Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components

We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.     

Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance

Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia.     

Effect of acute hyperglycemia on potassium (86Rb+) permeability and plasma lipid peroxidation in subjects with normal glucose tolerance

Hyperglycemia is likely to be one of the important determinants of ion transport as it is known to induce oxidative stress and may thus enhance non-specific permeability of membranes. The aim of the present study was to evaluate the effects of an acute increase in glycemia on 86Rb+ (a marker for K+) influx and lipid peroxidation. We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modification on 86Rb+ influx and plasma lipid peroxidation in 20 subjects with normal glucose tolerance (NGT). After 2-hour glucose loading, the levels of passive 86Rb+ influx and plasma lipid peroxidation were significantly increased, whereas the active influx of 86Rb+ was unchanged. The total and passive influx of 86Rb+ into erythrocytes was significantly correlated with the level of plasma lipid peroxidation. This study demonstrates that acute hyperglycemia induces an increase in the passive influx of 86Rb+ in subjects with NGT, suggesting that acute hyperglycemia may produce an oxidative stress in plasma. These changes may be among the earliest changes occurring in response to hyperglycemia.     

Hormonal regulation of inhibin production by cultured Sertoli cells

The hormonal regulation of inhibin production by cultured rat Sertoli cells was examined using a specific radioimmunoassay (RIA) which detects the N-terminal portion of the porcine inhibin alpha chain. FSH, but not hCG or prolactin caused a dose-dependent increase in inhibin production (EC50 for FSH = 2.4 ng/ml); both secreted and intracellular levels of inhibin were increased, but the secreted form represented one-half to two-thirds of the total. The FSH-stimulated production of inhibin was augmented by addition of a phosphodiesterase inhibitor, and could be mimicked by cholera toxin, forskolin, or dibutyryl cAMP, all of which are known to increase intracellular cAMP levels. Inclusion of either dihydrotestosterone or estradiol in the cultures had no effect on inhibin production, both in the presence and absence of FSH. Examination of the conditioned media from forskolin-treated Sertoli cells by gel filtration chromatography revealed a single peak of bioactive and immunoreactive inhibin, at a molecular weight of approximately 32,000, similar to that observed for the porcine and bovine follicular fluid inhibins. Thus, FSH activated the cAMP pathway to stimulate Sertoli cell production of inhibin which in turn suppresses pituitary FSH release to form a closed-loop feedback system.     

Metabolism of amino acids by cultured rat Sertoli cells

Sertoli cells support spermatogenesis both spatially and energetically; for this reason, these cells have important adaptations. The energetic metabolism of Sertoli cells was adapted to provide lactate and pyruvate to developing germ cells, because these substrates are essential for spermatocytes and spermatids. In this study, we investigated whether Sertoli cells use alanine, leucine, valine, and glycine as energetic substrates and whether the simultaneous addition of other nutrients, such as glucose and glutamine, might affect the metabolism of these amino acids. Alanine, leucine, valine, and glutamine are almost totally oxidized to CO2 by these cells. In contrast, glycine has been demonstrated to be a poor energetic substrate, being mainly incorporated into proteins, and their metabolism did not change in the presence of palmitic acid, glucose, and/or glutamine. The metabolism of the 3 other amino acids was modified by palmitic acid; besides, glucose changed alanine, leucine, and valine oxidation. Glutamine decreased the oxidation of alanine, leucine, and valine to CO2. Conversely, both alanine and leucine decreased the oxidation of glutamine. Our present findings show that Sertoli cells can adapt its energy metabolism to the oxidative substrates available to fulfill their role in spermatogenic energetic supply.     

Vectorial secretion of inhibin by immature rat Sertoli cells in vitro: reexamination of previous results

