Fluorescent sensors based on BODIPY derivatives for aluminium ion recognition: an experimental and theoretical study

Two BODIPY derivative sensors for metal ion recognition containing 10-(4-hydroxyphenyl) (L1) and 10-(3,4-dihydroxyphenyl) (L2) were synthesized in a one-pot reaction of benzaldehyde derivative and 2,4-dimethylpyrrole in the presence of trifluoroacetic acid as catalyst. The binding abilities between these sensors and 50 equivalents of Na⁺, K⁺, Ag⁺, Ca²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Al³⁺ and Cr³⁺ ions were studied using UV–vis and fluorescent spectroscopic methods. Of all the metal ions tested, Al³⁺ ion showed the greatest decrease in intensity in the spectra of the sensors, and therefore Al³⁺ ion forms the strongest complex. The binding abilities of BODIPY receptors with Na⁺, Ag⁺, Ca²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ and Al³⁺ ions were also investigated using density functional theory (DFT) calculations at B3LYP/LanL2DZ theoretical level. The calculated results point to the same conclusion. DFT calculations also provided the HOMO–LUMO energy levels, which can explain the spectrum change upon complexation.

Do Ca2+-chelating polysaccharides reduce calcium ion release from gypsum-based biomaterials?

Background

Calcium sulphate, a widely used bone filler, may negatively affect human osteoblasts due to release of high quantities of calcium ions. To reduce this effect, an attempt was made to enrich calcium sulphate with Ca²⁺-chelating plant and rhizobial exopolysaccharides (EPS).

Methodology

Incubation of polysaccharide-enriched calcium sulphate composites was performed in DMEM/F12 medium. Ca²⁺ (and Mg²⁺ and Pi) levels were estimated using standardised, spectrophotometry-based kits. Composite surface morphology was tested using SEM technique.

Results

Rhizobial EPS was found slightly less effective at Ca²⁺ chelation than sodium alginate. Both polysaccharides may be used as gypsum supplements in the form of setting liquids (0.3% total mass), but only sodium alginate may be used as a powder (up to 5% total mass of the composite). Polysaccharide-triggered reduction of Ca²⁺ release reached the level of 50% during the first 2.5 h of incubation, then decreased significantly.

Conclusions

Both tested polysaccharides possess calcium-chelating properties. However, although alginate caused a reduction in Ca²⁺ levels in the media incubated with the gypsum samples, the reduction was too short lived to provide a long-term effect. Further modification of the composite content using calcium-deficient hydroxyapatite and low-molecular weight rhizobial EPS with higher solubility could bring more satisfactory results.     

Apoplastic calmodulin promotes self-incompatibility pollen tube growth by enhancing calcium influx and reactive oxygen species concentration in Pyrus pyrifolia

Key message

This study indicated that Ca ²⁺ , ROS and actin filaments were involved with CaM in regulating pollen tube growth and providing a potential way for overcoming pear self-incompatibility.

Abstract

Calmodulin (CaM) has been associated with various physiological and developmental processes in plants, including pollen tube growth. In this study, we showed that CaM regulated the pear pollen tube growth in a concentration-dependent bi-phasic response. Using a whole-cell patch-clamp configuration, we showed that apoplastic CaM induced a hyperpolarization-activated calcium ion (Ca²⁺) current, and anti-CaM largely inhibited this type of Ca²⁺ current. Moreover, upon anti-CaM treatment, the reactive oxygen species (ROS) concentration decreased and actin filaments depolymerized in the pollen tube. Interestingly, CaM could partially rescue the inhibition of self-incompatible pear pollen tube growth. This phenotype could be mediated by CaM-enhanced pollen plasma membrane Ca²⁺ current, tip-localized ROS concentration and stabilized actin filaments. These data indicated that Ca²⁺, ROS and actin filaments were involved with CaM in regulating pollen tube growth and provide a potential way for overcoming pear self-incompatibility.     

