Saswati Das

Calmodulin and protein kinase C activation duplicates the biphasic secretion of luteinizing hormone

Ionomycin, which activates the Ca2+-calmodulin system, and phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), were used to investigate potential roles of these systems as mediators of the biphasic secretion of luteinizing hormone. Quartered pituitaries from diestrous II female rats were perifused at 37 degrees C, and sequential effluent fractions collected every 10 min. Gonadotropin-releasing hormone administration resulted in a biphasic response: an initial, protein synthesis-independent secretion, followed 60 min later by a secondary, augmented, protein synthesis-dependent component. Ionomycin-stimulated gonadotropin secretion was immediate and partially independent of protein synthesis, whereas the PMA-induced secretion was delayed (approximately 70 min), and was completely dependent on protein synthesis. Simultaneous infusions of ionomycin and PMA resulted in an initial, protein synthesis-independent response followed by the secondary, augmented, protein synthesis-dependent component, which exhibited synergistic interactions between calmodulin and PKC. These results suggest that calmodulin mediates the initial, protein synthesis-independent secretion, PKC mediates part of the secondary, augmented response, while calmodulin and PKC synergize to mediate the remaining component of the secondary response.

Validation of Bone Marrow Derived Dendritic Cells as an Appropriate Model to Study Tumor-Mediated Suppression of DC Maturation through STAT3 Hyperactivation

PURPOSE Tumors can escape immune eradication by harnessing dendritic cell (DC) maturation. However, DC types used as in vitro models to study tumor-mediated immunosuppression possess fundamental variability that could influence research outcomes. Therefore, we assessed the behavior of two distinct murine DC models upon exposure to tumor-conditioned medium of B16.F10 melanoma (B16-CM). METHODS Using primary bone-marrow derived dendritic cells (BMDCs) or immortalized DC2.4 cell line, we evaluated the level of signal transducer and activator of transcription 3 (STAT3) phosphorylation by Western blot as a molecular parameter. We also examined the surface expression of co-stimulatory molecules on DCs by flow cytometry as a phenotypic parameter. RESULTS Our results revealed critical discrepancies between the two models in response to tumor-conditioned medium. While conditioned medium was able to induce STAT3 phosphorylation in BMDCs, it did not significantly induce STAT3 phosphorylation in DC2.4 cell line. Moreover, only in BMDCs, the expression of CD86 and CD40 was remarkably downregulated by B16-CM and was not totally recovered after LPS stimulation. In contrast, DC2.4 cells did not show any signs of harnessed maturation upon exposure to B16-CM. CONCLUSIONS In order to study the effect of tumor-mediated immunosuppression on DC maturation in vitro via tumor-induction of STAT3 activation, primary BMDCs are more reliable as a model than DC2.4.

Sex differences in the extracellular Ca2+-independent release of LH and FSH

The present study was undertaken to investigate the effects of sex and estrous cycle on the manifestation of the extracellular Ca2+-independent component of gonadotropin secretion. Quartered pituitaries from male, ovariectomized (OVX) females +/- estradiol (E2) implants, and mature females at each stage of the estrous cycle were perifused with Ca2+-free medium. Gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from male and OVX pituitaries was inhibited in Ca2+-free medium. In contrast, only a partial inhibition was obtained from OVX + E2 or regularly cycling female pituitaries. This extracellular Ca2+-independent component of gonadotropin secretion was lowest at estrus and increased progressively during the estrous cycle. Estradiol replacement in OVX animals resulted in a response similar to that obtained on proestrus. These results indicate that the extracellular Ca2+-independent component of LH and FSH release is only manifest from intact female and not male pituitaries, and is dependent on estradiol.

The characterization of phospholipase D in FRTL-5 thyroid cells.