The vectorial secretion of immunoactive and bioactive inhibin by immature rat Sertoli cells (Sc) cultured in a two-compartment system was investigated using various culture supports. When Sc were cultured on Millipore-HA filters (used in all previous studies on vectorial secretion of inhibin), both immuno- and bioactive inhibin were found almost exclusively in the apical compartment, suggesting predominantly apical secretion of the glycoprotein. However, the cell-free Millipore-HA filters completely blocked the passage of Sc-conditioned medium (SCCM) inhibin, even after pretreatment with BSA and SCCM to saturate the protein-binding sites. On the other hand, polycarbonate Nucleopore filters or Millicell-CM membranes, both exhibiting extremely low protein-binding capacity, did not significantly block the passage of SCCM inhibin. When Sc were cultured on Nucleopore filters, the immunoactive inhibin was detected in both culture compartments; the basal compartment/apical compartment (BC/AC) ratio was about 1.5 (range, 1.2-1.9). The maximal effective dose of FSH or (Bu)2cAMP caused a 6- to 9-fold increase in the total (BC plus AC) secretion of immunoactive inhibin, but only a 60% increase in the secretion of bioactive inhibin, as evaluated by RIA and pituitary cell bioassay, respectively. The latter phenomenon was not accompanied by any significant change in the basal/apical distribution of either bioactive nor immunoactive inhibin. The presence of testosterone alone (10(-6) M) did not affect either total immunoactive inhibin secretion or its BC/AC ratio. The effects of the concomitant presence of FSH and testosterone did not differ significantly from those of FSH alone. Similarly to testosterone, the lack of any significant effect was observed for 17 beta-estradiol, dihydrotestosterone, androstenediol, and androstenedione regardless of the presence or absence of FSH. The striking dissimilarity of BC/AC ratios of inhibin noted in cultures maintained on Millipore-HA and Nucleopore filters was not due to differences in permeability barrier or Sc functional polarity. When cultured on either support, Sc monolayers developed comparable permeability barriers, as evaluated by measuring the passage of [3H]inulin and development of electrical resistance. The maximal electrical resistance (130-150 omega cm2) developed after 6-8 days of culture on either support. Also, total transferrin secretion and transferrin BC/AC ratio were similar on both supports, suggesting comparable cell numbers and functional polarities. These findings demonstrate that immature Sc in vitro secrete inhibin bidirectionally (BC/AC ratio, approximately 1.5); the polarity of secretion is unaffected by either FSH or various naturally occurring steroids, including testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Net K+(86Rb+) uptake of human lymphocytes following phytohemagglutinin stimulation

We tried to develop a short-time incubation method for the early detection of lymphocyte stimulation due to lectins and antigens. For this purpose the netto-K+(86Rb+)- uptake of (1) nonstimulated, (2) phytohaemagglutinin (PHA) activated and (3) potassium dichromate (K2Cr2O7) activated human lymphocytes was examined by means of 86Rb+ as a marker. The relative netto-K+-uptake was reduced by 38 per cent (p less than or equal to 0.01) on average after the incubation with PHA. After the incubation with K2Cr2O7 the 86Rb+-values differed, however, only unessentially from the nonstimulated controls in one patient sensibilized by K2Cr2O7. We concluded from our results that this method is not suitable for the early detection of cell mediated immune reactions.     

Regulation of gonadotropin receptors on cultured porcine Leydig and Sertoli cells: effect of potassium depletion

We have examined the role of the NaK-ATPase pump activity on the ligand-induced down-regulation of gonadotropin receptors in cultured porcine Leydig and Sertoli cells. In both cells, inhibition of the NaK pump by ouabain produced a depletion of intracellular K+ levels (ID50, 10(-7) M) after a lag period of about 8 h. In the absence of ligand, the number of FSH receptors in ouabain-treated Sertoli cells was unaffected or slightly reduced, whereas a 2-fold increase in the number of human CG (hCG)/LH receptors with small changes in the binding affinity was observed in Leydig cells treated by ouabain. The effect of ouabain was dose dependent. Differences were also observed in the down-regulation process of gonadotropin receptors in ouabain-treated cells. The hCG-induced receptor loss in Leydig cells was completely reversed by ouabain whereas the drug had no effect on ligand-induced loss of FSH receptors in Sertoli cells. Similar results were observed when the cells were incubated in K+-free medium. Kinetics studies with labeled hCG have shown that ouabain treatment slows down significantly the rate of [125I]iodo-hCG internalization (t 1/2, 18 h; control cells, t 1/2, 6 h), but had no effect on the degradation of internalized hormone. The internalization of receptor-bound [125I]iodo-hCG was also reduced when Leydig cells were incubated in K+-free medium, but was restored when this medium was supplemented with rubidium. The influence of the NaK pump on the receptor regulation of a ligand common to both types of cells, such as epidermal growth factor, was studied under the same experimental conditions. Neither ouabain nor K+-free medium were able to prevent the epidermal growth factor-induced reduction of receptor levels in Leydig and Sertoli cells. Thus, it appears that modulation of ligand-induced receptor loss by depletion of cellular K+ levels is not dependent on the cell type, but on the ligand-receptor complex. The data also show a striking difference in the dynamics of gonadotropin-receptor interaction of two structurally related hormones.     

