Cytoplasmic Calcein Research Articles

Effects of N-acetylcysteine on Growth, Viability, and Ultrastructure of In Vitro Cultured Bovine Secondary Follicles.

This study aimed to investigate the effects of different concentrations of N-acetylcysteine (NAC) on the growth, antrum formation, viability, and ultrastructure of bovine secondary follicles cultured in vitro for 18 days. To this end, the follicles were cultured in TCM-199+ medium alone or supplemented with 1.0, 5.0, or 25.0 mM NAC. Follicular growth, antrum formation, viability (calcein-AM and ethidium homodimer-1) and ultrastructure were evaluated at the end of culture period. The results showed that 1.0 mM NAC increased the percentage of growing follicles and the fluorescence intensity for calcein-AM when compared to other treatments (p < 0.05). On the other hand, follicles cultured with 25.0 mM NAC had higher fluorescence intensity for ethidium homodimer-1, which is a sign of degeneration. Ultrastructural analysis showed that oocytes from follicles cultured in control medium alone or with 1 mM NAC had intact zonae pellucidae in close association with oolemmae, but the ooplasm showed mitochondria with a reduced number of cristae. On the other hand, oocytes from follicles cultured with 5 or 25 mM NAC had extremely vacuolated cytoplasm and no recognizable organelles. In conclusion, 1 mM NAC increases cytoplasmic calcein staining and the growth rate in bovine secondary follicles cultured in vitro, but the presence of 5 or 25 mM NAC causes damage in cellular membranes and organelles, as well as reducing the percentages of growing follicles.

Amplification of human platelet activation by surface pannexin‐1 channels

BackgroundPannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ∼1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown.ObjectiveTo determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques.MethodsPanx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca2+ influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL−1 apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels.ResultsPANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca2+ influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors.ConclusionsPanx1 is expressed on human platelets and amplifies Ca2+ influx, ATP release and aggregation through the secondary activation of P2X1 receptors. We propose that Panx1 represents a novel target for the management of arterial thrombosis.

Very high aquaporin-1 facilitated water permeability in mouse gallbladder

Water transport across gallbladder epithelium is driven by osmotic gradients generated from active salt absorption and secretion. Aquaporin (AQP) water channels have been proposed to facilitate transepithelial water transport in gallbladder and to modulate bile composition. We found strong AQP1 immunofluorescence at the apical membrane of mouse gallbladder epithelium. Transepithelial osmotic water permeability (Pf) was measured in freshly isolated gallbladder sacs from the kinetics of luminal calcein self-quenching in response to an osmotic gradient. Pf was very high (0.12 cm/s) in gallbladders from wild-type mice, cAMP independent, and independent of osmotic gradient size and direction. Although gallbladders from AQP1 knockout mice had similar size and morphology to those from wild-type mice, their Pf was reduced by approximately 10-fold. Apical plasma membrane water permeability was greatly reduced in AQP1-deficient gallbladders, as measured by cytoplasmic calcein quenching in perfluorocarbon-filled, inverted gallbladder sacs. However, neither bile osmolality nor bile salt concentration differed in gallbladders from wild-type vs. AQP1 knockout mice. Our data indicate constitutively high water permeability in mouse gallbladder epithelium involving transcellular water transport through AQP1. The similar bile salt concentration in gallbladders from AQP1 knockout mice argues against a physiologically important role for AQP1 in mouse gallbladder.

Mechanisms of secretion-associated shrinkage and volume recovery in cultured rabbit parietal cells

We have previously shown that stimulation of acid secretion in parietal cells causes rapid initial cell shrinkage, followed by Na(+)/H(+) exchange-mediated regulatory volume increase (RVI). The factors leading to the initial cell shrinkage are unknown. We therefore monitored volume changes in cultured rabbit parietal cells by confocal measurement of the cytoplasmic calcein concentration. Although blocking the presumably apically located K(+) channel KCNQ1 with chromanol 293b reduced both the forskolin- and carbachol-induced cell shrinkage, inhibition of Ca(2+)-sensitive K(+) channels with charybdotoxin strongly inhibited the cell volume decrease after carbachol, but not after forskolin stimulation. The cell shrinkage induced by both secretagogues was partially inhibited by blocking H(+)-K(+)-ATPase with SCH28080 and completely absent after incubation with NPPB, which inhibits parietal cell anion conductances involved in acid secretion. The subsequent RVI was strongly inhibited with the Na(+)/H(+) exchanger 1 (NHE1)-specific concentration of HOE642 and completely by 500 muM dimethyl-amiloride (DMA), which also inhibits NHE4. None of the above substances induced volume changes under baseline conditions. Our results indicate that cell volume decrease associated with acid secretion is dependent on the activation of K(+) and Cl(-) channels by the respective secretagogues. K(+), Cl(-), and water secretion into the secretory canaliculi is thus one likely mechanism of stimulation-associated cell shrinkage in cultured parietal cells. The observed RVI is predominantly mediated by NHE1.

