, the maximal insulin-induced increase (approximately 3.5-fold) in uptake was cycloheximide-sensitive and was paralleled by equivalent increases in the levels of GLUT-1 immunoreactive protein and mRNA. (ii) By contrast, under similar conditions, CHO-Y2 cells exhibited a marked decrease in their response to insulin for [U-14C]glucose incorporation into glycogen (decreased sensitivity and maximal responsiveness) and for [U-14C]leucine incorporation into protein (decreased sensitivity) as compared with CHO-R cells. (iii) After a 24-h treatment with 10(-7) M insulin, CHO-R (but not CHO-Y2) cells showed a decreased ability to respond to a subsequent acute insulin stimulation of either receptor exogenous kinase activity or 2-deoxyglucose uptake as compared with respective untreated controls. These results indicate that (i) insulin receptors mutated at Tyr1162 and Tyr1163 retain normal signaling of the long-term stimulatory effect of insulin on glucose transport activity and GLUT-1 expression, but not on glycogenesis and overall protein synthesis; (ii) these three insulin signaling pathways may be triggered by distinct domains of the insulin receptor beta-subunit; and (iii) wild-type (but not twin-tyrosine mutant) receptors undergo negative regulation by chronic insulin treatment for subsequent signaling of acute biological actions of insulin.     

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.     

Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance

We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37 degrees C) and cell surface insulin binding (16 degrees C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (Ki = 2.2 +/- 0.4 mM) compared with maximal transport rates (Ki = 4.1 +/- 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cycloheximide inhibitation on hypoxanthine transport in cultured cells

Cycloheximide preincubation inhibits hypoxanthine uptake into the acid-soluble fractions of cultured rat hepatoma cells (MH₁C₁) and human skin epithelial cells (NCTC 2544, HE cells) in a time- and dose-dependent manner 50% inhibition is seen after 4 h preincubation with 10^?4 M cycloheximide of MH₁C₁ cells and after 2.5 h of HE cells. Adenine uptake is much less affected, after 10 h preincubation with 10^?4 M cycloheximide it was reduced to 83% and 67% of controls in MH₁C₁ cells and HE cells respectively. Cycloheximide inhibits hypoxanthine uptake in a dose-dependent manner above 10^?7 M, with 50% inhibition in MH₁C₁ cells at 4 · 10^?7 M after 12 h preincubation and at 10^-6 M in HE cells after 6 h preincubation. Puromycin mimics the action of cycloheximide. The inhibition of hypoxanthine uptke is not caused by reduction of the activity of hypoxanthine phosphoribosyltransferase in the two cell lines. 10^?4 M cycloheximide preincubation for 10 h does not significantly reduce the uptake of the two non-metabolizable amino acids α-aminoisobutyric acid or 1-aminocyclopentane-1-carboxylic acid (cycloleucine). It is suggested that cycloheximide inhibits the synthesis of a rapidly turning over the protein involved in hypoxanthine transport.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1.25-dihydroxyvitamin D-3

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 · 10^?5 M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in V_max of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50–70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.     

R-alpha-lipoic acid action on cell redox status, the insulin receptor, and glucose uptake in 3T3-L1 adipocytes.

The insulin signaling pathway has been reported to mediate R-alpha-lipoic acid- (R-LA-)-stimulated glucose uptake into 3T3-L1 adipocytes and L6 myotubes. We investigated the role of the thiol antioxidant dihydrolipoic acid (DHLA) and intracellular glutathione (GSH) in R-LA-stimulated glucose transport and explored the hypothesis that R-LA could increase glucose uptake into 3T3-L1 adipocytes in an oxidant-mimetic manner. R-LA pretreatment of 3T3-L1 cells stimulated glucose transport at early time points (30 min - 6 h), whereas it inhibited glucose uptake at later time points. Analysis of the oxidized and reduced content of LA in cells and medium showed that >90% of lipoic acid present was in its oxidized form. Furthermore, all oxidized forms of LA (S-, R-, and racemic LA) stimulated glucose uptake, whereas the reduced form, dihydrolipoic acid, was ineffective. Intracellular GSH levels were not changed at the early time points (before 12 h), while longer preincubation (24 - 48 h) of cells with R-LA significantly increased intracellular GSH. Pretreatment of adipocytes with R-LA increased intracellular peroxide levels at early time points (30 min - 6 h), after which it was decreased (12 - 48 h). R-LA also increased tyrosine phosphorylation of immunoprecipitated insulin receptors from 3T3-L1 adipocytes. These results indicate that (i) 3T3-L1 adipocytes have a low capacity to reduce R-LA and the oxidized form of lipoic acid is responsible for stimulating glucose uptake, (ii) R-LA modulates glucose uptake by changing the intracellular redox status, and (iii) the insulin receptor is a potential cellular target for R-LA action.     