Desensitization of cultured rat Sertoli cells by follicle-stimulating hormone and by L-isoproterenol

When Sertoli cell-enriched cultures derived from 19-day-old Wistar rats are exposed to FSH or L-isoproterenol, cAMP accumulates in the medium. Previous contact of cultured cells with either one of these agents results in a reversible state of desensitization. During secondary stimulation up to 20 times less cAMP accumulates in the medium and in the cells. The extent of desensitization depends on the concentration of FSH or L-isoproterenol to which the cells have been exposed and on the duration of this previous contact. Pre-incubation with FSH results in cross-desensitization for L-isoproterenol. Comparable heterologous desensitization is not observed after pre-incubation with L-isoproterenol. This suggests that down-regulation of the respective receptors cannot be the only explanation. Refractoriness can also be induced by dbcAMP. This opens the possibility that cAMP itself contributes to the homologous and heterologous desensitization process.     

ATP-Sensitive potassium channels modulate glucose transport in cultured human skeletal muscle cells

Several lines of evidence suggest that ATP-sensitive potassium (KATP) channels are involved in glucose uptake by insulin target tissues. The aim of the present study was to prove directly the effect of KATP channel activity on glucose transport into cultured human skeletal muscle cells. We used potassium channel openers PCO-400 and nicorandil alone or in combination with channel blockers glibenclamide and gliclazide to examine their effects on insulin- or high glucose concentration-induced glucose uptake using 2-deoxy-D-3H-glucose or 3-O-methyl-D-3H-glucose as tracer, respectively. PCO-400 inhibited the basal (non-stimulated) uptake of 2-DG or 3-OMG at the glucose concentration of 5 mM. PCO-400 and nicorandil dose-dependently inhibited insulin-stimulated glucose uptake, and their inhibitory effects were reversed by glibenclamide or gliclazide. In addition, PCO-400 inhibited high glucose concentration-facilitated glucose transport in the absence of insulin, and this effect was also antagonized by both sulfonylurea drugs. Regarding the mechanism by which KATP channels modulate glucose transport, we focused on protein kinase C (PKC), because PKC has been supposed to participate in both insulin- and high glucose concentration-stimulated glucose transport. PMA (phorbol 12-myristate 13-acetate) dose-dependently reversed the PCO-400-induced suppression of insulin-stimulated glucose uptake. On the other hand, PCO-400 at the concentration that inhibited glucose uptake caused no alteration of membrane-associated PKC activity in the presence of insulin or PMA. From these results we conclude that KATP channels modulate the basal and insulin-or high glucose level-stimulated glucose transport in skeletal muscle through a mechanism independent of PKC.     

Altered biochemical responses by rat Sertoli cells and peritubular cells cultured under simulated diabetic conditions

Summary The purpose of the present study was to determine whether the secretion of androgen-binding protein and lactate by 16-day-old rat Sertoli cells is altered when these cells are cultured in simulated diabetic conditions. Incorporation of ³H-uridine into RNA by peritubular cells cultured in simulated diabetic conditions was also studied. The cells were exposed to various concentrations of glucose, -hydroxybutyrate, sodium bicarbonate (to alter the pH of the medium), mannitol (to influence the osmolarity of the medium), or a combination of these compounds. All of the metabolic parameters when tested alone or in combination were capable of increasing lactate secretion by Sertoli cells above control values. Basal secretion of androgen-binding protein, however, was not influenced by any individual component or when all components were tested together. FSH-stimulated levels of androgen-binding protein secretion was depressed only when the Sertoli cells were exposed to all the components simultaneously. Incorporation of uridine by peritubular cells was decreased by exposing the cells to butyrate or mannitol, while no effect was observed with glucose treatment. These results indicate that Sertoli cell and peritubular cell function can be directly altered by several specific metabolic parameters associated with diabetes.     