Xylem sap calcium concentrations do not explain liming-induced inhibition of legume gas exchange

Background and aims

Liming is considered normal agricultural practise for remediating soil acidity and improving crop productivity; however recommended lime applications can reduce yield. We tested the hypothesis that elevated xylem sap Ca²⁺ limited gas exchange of Phaseolus vulgaris L. and Pisum sativum L. plants that exhibited reduced shoot biomass and leaf area when limed.

Methods

We used Scholander and whole-plant pressure chamber techniques to collect root and leaf xylem sap, a calcium-specific ion-selective electrode to measure xylem sap Ca²⁺, infra-red gas analysis to measure gas exchange of limed and unlimed (control) plants, and a detached leaf transpiration bioassay to determine stomatal sensitivity to Ca²⁺.

Results

Liming reduced shoot biomass, leaf area and leaf gas exchange in both species. Root xylem sap Ca²⁺ concentration was only increased in P. vulgaris and not in P. sativum. Detached leaves of both species required 5 mM Ca²⁺ supplied to via the transpiration stream to induce stomatal closure, however, maximum in vivo xylem sap Ca²⁺ concentrations of limed plants was only 1.7 mM and thus not high enough to influence stomata.

Conclusion

We conclude that an alternative xylem-borne antitranspirant other than Ca²⁺ decreases gas exchange of limed plants.     

Programmed cell death of secretory cavity cells of citrus fruits is associated with Ca2+ accumulation in the nucleus

Key message

An increase in Ca ²⁺ concentration in the nucleus may activate the PCD of secretory cavity cells, and further Ca ²⁺ accumulation contributes to the regulation of nuclear DNA degradation.

Abstract

Calcium plays an important role in plant programmed cell death (PCD). Previously, we confirmed that PCD was involved in the degradation of secretory cavity cells in Citrus sinensis (L.) Osbeck fruits. To further explore the function of calcium in the PCD of secretory cavity cells, we used potassium pyroantimonate precipitation to detect and locate calcium dynamics. At the precursor cell stage of the secretory cavity, Ca²⁺ was only distributed in the cell walls. At the early stage of secretory cavity initial cells, Ca²⁺ in the cell walls was gradually transported into the cytoplasm via pinocytotic vesicles. Although a small amount of Ca²⁺ was present in the nucleus, the TUNEL signal was scarcely observed. At the middle stage of initial cells, a large number of pinocytotic vesicles were transferred to the nucleus, where the vesicle membrane fused with the nuclear membrane to release calcium into the nucleoplasm. In addition, abundant Ca²⁺ aggregated in the condensed chromatin and nucleolus, where the TUNEL signal appeared the strongest. At the late stage of initial cells, the chromatin and nucleolus gradually degraded and disappeared, and the nucleus appeared broken-like, as Ca²⁺ in the cell wall had nearly completely disappeared, and Ca²⁺ in the nucleus was also rapidly reduced. Furthermore, the TUNEL signal also disappeared. These phenomena indicated that an increase in Ca²⁺ concentration in the nucleus might activate the PCD of secretory cavity cells, and further Ca²⁺ accumulation contributed to the regulation of nuclear DNA degradation.     

Identification of novel DNA methylation inhibitors via a two-component reporter gene system

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

The plasma membrane-localised Ca2+-ATPase ACA8 plays a role in sucrose signalling involved in early seedling development in Arabidopsis

Key message

Arabidopsis Ca ²⁺ -ATPase ACA8 plays a role in sucrose signalling during early seedling development by integrating developmental signals with carbon source availability.