Adenosine cyclic 3',5'-monophosphate uptake and regulation of membrane protein kinase in intact human erythrocytes

The uptake of adenosine cyclic 3',5'-monophosphate (cAMP) and stimulation of membrane-associated protein kinase in mature human erythrocytes were investigated. cAMP transport across the membrane was temperature dependent, and cAMP binding to the isolated membrane had less temperature dependence. More than 99% of the [3H]-cAMP taken up by erythrocytes was nonmembrane bound. Maximal stimulation of membrane protein kinase and maximal occupancy of membrane cAMP binding sites by extracellular cAMP cccurred at 30 degrees C within 30 min after initiation of the incubation of erythrocytes with cAMP. The concentration of extracellular cAMP that gave half-maximal stimulation of membrane protein kinase was 5.4 X 10-4 M, a value consistent with the concentrations of cAMP (5.2 X 10-4 M) found to occupy half-maximally the membrane cAMP binding sites in erythrocytes. Extracellular cAMP and to a lesser extent guanosine cyclic 3',5'-monophosphate and inosine cyclic 3',5'-monophosphate stimulated membrane protein kinase in erythrocytes. The cAMP uptake by human erythrocytes as well as cAMP binding to membranes in the erythrocyte was blocked by an inhibitor [4,4'-bis(isothiocyano)stilbene-2,2-disulfonate] of the anion channel. These studies indicate that cAMP can be transported across membranes into human erythrocytes and can bind to membranes to activate membrane protein kinase. It appears that there is a shared transport channel for cAMP and anion transport.     

Interaction of 2-deoxy-d-glucose and adenine with phosphate anion uptake in yeast

The transport of inorganic phosphate anions into yeast cells (after preincubation with glucose; fructose or another metabolizable sugar, and in the presence of glucose) shows two kinetic components with half-saturation constants of 40 μmol/L and 2.4 mmol/L. The uptake was strikingly stimulated by 2-deoxy-d-glucose (2-dGle) at lower concentrations but inhibited above, 100 mmol/L. A similar stimulation was caused by adenine (0.01–1 mmol/L) and a very small one by uracil and inorganic sulfate. It is suggested that either a phosphorylation reaction accompanies the transport (2-dGlc) or that some compounds stimulate the H⁺-ATPase more than inorganic phosphate itself and thus increase its rate of transport.     

Silicon metabolism in diatoms. III. Respiration and silicon uptake in Navicula pelliculosa

1. Evidence is presented that silicon uptake in the diatom Navicula pelliculosa is linked with aerobic respiration. 2. Cyanide, fluoride, iodoacetate, arsenite, azide, and fluoroacetate, at concentrations inhibitory to respiration, were also inhibitory to silicon uptake. 3. 2,4-Dinitrophenol (1 to 2 x 10(-5)M) stimulated respiration by 100 per cent, but almost completely inhibited silicon uptake. 4. The respiratory quotient of non-Si-deficient cells decreased from 0.93 to 0.75 after 4 days of starvation in darkness. Glucose (1 per cent) raised the respiratory quotient of such starved cells to 1.05. 5. Silicate (20 mg. Si/liter) stimulated respiration of unstarved Si-deficient cells by about 40 per cent. The effect of silicate on the respiration of Si-deficient cells which had been starved in darkness for 4 days was less marked. 6. The respiratory quotient of Si-deficient cells decreased from 0.8-0.9 to 0.3 after 4 days of starvation in darkness. The addition of silicate to starved cells raised the quotient to 0.5. This represented a 25 per cent stimulation of oxygen uptake concomitant with a 90 per cent stimulation of carbon dioxide evolution. 7. Glucose (1 per cent) caused an increase of respiratory quotient in starved cells from 0.3 to 0.7-0.8. The addition of silicate had no effect on the R.Q. during the oxidation of exogenous glucose. 8. Substrates (glucose, fructose, galactose, lactate, succinate, citrate, glycerol), which caused a stimulation of respiration in starved cells, also stimulated silicon uptake by those cells. However, the stimulation of silicon uptake (50 to 100 per cent) was not proportional to the respiratory stimulation by these substrates (30 to 300 per cent).     