Hormonal control of phosphodiesterase activity in cultured rat Sertoli cells

Phosphodiesterase activity was studied in cultures derived from 19-day-old rats and enriched with Sertoli cells. Pretreatment of such cultures with follicle-stimulating hormone or L-isoproterenol increased cAMP-phosphodiesterase activity 5.2 and 2.0 times, respectively. cGMP-phosphodiesterase activity was not affected. Similar effects were observed in freshly isolated cells. The stimulatory effect was enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was mimicked by cholera toxin and dbcAMP. Increased activity was observed after a latent period of 1 h. Stimulation was blocked by cycloheximide and actinomycin D. The enzyme had an apparent Km for cAMP of 1.4 micro M. Its activity was enhanced by Mg2+ but not by Ca2+. It is concluded that phosphodiesterases play an important role in the hormonal control of Sertoli-cell function and may contribute to the refractory state of these cells after stimulation with various agonists.     

Lymphocyte (Na,K) ATPase-dependent 86Rb+ uptake in human obesity

Sodium and potassium ion-activated adenosinetriphosphatase (EC number 3.6.1.3) activity, measured as the uptake of 86 rubidium (an analogue of potassium) was determined on peripheral lymphocytes isolated from 20 normotensive obese subjects and 20 normal weight subjects. No difference in the total uptake of 86Rb or in the Na, K-ATPase-dependent uptake was observed in either group. Furthermore, no correlation between the body mass index (BMI) and the Na,K-ATPase-dependent 86Rb uptake was observed. However the Na,K-ATPase mediated 86Rb uptake was always positively correlated with basal blood insulin levels and the insulin sensitivity index. It may be concluded that no lymphocyte dysfunction of Na,K-ATPase was present in our obese patients and that its activity is controlled by insulin in both normal-weight and obese subjects.     

The influence of temperature on the functions of cultured Sertoli cells

Three functions of Sertoli cells were examined with cells from rats aged 13 and 25 days at 34 C, 38 C, and 40 C, namely protein synthesis (incorporation of amino acids into material precipitated by trichloroacetic acid), transport of alpha-aminoisobutyric acid, and production of lactate. A fourth function, namely production of cAMP was examined at two temperatures (34 C and 38 C) in cells from rats aged 25 days. In all cases, activity was greater at 38 C than at 34 C and greater at 40 C than at 38 C. Lysate of Sertoli cells showed similar differences in protein synthesis at the three temperatures, again at both ages. Protein synthesis was higher at 38 C than at 34 C in Sertoli cell-enriched tubules from rats aged 25 days and in Sertoli cells from normal rats of the same age cultured on rat tail collagen. The difference in protein synthesis at the two temperatures was seen whether the cells were cultured, before the experiment, for 7 days at 38 C or at 34 C. Production of cAMP by Sertoli cells was greater at 38 C than at 34 C. It is concluded that Sertoli cells do not show in vitro any inhibitory effect of body temperature on any of the functions tested, but on the contrary, are more active at body than at scrotal temperature. The possible significance of the differences seen is discussed.     

The influence of insulin and insulin-like growth factor-I on hexose transport by Sertoli cells

Insulin stimulates the synthesis of lactate by cultured Sertoli cells prepared from rats aged 13 days, to a much greater extent if glucose is present in the incubation medium than when it is absent. Insulin also stimulates the transport of 3-O-methyl-D-[14 C]glucose into cultured Sertoli cells. This increased transport results from a decrease in the Michaelis-Menten constant (Km) of methylglucose for the transport system without change in maximum velocity (Vmax). Moreover, insulin stimulates influx of the glucose analog and is without effect on efflux. Insulin does not alter transport of methylglucose in peritubular fibroblasts or in mixed populations of germ cells. It was observed that whereas insulin stimulates transport of methylglucose at micromolar concentrations, insulin-like growth factor I (IGF I) exerts the same effect at nanomolar (physiological) concentrations. Moreover, the response to insulin plus IGF I is the same as the maximal responses to either hormone alone. In view of the effects of insulin and IGF I on glucose transport by Sertoli cells and in view of the importance of lactate as a substrate for germ cells, it is suggested that IGF I may be important for the development of normal germinal epithelium in rat testes.     

