Francesco Michelangeli

Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: A comparison with the human bladder cancer derived epithelial cell line t24/83

Early reports indicated that ECV304 was a spontaneously-transformed line derived from a Japanese human umbilical vein endothelial cells (HUVEC) culture. Many morphological, immunochemical, and genetic studies provided further evidence that ECV304 was a valuable biomedical research tool and could be used to study processes that include angiogenesis in vitro and signal transduction by a variety of G protein-coupled receptors. However, several distinct differences between ECV304 and HUVEC are now apparent and recent reports have indicated genetic similarity between ECV304 and T24/83, a human bladder cancer cell line. To further assess the utility of ECV304 as a human endothelial cell model, we compared the functional responses of ECV304 and T24/83 to a range of G protein-coupled receptor agonists. We also used DNA fingerprinting to karyotype both ECV304 and T24/83. Both ATP and uridine triphosphate (UTP) stimulated inositol phosphate metabolism in ECV304 without alteration of cAMP levels. Comparative data using selective P2Y receptor agonists indicated that this response, leading to calcium mobilization from intracellular stores, was predominantly mediated by the activation of P2Y2 receptors. Similar responses were recorded from both ECV304 and T24/83 cells. ECV304 expressed a relatively high basal activity of NOS that was reduced by L-NAME and stimulated by P2Y2 receptor agonists. In contrast, P2Y2 receptor activation did not induce prostaglandin synthesis in ECV304. Both ECV304 and T24/83 express receptors for adenosine, adrenaline, and calcitonin, which stimulate adenylate cyclase. Proliferation of ECV304 and T24/83 cells, measured by the incorporation of [3H]thymidine into DNA, was largely serum-independent. This was in contrast to parallel experiments with porcine and bovine aortic endothelial cells that indicated a marked serum-dependent increase in DNA synthesis. Genetic analysis confirmed that ECV304 and T24/83 are identical. ECV304 displays some endothelial characteristics and is useful for the study of receptor pharmacology. However, ECV304 is not of HUVEC origin and is therefore an inappropriate cell line to study endothelial cell biology.

Ca2+-stores in sperm: their identities and functions

Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+](i) and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles that serve as Ca2+ stores in somatic cells. Here, we review i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and ii) the evidence for the existence of functional Ca2+ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the probable identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally, we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca2+ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm.

Alisol B, a Novel Inhibitor of the Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase Pump, Induces Autophagy, Endoplasmic Reticulum Stress, and Apoptosis

Emerging evidence suggests that autophagic modulators have therapeutic potential. This study aims to identify novel autophagic inducers from traditional Chinese medicinal herbs as potential antitumor agents. Using an image-based screen and bioactivity-guided purification, we identified alisol B 23-acetate, alisol A 24-acetate, and alisol B from the rhizome of Alisma orientale as novel inducers of autophagy, with alisol B being the most potent natural product. Across several cancer cell lines, we showed that alisol B–treated cells displayed an increase of autophagic flux and formation of autophagosomes, leading to cell cycle arrest at the G1 phase and cell death. Alisol B induced calcium mobilization from internal stores, leading to autophagy through the activation of the CaMKK-AMPK-mammalian target of rapamycin pathway. Moreover, the disruption of calcium homeostasis induces endoplasmic reticulum stress and unfolded protein responses in alisol B–treated cells, leading to apoptotic cell death. Finally, by computational virtual docking analysis and biochemical assays, we showed that the molecular target of alisol B is the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase. This study provides detailed insights into the cytotoxic mechanism of a novel antitumor compound. Mol Cancer Ther; 9(3); 718–30

A diversity of SERCA Ca2+ pump inhibitors

The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.

The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca2+, Mg2+-ATPase

2,5‐Di(tert‐butyl)‐1,4‐benzohydroquinone has been shown to inhibit the Ca2+, Mg2+‐ATPase of sarcoplasmic reticulum with an affinity of 0.4 μM. It has been shown to shift the E2‐E1 equilibrium for the ATPase towards E2, as shown previously for the inhibitor thapsigargin. The shift towards E2 results in a decrease in affinity for Ca2+, as also observed for thapsigargin. A marked decrease in the rate of the E2‐E1 transition is observed for both BHQ and thapsigargin. A decrease in the equilibrium level of phosphorylation by Pi and of the steady‐state lever of phosphorylation by ATP are consistent with a decrease in the equilibrium constant for phosphorylation by Pi and an increase in the rate of dephosphorylation.

Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

Background: Ca2+ signals, elicited by cues from the oocyte and female tract, regulate human sperm behavior. Results: CatSper channel activation (flagellum) and Ca2+ store mobilization (neck) caused similar [Ca2+]i elevation but induced functionally different behaviors. Conclusion: Sperm motility pattern is determined by the site of Ca2+ mobilization. Significance: Selection of Ca2+ signaling components and/or regulation of their availability for activation controls human sperm behavior. [Ca2+]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca2+ signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca2+]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca2+ caused sustained [Ca2+]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca2+ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca2+-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca2+]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca2+ at the sperm neck can be mobilized by Ca2+-induced Ca2+ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca2+ store, which may be regulated by capacitation and NO from the cumulus.

Environmental endocrine disrupters dysregulate estrogen metabolism and Ca2+ homeostasis in fish and mammals via receptor-independent mechanisms

Xenoestrogen endocrine disrupters (EDs) in the environment are thought to be responsible for a number of examples of sexual dysfunction that have recently been reported in several species. There is growing concern that these compounds may also cause abnormalities of the male reproductive tract and reduced spermatogenesis in man. Whilst some effects of EDs may be receptor-mediated, there is growing evidence that these compounds can exert potent effects in vivo by directly interacting with cellular enzyme targets. Here we report on, and review, the effects of alkylphenols and other EDs on two such enzymes: (1) sulfotransferases, which convert active estrogenic steroids to inactive steroid sulfates; and (2) Ca(2+)-ATPases, which are responsible for maintaining low, physiological, intracellular Ca(2+) concentrations. These enzymes are potently inhibited by EDs in both fish and mammalian species. The increased concentrations of active estrogens and the likely cytotoxic effects of elevated concentrations of intracellular Ca(2+) arising from these effects may underlie some of the endocrine disrupting potential of these widespread industrial pollutants.

Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump, leading to autophagy induction through the activation of the Ca2+/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.

Secretory pathway Ca(2+)-ATPase (SPCA1) Ca(2)+ pumps, not SERCAs, regulate complex [Ca(2+)](i) signals in human spermatozoa.

The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin (0.1-1 μM) and cyclopiazonic acid (10 μM), failed to affect resting [Ca2+] in human spermatozoa. Slow progesterone-induced [Ca2+ i]i oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca2+ store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 μM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca2+]i and partial, dose-dependent disruption of oscillations. A 10-40 μM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca2+ ATPases, caused elevation of resting [Ca2+]i and inhibition of [Ca2+]i oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca2+. Low doses of bis-phenol had marked effects on [Ca2+]i oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca2+ influx induced by progesterone is not mediated via mobilisation of Ca2+ stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca2+ ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca2+ store(s) and store-dependent [Ca2+]i oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca2+ pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca2+ ATPases contribute at least part of this non-SERCA Ca2+ pump activity.

Expression and distribution of the type 1 and type 3 inositol 1,4, 5-trisphosphate receptor in developing vascular smooth muscle.

The recent discoveries of inositol 1,4,5-trisphosphate (IP3) receptor subtypes with different affinities for IP3 and their potential involvement in development has important consequences for vascular smooth muscle. This study has examined the expression and distribution of the type 1 and type 3 IP3 receptor subtypes in developing rat vascular smooth muscles. Immunoblotting of portal vein and aorta from neonatal (2 to 4 days) and fully developed (6 weeks) rats revealed significantly higher levels of the type 3 IP3 receptor expression in neonatal, compared with developed, vascular smooth muscles. In contrast, expression of the type 1 IP3 receptor in neonates was lower compared with developed vascular smooth muscles. Immunolocalization of the type 3 IP3 receptors in neonatal tissues revealed that staining corresponded to the distribution of the sarcoplasmic reticulum (visualized by osmium ferricyanide staining of thin tissue sections), which suggested localization of the type 3 IP3 receptor throughout the sarcoplasmic reticulum network. We conclude that type 3 IP3 receptors are the predominant subtype in the development of vascular smooth muscle and are distributed throughout the sarcoplasmic reticulum in these cells. The switch in isoforms of the IP3 receptor during development from the type 3 with low affinity for IP3 to the higher-affinity type 1 receptor may play a role in calcium-mediated regulation of developing vascular smooth muscle.