Mitochondrial permeability transition can be directly monitored in living neurons

Mitochondria have been suggested as key players in apoptotic cell death of neurons and many other tissues, since the release of proapoptotic molecules from mitochondria is implicated in caspase activation. As a potential release mechanism, the occurrence of a large pore opening in the inner membrane (mitochondrial permeability transition pore, PTP) has been proposed, but has not yet been observed directly in neurons. We investigated whether the calcein/Co 2+-quenching technique introduced by Petronilli et al. 〚Biofactors 8 (1998) 263〛, which allows direct observation of PTP opening, can be applied to neurons. Exposure of calcein-loaded neurons to Co 2+ ions resulted in the fading of diffuse cytoplasmic calcein fluorescence, with organelle-restricted fluorescent spots remaining. These spots were colocalized with mitochondrially-entrapped tetramethylrhodamineethylester (TMRE) fluorescence and corresponded to colocalization of calcein and TMRE fluorescence in digitonin-permeabilized neurons. Importantly, extensive neuronal calcium loading, which is assumed to induce PTP opening, resulted in significant fading of mitochondrial fluorescence, suggesting the occurrence of permeability transition. This fluorescence decrease could be completely prevented by the PTP blocker cyclosporin A.

Visualization of the secretory process involved in Ca 2+-activated fluid secretion from rat submandibular glands using the fluorescent dye, calcein

The central feature of fluid and electrolyte secretion by salivary acinar cells is transepithelial Cl- movement as a driving force for the secretion. However, little is known about the membrane localization and regulation by agonists of various anion channels. To characterize the anion transport and fluid secretion, we visualized the secretory process induced by the cholinergic agonist, carbachol (CCh), using the anionic fluorescent dye, calcein, under a confocal laser scanning microscope. The fluorescence of calcein loaded into the isolated acini was spread diffusely throughout the cytoplasm and was less intense in the secretory vesicles which occupied the apical pole. Cytoplasmic calcein was released into intercellular canaliculi just after the addition of CCh, depending upon a rise in [Ca2+]i by Ca2+ release from intracellular stores. Thereafter, the formation of watery vacuoles connected with intercellular canaliculi was visualized in the calcein-loaded acini, depending upon external Ca2+. Both the calcein release and vacuole formation were inhibited by suppressing the Ca(2+)-activated K+ efflux. The calcein release was also affected by the external anion substitution, suggesting that calcein is released through an anion channel. In the isolated, perfused glands, CCh-induced fluid secretion was sustained in two phases, whereas the loaded calcein was initially and transiently released into the saliva. By revealing the [Ca2+]i dependence and sensitivities to channel blockers, our results suggest that the initial phase of CCh-induced fluid secretion was evoked in association with the release of the organic anion, calcein, and the late phase of fluid secretion, during which calcein is less permeable, was associated with the formation of watery vacuoles. Thus, the anion channels possessing the distinct property of anion permeation may be activated in the initial phase and late phase. These results indicate that the anionic fluorescent dye, calcein, is useful for visualizing the process of Ca(2+)-dependent fluid secretion, and for clarifying the relation between fluid secretion and anion transport.