Passive transport pathways for Ca(2+) and Co(2+) in human red blood cells. (57)Co(2+) as a tracer for Ca(2+) influx

The passive transport of calcium and cobalt and their interference were studied in human red cells using (45)Ca and (57)Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1μM, the progress curve for (45)Ca uptake at 1mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca(T)](c) to reach at [Ca(T)](c) about 5μmol(lcells)(-1) a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca(T)](c). Moreover, vanadate markedly increases the passive Ca(2+) influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40-50μmol(lcells)(-1)h(-1). The apparent Ca(2+) affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the (45)Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1mM Ca(2+) estimated at 50-60μmol(lcells)(-1)h(-1). The Ca influx increases with the extracellular Ca(2+) concentration with a saturating component, with K(?(Ca)) about 0.3mM, plus a non-saturating component. From (45)Ca-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The (45)Ca efflux is markedly accelerated by external Ca(2+), both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange. The (57)Co uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide), consistent with the notion that Co(2+) is not transported by the Ca-pump. The transporter is thus neither SH-group nor ATP or phosphorylation dependent. The (57)Co uptake shows several similarities with the (45)Ca uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J(max) about 16μmol(lcells)(-1)h(-1) and K(?(Co)) about 0.1mM, plus a non-saturating component. The (57)Co and (45)Ca uptake shows mutual inhibition, and at least the stochastic Ca(2+) influx is inhibited by Co(2+). The (57)Co and (45)Ca uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100μM. The (57)Co uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The (57)Co influx amounts to about half the (45)Ca influx in ATP-depleted cells. It is speculated that the basal Ca(2+) and Co(2+) uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that (57)Co could be useful as a tracer for at least the channel-like Ca(2+) entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca(2+) buffering. The molecular identity of the putative common transporter(s) remains to be defined.     

Correlation of follicle-stimulating hormone (FSH)-receptor complex internalization with the sustained phase of FSH-induced calcium uptake by cultured rat Sertoli cells.

We have previously reported that synthetic peptide amides corresponding to regions of the beta-subunit of human FSH [hFSH-beta-(1-15) and hFSH-beta-(51-65)] have the ability to bind calcium and to facilitate its entry into liposomes. In the present study, we have examined the ability of synthetic peptides corresponding to the entire primary structure of hFSH-beta-subunit, to induce calcium influx in cultured rat Sertoli cells. Calcium (as 45Ca2+) uptake in response to 50 microM hFSH-beta-(1-15), hFSH-beta-(21-35), or hFSH-beta-(51-65) peptide amides was 2.5-, 2.4-, and 2.0-fold higher, respectively, than basal uptake. Pretreatment of Sertoli cells for 5 min with phenylarsine oxide (PAO, 80 microM), an inhibitor of receptor-mediated endocytosis, significantly (P < 0.05) reduced 45Ca2+ influx in response to hFSH-beta-(1-15), hFSH-beta-(21-35), and hFSH-beta-(51-65). A delay of 20 min was required, however, before the inhibitory effect of PAO on 45Ca2+ uptake was observed. Specific binding of [125I] hFSH to receptor at 4 C was unaffected by PAO. After 2 h at 37 C, however, approximately 1.6-fold more [125I]hFSH specifically bound at 4 C could be dissociated from the cell surfaces of PAO-pretreated Sertoli cell monolayers, compared to untreated monolayers. This result is consistent with an inhibitory effect of PAO on FSH receptor internalization. Chloroquine (at 100 microM), a lysosomotropic agent known to block FSH degradation, also significantly (P < 0.05) inhibited FSH-induced 45Ca2+ uptake. Extending our earlier studies, these results suggest that the sustained (> 20 min) phase of FSH-induced calcium uptake, also seen in response to synthetic hFSH-beta-(1-15), hFSH-beta-(21-35), and hFSH-beta-(51-65) peptide amides, may occur as a consequence of FSH-receptor complex internalization and FSH degradation. Vesicular uptake of extracellular calcium, which accompanies internalization of FSH-receptor complexes, and release of channel-forming peptides by lysosomal hydrolysis of FSH suggests a novel mechanism whereby FSH increases intracellular calcium levels in Sertoli cells.     