We previously demonstrated that TSH activates phospholipase D (PLD) in Fischer rat thyroid line (FRTL)-5 cells. To date, two types of mammalian phosphatidylcholine-specific PLD cDNAs, designated as PLD-1 and PLD-2, have been cloned. The present study determined the PLD isoform composition in FRTL-5 thyroid cells and which isoform is regulated by TSH. PLD-1 is activated by small molecular weight G-proteins, such as ADP-ribosylation factor (ARF) and RhoA family members, while PLD-2 is relatively independent of such stimuli. We established the presence of PLD-1 and PLD-2 by Western blot analysis and compared PLD activity in cytosol, membranes and combined fractions in the presence and absence of GTPgammaS. The membrane fraction showed very little activity in the absence of GTPgammaS, but this activity increased approximately 5-fold (P<0.05, ANOVA) in the presence of GTPgammaS. Maximal PLD activity was seen with the combination of membrane plus cytosolic fractions (which contained ARF and RhoA) where the addition of GTPgammaS increased PLD activity approximately 8-fold (P<0.05, ANOVA). To determine the relative activities of PLD-1 and PLD-2 in FRTL-5 thyroid cells, cell-free PLD assays were performed in the presence of GTPgammaS or GDPbetaS with varying concentrations of phosphatidylinositol 4,5-bisphosphate (PIP(2)). PLD-2 contributed only approximately 19% of the total amount of PLD activity in the membranes and PLD-1 was the predominant PLD isoform. TSH stimulated PLD-1 activity by up to 2. 3-fold over control values (P<0.01, ANOVA). To establish the dependence of PLD-1 on small molecular weight G-proteins, the translocations of ARF and RhoA to the membrane fractions was determined after stimulation by TSH. Both ARF and RhoA were maximally translocated to the membrane fraction after 10 min incubation with 100 microU/ml TSH by approximately 1.7- and 2.3-fold over control values, respectively (P<0.02 and P<0.03, ANOVA). It is concluded that TSH stimulates PLD-1 activity in FRTL-5 thyroid cells and this is accompanied by the translocation of ARF and RhoA to the membrane fraction.

Dependency of phorbol ester-induced gonadotropin secretion on estradiol

Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC) was used to investigate the estradiol dependency of PKC-stimulated luteinizing hormone (LH) secretion from perifused anterior pituitaries. Infusions of PMA stimulated LH secretion from diestrous II, ovariectomized + estradiol-treated, and orchidectomized + estradiol-treated quartered pituitaries, by protein synthesis-dependent mechanisms. In contrast, pituitaries from intact, orchidectomized males, or ovariectomized females were unresponsive to PMA. Interestingly, dispersed male pituitary cells differed from male pituitary tissue blocks, in that the dispersed cells responded to PMA with increased LH secretion. These results indicate that PKCs ability to directly stimulate LH secretion is dependent on de novo protein synthesis and estradiol. Moreover, the effects of estradiol on PKC-stimulated secretion form at least one basis for the estradiol-induced increased responsiveness of gonadotrophs to GnRH. Additionally, it appears that dispersed pituitary cells may not respond to activators of PKC in a physiological manner.

The phorbol ester-induced extracellular Ca2+-independent release of LH is dependent on estradiol and de novo protein synthesis

Phorbol 12‐myristate 13‐acetate (PMA) was used to determine whether the PMA‐induced extracellular Ca2+‐independent release of LH was dependent on sex, estradiol and de novo protein synthesis. Infusions of gonadotropin‐releasing hormone (GnRH) or PMA in a perifusion system stimulated a partial secretion of LH from diestrous II and ovariectomized + estradiol‐treated female pituitaries (responses inhibited by cycloheximide). In contrast, PMA was ineffective in stimulating PRL secretion from these pituitaries, as well as LH secretion from male or ovariectomized female pituitaries. These results indicate that the PMA‐stimulated extracellular Ca2+‐independent secretion of LH is a specific process which is dependent on sex, estradiol and de novo protein synthesis, and mimics the characteristics of the GnRH‐stimulated responses.