Stimulation of glucose transport in L6 muscle cells by long-term intermittent stretch-relaxation.

Skeletal muscle stretch increases resting metabolism and causes hypertrophy. We have examined the effect of mechanical stretch in vitro on glucose transport activity and transporter contents in L6 muscle cells. Long-term (24-48 h) stretch-relaxation (25% maximal elongation at 30 cycles per min) of cell monolayers significantly increased glucose uptake by 1.6- to 2-fold in myotubes but not in myoblasts. The presence of serum was required for the stretch-relaxation induced increase in glucose uptake. Cycloheximide inhibited the mechanical stimulation of glucose uptake, and the latter response was not additive to the stimulatory effect of long-term exposure to insulin. GLUT1 and GLUT4 glucose transporter contents were not changed in total cell membranes from mechanically stimulated cells relative to controls. These results indicate that mechanical stimulation through passive stretch may be an important regulation of nutrient uptake in fetal myotubes independent of innervation.     

Effects of epinephrine and norepinephrine on glucose release from chinook salmon (Oncorhynchus tshawytscha) liver incubated in vitro

The effects of two catecholamines, epinephrine (EP) and norepinephrine (NE), on carbohydrate metabolism were studied by incubating chinook salmon liver in vitro. Basal release of glucose over the course of a 5-h incubation was 7.93 +/- 1.70 mumol/g dry weight. Both EP and NE (2 X 10(-7) M) stimulated glucose release rapidly during the first hour. After 5 h, EP and NE significantly increased glucose release over basal levels to 43.55 +/- 9.01 and 32.75 +/- 6.17 mumol/g dry weight, respectively. Epinephrine- and NE-stimulated glucose release was dose dependent, with a minimum effective dose of 10(-9) M. ED50 for both agents was approximately 2 X 10(-7) M; maximal stimulation occurred at 10(-5) M. No difference in potency between the two catecholamines was found. The effects of adrenergic agonists and antagonists were also studied. Alpha-agonists, methoxamine and phenylephrine, had no effect on glucose release. Isoproterenol, a beta-agonist, stimulated glucose release in a manner similar to EP. The beta-antagonist, propranolol, inhibited both catecholamine- and isoproterenol-stimulated glucose release. Alpha-antagonists (phentolamine, prazosin, and yohimbine) had no effect on either catecholamine- or isoproterenol-stimulated glucose release. Epinephrine and NE stimulate glycogen phosphorylase activity; propranolol inhibits catecholamine-stimulated phosphorylase activity. These results indicate that catecholamines stimulate glucose mobilization in salmon liver by promoting glycogenolysis mediated through beta-adrenergic receptors.     

Characterization of the polyamine transport system in mouse neuroblastoma cells. Effects of sodium and system A amino acids

The biochemical properties of polyamine transport system have been studied in detail in NB-15 mouse neuroblastoma cells in culture by measuring the uptake of [14C]putrescine under various experimentally imposed pharmacological conditions. Putrescine uptake in the NB-15 mouse neuroblastoma cells appeared to be a sodium-dependent process. Iso-osmotic displacement of Na+ in the assay medium with either choline or Li+ resulted in a linear decrease of putrescine uptake. Gramicidin, a channel-former ionophore, inhibited putrescine uptake by more than 90% at 20 nM. N-Ethylmaleimide at 5 mM or p-chloromercuribenzene sulfonate at 50 microM completely abolished putrescine uptake. Conversely, oxidized glutathione at 10 mM or 5,5'-dithiobis-(2-nitrobenzoic acid) at 5 microM gave a 1.3-1.4-fold stimulation after a 1-h incubation. This polyamine transport system appeared to be subjected to adaptive regulation. Polyamine antimetabolites such as alpha-difluoromethyl ornithine stimulated putrescine uptake whereas preloading of cells with polyamines inhibited putrescine uptake. Preloading cells with neutral amino acids that belong to sodium-dependent transport System A stimulated putrescine uptake by more than 8-10-fold. These results suggested that the polyamine transport system in NB-15 mouse neuroblastoma cells was sodium dependent and shared some characteristics common to other known sodium-dependent transport systems. These characteristics included (a) sensitivity to ionophores, (b) sensitivity to sulfhydryl reagents, and (c) sensitivity to intracellular contents of substrate molecules. Our data also indicated that polyamine transport may be regulated by transport System A amino acids.     