CAMP-dependent potentiation of the Ca 2+-activated release of the anionic fluorescent dye, calcein, from rat parotid acinar cells

A recent study indicates that elevation of [Ca 2+] i enhances the release of calcein, an anionic fluorescent dye, from isolated exocrine acinar cells, so cytoplasmic calcein is useful for monitoring the secretion of organic anions. In this study, we investigated the effect of cAMP on the calcein release evoked by elevation of [Ca 2+] i. Isoproterenol, forskolin and dibutyryl cyclic AMP (dbcAMP) did not induce the release of calcein from isolated parotid acinar cells, but they potentiated the carbachol-induced release of calcein. Although cytoplasmic calcein is released through an increase in [Ca 2+] i, isoproterenol potentiated the carbachol-induced release of calcein without affecting the increase in [Ca 2+] i evoked by a high concentration of carbachol (10 −6 M). Charybdotoxin, a K + channel blocker, inhibited both the carbachol-induced release and the potentiation by isoproterenol. However, the calcein permeation pathways mediating the carbachol-induced release and the isoproterenol-potentiated release exhibited distinct sensitivities to anion channel blockers. Our results indicate that the calcein release induced by carbachol is potentiated through an increase in intracellular levels of cAMP. Although both the Ca 2+-activated release and the cAMP-potentiated release may be coupled to Ca 2+-activated K + efflux, increases in both [Ca 2+] i and [cAMP] i may activate the calcein conduction pathway which is not activated by an increase in [Ca 2+] i alone.

Cytotoxic effect of calcein acetoxymethyl ester on human tumor cell lines

Calcein acetoxymethyl ester (calcein/AM) and some related cellular dyes with a cytoplasmic distribution were investigated with respect to cellular hydrolysis, accumulation, efflux and cytotoxicity in a panel of established human cell lines, including multidrug resistant (MDR) phenotypes. At 0.1-1 micrograms/ml, calcein/AM was highly cytotoxic against several cell lines, even after short-term exposure (30 min). Calcein/AM induced no immediate loss (3 h) of membrane integrity and the drug was more active against low compared with high density plated cells. In cell lines with the MDR phenotype and in the renal carcinoma cell line ACHN, the drug was considerably less active. Non-esterified calcein had no effect and calcein/AM was significantly more potent than other structurally related fluorescein analogs and AM esters tested. Although MDR cell lines showed a decreased cellular hydrolysis and accumulation of the dye, there was no strict relationship between cytoplasmic calcein exposure and cytotoxic activity. The rate of efflux was low in the two most sensitive cell lines, the human lymphoma U-937-GTB and its vincristine (vcr) resistant subline U-937/vcr10, while the remaining cell lines showed similar biphasic efflux patterns, including cell lines of the MDR phenotype. The results show that calcein/AM has cytotoxic activity against human tumor cell lines at low concentrations. The effect appears dependent on the intracellular trapping of the drug, although the specific cellular target remains unknown. Due to its cytotoxic efficacy and unique principle of cellular drug delivery, further investigation of calcein/AM and related compounds as potentially new anticancer agents seems warranted.

Efficient cytoplasmic delivery of a fluorescent dye by pH-sensitive immunoliposomes.

We previously showed that liposomes composed of dioleoylphosphatidyl-ethanolamine and palmitoyl-homocysteine (8:2) are highly fusion competent when exposed to an acidic environment of pH less than 6.5. (Connor, J., M. B. Yatvin, and L. Huang, 1984, Proc. Natl. Acad. Sci. USA. 81:1715-1718). Palmitoyl anti-H2Kk was incorporated into these pH-sensitive liposomes by a modified reserve-phase evaporation method. Mouse L929 cells (k haplotype) treated with immunoliposomes composed of dioleoylphosphatidylethanolamine/palmitoyl-homocysteine (8:2) with an entrapped fluorescent dye, calcein, showed diffused fluorescence throughout the cytoplasm. Measurements by use of a microscope-associated photometer gave an approximate value of 50 microM for the cytoplasmic calcein concentration. This concentration represents an efficient delivery of the aqueous content of the immunoliposome. Cells treated with immunoliposomes composed of dioleoylphosphatidylcholine (pH-insensitive liposomes) showed only punctate fluorescence. The cytoplasmic delivery of calcein by the pH-sensitive immunoliposomes could be inhibited by chloroquine or by incubation at 20 degrees C. These results suggest that the efficient cytoplasmic delivery involves the endocytic pathway, particularly the acidic organelles such as the endosomes and/or lysosomes. One possibility is that the immunoliposomes fuse with the endosome membranes from within the endosomes, thus releasing the contents into the cytoplasm. This nontoxic method should be widely applicable to the intracellular delivery of biomolecules into living cells.