Hormonal regulation of proteins secreted by cultured pig Sertoli cells: characterization by two-dimensional polyacrylamide gel electrophoresis

Using a primary culture of immature porcine Sertoli cells, we studied the effect of porcine FSH (pFSH), testosterone and retinoic acid on the labelled secreted protein. Cells were cultured in a chemically defined medium for 3 days and, on day 3, they were incubated for different times in another medium containing labelled amino acids either in the presence or absence of pFSH (50 ng/ml or 2 micrograms/ml), or testosterone (10(-6) M), or retinoic acid (10(-7) M), either alone or in several combinations. After 4 and 8 h of incubation, 20 and 30 secreted peptides were detected respectively by a two-dimensional polyacrylamide gel electrophoresis of the radiolabelled secreted proteins. During these 2 periods, the effect of pFSH was negligible. After 25 h, about 84 spots (pI in the range of 5-8) were identified on the autoradiograms. pFSH (2 micrograms/ml) induced an increase of 14 spots, and a decrease of 7, but at 50 ng/ml only 5 spots were increased and one decreased. Retinoic acid alone induced the increase of one peptide, while testosterone alone or in combination with pFSH (50 ng/ml) did not modify the electrophoretic pattern. When pig Sertoli cells were treated with retinoic acid, testosterone and pFSH (50 ng/ml), the effects on secreted proteins were higher than those induced by pFSH (50 ng/ml) alone.     

Sibutramine metabolites increase glucose transport by cultured rat muscle cells

BACKGROUND: The anti-obesity agent sibutramine, a serotonin and noradrenaline reuptake inhibitor (SNRI), has been shown to reduce insulin resistance and improve glycaemic control in obese-diabetic ob/ob mice and overweight type 2 diabetic patients. OBJECTIVE: To investigate whether sibutramine or its metabolites act directly on muscle cells to improve glucose uptake and insulin action. DESIGN: Uptake of the non-metabolized glucose analogue 2-deoxyglucose was measured in cultured L6 rat muscle cells after incubation with sibutramine, its two pharmacologically active metabolites and related agents. RESULTS: Sibutramine itself (10(-8)-10(-6) M) did not significantly affect 2-deoxyglucose uptake during incubations up to 72 h. The primary amine metabolite M2 (10(-7) and 10(-6) M) increased basal and insulin-stimulated 2-deoxyglucose uptake (by 12% and 34%) after 24 h incubation. These effects of M2 were lost by 72 h incubation. However, the secondary amine metabolite M1 (10(-6) M) increased basal and insulin-stimulated 2-deoxyglucose uptake (by 50%) after 72 h incubation, although M1 was ineffective after 24 h. M2 stimulated 2-deoxyglucose uptake in the presence of LY-294,002 (an inhibitor of phosphatidylinositol 3-kinase) but the effect of M2 was inhibited by cytochalasin B, which acutely blocks glucose transporters. Incubations with serotoninergic, noradrenergic and dopaminergic agents, or agents known to stimulate release or inhibit reuptake of these substances in nervous tissues indicated that the sibutramine metabolites were not affecting 2-deoxyglucose uptake via mechanisms associated with their SNRI properties. CONCLUSIONS: Sibutramine metabolites can improve insulin-sensitive 2-deoxyglucose uptake by cultured muscle cells independently of SNRI effects.     

Adenosine receptor-dependent modulation of inhibin secretion in cultured immature rat Sertoli cells

Inhibitory (A1) adenosine receptors that attenuate adenylate cyclase activity are present in cultured Sertoli cells. To investigate the possible effect of activating these receptors on the secretion of inhibin by the Sertoli cell, immature rat Sertoli cells were incubated for 24 h with follicle-stimulating hormone (FSH) in the absence or presence of the non-metabolizable, adenosine agonist phenyl-isopropyl-adenosine (PIA), and the accumulation of alpha-inhibin immunoreactivity was measured in the medium. Although devoid of effects when added alone, PIA inhibited the FSH-dependent secretion of alpha-inhibin in a concentration-dependent manner (ED50 = 1-1.5 nM). PIA treatment of the Sertoli cells also rendered the cells less sensitive to FSH in terms of alpha-inhibin secretion. The concentration-response curve to FSH was shifted to the right when cells were incubated in the presence of 100-1000 nM PIA. In contrast, dibutyryl cAMP stimulation of alpha-inhibin accumulation was unaffected by treatment with PIA, indicating that the site of PIA action is at the level of cAMP synthesis. These data provide experimental evidence of adenosine modulation of inhibin secretion by the Sertoli cell and suggest that adenosine may act as a local modulator within the pituitary-testicular axis.