Gabriela Picotto

Acute stimulation of intestinal cell calcium influx induced by 17β-estradiol via the cAMP messenger system

Recent studies have provided evidence for nuclear estrogen receptor-mediated calcium transport in intestinal mucosal cells. The possibility that, in addition, estrogens directly stimulate intestinal Ca2+ fluxes through second-messenger pathways was investigated. Exposure of enterocytes isolated from female rat duodenum to low physiological levels of 17 beta-estradiol (10(-11), 10(-10) and 10(-8) M) rapidly (1-10 min) increased (50-170%) cell 45Ca2+ influx. 17 alpha-Estradiol, dihydrotestosterone and progesterone were devoid of activity, suggesting specificity of the estrogen effect. Maximum responses induced by 17 beta-estradiol (5 min at 10(-10) M) could be abolished to a great extent (84%) by pretreating the cells with verapamil (10 microM) and nitrendipine (1 microM), involving the activation of voltage-dependent Ca2+ channels in the fast increase of rat duodenal calcium uptake by the hormone. Evidence was obtained indicating that the acute estrogen stimulation of enterocyte Ca2+ influx is mediated by the cyclic AMP/PKA pathway. 17 beta-Estradiol rapidly increased cAMP content of rat duodenal cells in parallel to the changes in Ca2+ uptake. In addition, forskolin, dibutyryl cAMP and Sp-cAMPS mimicked and Rp-cAMPS suppressed the prompt 17 beta-estradiol-induced stimulation of Ca2+ influx. These results are consistent with a direct action of estrogens in the enterocyte, presumably a non-genomic one, initiated on the cell surface and resulting in rapid activation of the cAMP pathway and Ca2+ channels, which may be relevant for regulation of intestinal calcium transport.

Involvement of the PLC/PKC pathway in Chitosan-induced anthraquinone production by Rubia tinctorum L. cell cultures

Abstract The signal transduction pathway by which the plant elicitor Chitosan affects secondary metabolite formation in Rubia tinctorum was investigated. Chitosan significantly stimulated (∼100%) anthraquinone (Aqs) synthesis in cultured R. tinctorum. The action of the elicitor could be greatly reduced by inhibition of phospholipase C (neomycin, U-73122) and protein kinase C (PKC) (calphostin C, bisindolylmaleimide, PKC down-regulation). The phorbol ester PMA mimicked the effects of Chitosan on Aqs production. Furthermore, a marked increase in PKC activity and PKC α associated to the cell membranes was observed in response to the elicitor. Compound 2-APB, a blocker of IP3 receptor-mediated release of Ca2+ from inner stores, inhibited Chitosan stimulation of Aqs formation. These results indicate that Chitosan modulation of Aqs levels in R. tinctorum involves activation of the phopholipase C/PKC pathway, with the Ca2+-dependent PKC α isoform playing a major role.

Effect of aging on the mechanisms of PTH‐induced calcium influx in rat intestinal cells

We have investigated the effects of aging on parathyroid hormone (PTH) modulation of intracellular calcium homeostasis and their relationship to signal transduction pathways in isolated rat duodenal cells (enterocytes). PTH (10−8–10−9 M) increased enterocyte 45Ca2+ influx and intracellular Ca2+ concentration ([Ca2+]i) to a greater extent (twofold and 50%, respectively) in aged (24 months) than in young (3 months) animals. The [Ca2+]i response of old cells to the hormone was slower, lacking the early phase of changes in cytosolic Ca2+. Ca2+ influx induced by PTH was prevented by the protein kinase A antagonist Rp‐cAMPS in both young and aged enterocytes, whereas neomycin and compound U73122, inhibitors of PLC‐catalyzed phosphoinositide hydrolysis, abolished hormone‐dependent Ca2+ influx in young but had no effect on aged cells. Higher basal adenylyl cyclase (AC) activity and cAMP content were detected in old enterocytes. PTH increased the absolute levels of cAMP in aged cells and AC activity of microsomes isolated therefrom to a greater extent (≥ twofold) than in young enterocytes/membranes. In young cells, the hormone also induced a rapid and transient release of inositoltrisphosphate (IP3) and diacylglycerol (neomycin‐sensitive) at 45 sec, and a delayed phase of DAG at 5 min (neomycin‐insensitive). The early formation of IP3 and DAG was blunted in aged animals. These results suggest that both the PLC and adenylyl cyclase cascades are involved in PTH stimulation of Ca2+ influx in duodenal cells. During aging, however, only the cAMP pathway is operative, mediating a potentiation of the effects of the hormone. Additional studies are required to establish the relative role of PTH‐dependent messenger systems in the regulation of intestinal calcium absorption and age‐related abnormalities. J. Cell. Physiol. 182:429–437, 2000.