Hormonal regulation of prolactin release by human prolactinoma cells cultured in serum-free conditions

Thyrotropin-releasing hormone (TRH) stimulates the prolactin (PRL) release from normal lactotrophs or tumoral cell line GH3. This effect is not observed in many patients with PRL-secreting tumors. We examined in vitro the PRL response to TRH on cultured human PRL-secreting tumor cells (n = 10) maintained on an extracellular matrix in a minimum medium (DME + insulin, transferrin, selenium). Addition of 10(-8) M TRH to 4 X 10(4) cells produced either no stimulation of PRL release (n = 6) or a mild PRL rise of 32 +/- (SE) 11% (n = 4) when measured 1, 2 and 24 h after TRH addition. When tumor cells were preincubated for 24 h with 5 X 10(-11) M bromocriptine, a 47 +/- 4% inhibition of PRL release was obtained. When TRH (10(-8) M) was added, 24 h after bromocriptine, it produced a 85 +/- 25% increase of PRL release (n = 8). This stimulation of PRL release was evident when measured 1 h after TRH addition and persisted for 48 h. The half maximal stimulatory effect of TRH was 2 X 10(-10) M and the maximal effect was achieved at 10(-9) M TRH. When tumor cells were pretreated with various concentrations of triiodothyronine (T3), the PRL release was inhibited by 50% with 5 X 10(-11) M T3 and by 80% with 10(-9) M T3. Successive addition of TRH (10(-8) M) was unable to stimulate PRL release at any concentration of T3. The addition of 10(-8) M estradiol for up to 16 days either stimulated or had no effect upon the PRL basal release according to the cases. In all cases tested (n = 4), preincubation of the tumor cells with estradiol (10(-8) M) modified the inhibition of PRL release induced by bromocriptine with a half-inhibitory concentration displaced from 3 X 10(-11) M (control) to 3 X 10(-10) M (estradiol). These data demonstrate that the absence of TRH effect observed in some human prolactinomas is not linked to the absence of TRH receptor in such tumor cells. TRH responsiveness is always restored in the presence of dopamine (DA) at appropriate concentration. This TRH/DA interaction seems specific while not observed under T3 inhibition of PRL. Furthermore, estrogens, while presenting a variable stimulatory effect upon basal PRL, antagonize the dopaminergic inhibition of PRL release.     

Opioid agonist modulation of cytoplasmic free Ca2+ level in concanavalin A-stimulated mouse lymphocytes.

In this study the influence of mu-, delta-, and kappa-selective opioid agonists (DAMGO, DSLET, and dynorphin A (1-13)) on cytoplasmic free Ca2+ ([Ca2+]i) level in normal and concanavalin-A (Con A)-activated mouse lymphocytes was investigated. [Ca2+]i was measured using the fluorescent dye FURA-2AM. The opioid peptides at 10-12-10-7 M induced some increase in [Ca2+]i in non-activated lymphocytes. However, DAMGO and DSLET (10-13-10-7 M) considerably inhibited a Con A-induced increase in [Ca2+]i. The inhibiting effect of both peptides was higher after 20-min preincubation compare to 2-h preincubation. The effect of the kappa-agonist dynorphin A (1-13) was significantly different depending on the duration of cell pretreatment and the concentration of the peptide used. After preincubation for 20 min at low concentrations (10-12-10-11 M) it slightly stimulated, while at higher (10-10-10-7 M) concentrations it inhibited lymphocyte response to Con A. After preincubation for 2 h, pronounced stimulation of mitogen-induced Ca2+ flux was observed at peptide concentration 10-9 M. The effects of opioids were antagonized by naloxone. These data indicate that functionally active opioid receptors expressed on lymphocytes could be involved in early stages of mitogen activation.