Inhibition of serum-stimulated mitogen activated protein kinase by 1α, 25(OH)2-Vitamin D3 in MCF-7 breast cancer cells

1α,25‐Dihydroxyvitamin D3 [1α,25(OH)2D3], the hormonally active form of vitamin D3, has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo. 1α,25(OH)2D3 acts through two different mechanisms. In addition to regulating gene transcription via its specific intracellular receptor (vitamin D receptor, VDR), 1α,25(OH)2D3 induces rapid, non‐transcriptional responses involving activation of transmembrane signal transduction pathways, like growth factors and peptide hormones. The mechanisms that mediate the antiproliferative effects of 1α,25(OH)2D3 in breast cancer cells are not fully understood. Particularly, there is no information about the early non‐genomic signal transduction effectors modulated by the hormone. The present study shows that 1α,25(OH)2D3 rapidly inhibits serum induced activation of ERK‐1 and ERK‐2 MAP kinases. The tyrosine kinase Src is involved in the pathway leading to activation of ERK 1/2 by serum. Furthermore, 1α,25(OH)2D3 increases the tyrosine‐phosphorylated state of Src and inhibits its kinase activity, while induces the association of the VDR with Src, either in the presence or absence of serum. In parallel, the hormone rapidly increases the amounts of VDR associated to plasma membranes (PM). Pretreatment with the tyrosine phosphatase inhibitors orthovanadate or bpV (phen) prevented mitogen‐activated protein kinase (MAPK) inhibition by 1α,25(OH)2D3. These data altogether suggest that 1α,25(OH)2D3 inhibits the MAPK cascade by inactivating Src tyrosine kinase through a mechanism mediated by the VDR and tyrosine phosphatases.

Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17β-estradiol

Little is known about the regulation of cytosolic calcium Ca2+ levels ([Ca2+]i) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF‐7 and its responsiveness to ATP. MCF‐7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 μM) in the absence of extracellular Ca2+, resulted in a rapid and transient elevation in [Ca2+]i. After recovery of basal levels, Ca2+ readmission (1.5 mM) to the medium increased Ca2+ influx (twofold over basal), reflecting pre‐activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca2+ store is involved in their response. Moreover, IP3‐dependent ATP‐induced calcium mobilization and CCE were completely blocked using compound U‐73122, an inhibitor of phospholipase C. Compound 2‐APB (75 μM) and Gd3+ (10 μM), antagonists of the CCE pathway, completely prevented ATP‐stimulated capacitative Ca2+ entry. CCE in MCF‐7 cells was highly permeable to Mn2+ and to the Ca2+ surrogate Sr2+. Mn2+ entry sensitivity to Gd3+ matched that of the Ca2+ entry pathway. 17β‐estradiol blocked ATP‐induced CCE, but was without effect on TG‐induced CCE. Besides, the estrogen blockade of the ATP‐induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER α immunoreactivity could also be detected in a purified plasma membrane fraction of MCF‐7 cells. These results represent the first evidence on the operation of a ATP‐responsive CCE pathway in MCF‐7 cells and also indicate that 17β‐estradiol interferes with this mechanism by acting at the cell surface level. J. Cell. Biochem. 87: 324–333, 2002.

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM). In cells from aged rats, hormone concentrations higher than physiological (>/=1 nM) were required to observe significant increases in [Ca(2+)](i). Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca(2+)](i) biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca(2+)](i) phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10(-8) M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85alpha, the regulatory subunit of PI3K, as well as the phosphorylation on Thr(308) of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca(2+) chelation (BAPTA-AM, 5 microM) affected the tyrosine phosphorylation of p85alpha and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca(2+) is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca(2+). Taken together, these results suggest that PTH regulation of enterocyte [Ca(2+)](i) involves Ca(2+) mobilization from IP(3)-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr(308) is impaired with ageing.

MAPK inhibition by 1α,25(OH)2–Vitamin D3 in breast cancer cells.: Evidence on the participation of the VDR and Src☆

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the hormonally active form of Vitamin D(3), has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo. 1alpha,25(OH)(2)D(3) acts through two different mechanisms. In addition to regulating gene transcription via its specific intracellular receptor (Vitamin D receptor, VDR), 1alpha,25(OH)(2)D(3) induces, rapid, non-transcriptional responses involving activation of transmembrane signal transduction pathways. The mechanisms that mediate the antiproliferative effects of 1alpha,25(OH)(2)D(3) in breast cancer cells are not fully understood. Particularly, there is no information about the early non-genomic signal transduction effectors modulated by the hormone. The present study shows that 1alpha,25(OH)(2)D(3) rapidly inhibits serum induced activation of ERK-1 and ERK-2 MAP kinases. The non-receptor tyrosine kinase Src is involved in the pathway leading to activation of ERK 1/2 by serum. Furthermore, 1alpha,25(OH)(2)D(3) increases the tyrosine-phosphorylated state of Src as well as it inhibits its kinase activity and induces the association of the VDR with Src. These data suggest that 1alpha,25(OH)(2)D(3) inhibits MAPK by inactivating Src tyrosine kinase through a so far unknown mechanism that seems to be mediated by the VDR.