Abstract

Calcium (Ca²⁺) is an essential signal transduction element in eukaryotic organisms. Changes in the levels of intracellular Ca²⁺ affect multiple developmental processes in plants, including cell division, polar growth, and organogenesis. Here, we report that the plasma-membrane-localised Arabidopsis Ca²⁺-ATPase ACA8 plays a role in sucrose signalling during early seedling development. Disruption of the ACA8 gene elevated the expression of genes that encode transporters for Ca²⁺ efflux. The seedlings that carried a T-DNA insertion mutation in ACA8 experienced water stress during early development. This response was unrelated to inadequate osmoregulatory responses and was most likely caused by disruption of cell membrane integrity and severe ion leakage. In addition, aca8-1 seedlings displayed a significant decline in photosynthetic performance and arrested root growth after removal of sucrose from the growth medium. The two phenomena resulted from impaired photosynthesis, reduced cell proliferation in the root meristem and the sucrose control of cell-cycle events. All of the stress-response phenotypes were rescued when expression of ACA8 was restored in aca8-1 mutant. Taken together, our results indicate that ACA8-mediated Ca²⁺ signalling contributes to modulate early seedling development and coordinates root development with nutrient availability.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K⁺ and Ca²⁺ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K⁺ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K⁺ current, but also induces a very large noninactivating K⁺ current. At 2 or 3 μm, AA enhances the T-type Ca²⁺ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca²⁺ current. Thus, the effect of AA on Ca²⁺ entry through voltage-gated Ca²⁺ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca²⁺ entry through T channels, both by lowering the voltage-gated K⁺ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels, both by inducing a K⁺ conductance and by blocking Ca²⁺ channels.     

Selenium Modulates Oxidative Stress-Induced Cell Apoptosis in Human Myeloid HL-60 Cells Through Regulation of Calcium Release and Caspase-3 and -9 Activities

Selenium is an essential chemopreventive antioxidant element to oxidative stress, although high concentrations of selenium induce toxic and oxidative effects on the human body. However, the mechanisms behind these effects remain elusive. We investigated toxic effects of different selenium concentrations in human promyelocytic leukemia HL-60 cells by evaluating Ca²⁺ mobilization, cell viability and caspase-3 and -9 activities at different sample times. We found the toxic concentration and toxic time of H₂O₂ as 100 μm and 10 h on cell viability in the cells using four different concentrations of H₂O₂ (1 μm–1 mm) and six different incubation times (30 min, 1, 2, 5, 10, 24 h). Then, we found the therapeutic concentration of selenium to be 200 nm by cells incubated in eight different concentrations of selenium (10 nm–1 mm) for 1 h. We measured Ca²⁺ release, cell viability and caspase-3 and -9 activities in cells incubated with high and low selenium concentrations at 30 min and 1, 2, 5, 10 and 24 h. Selenium (200 nm) elicited mild endoplasmic reticulum stress and mediated cell survival by modulating Ca²⁺ release, the caspases and cell apoptosis, whereas selenium concentrations as high as 1 mm induced severe endoplasmic reticulum stress and caused cell death by activating modulating Ca²⁺ release, the caspases and cell apoptosis. In conclusion, these results explained the molecular mechanisms of the chemoprotective effect of different concentrations of selenium on oxidative stress-induced apoptosis.     

Protocol for the BAG-RECALL clinical trial: a prospective, multi-center, randomized, controlled trial to determine whether a bispectral index-guided protocol is superior to an anesthesia gas-guided protocol in reducing intraoperative awareness with explicit recall in high risk surgical patients

Background

Sevoflurane has been demonstrated to vasodilate the foeto-placental vasculature. We aimed to determine the contribution of modulation of potassium and calcium channel function to the vasodilatory effect of sevoflurane in isolated human chorionic plate arterial rings.

Methods

Quadruplicate ex vivo human chorionic plate arterial rings were used in all studies. Series 1 and 2 examined the role of the K⁺ channel in sevoflurane-mediated vasodilation. Separate experiments examined whether tetraethylammonium, which blocks large conductance calcium activated K⁺ (K_Ca++) channels (Series 1A+B) or glibenclamide, which blocks the ATP sensitive K⁺ (K_ATP) channel (Series 2), modulated sevoflurane-mediated vasodilation. Series 3 – 5 examined the role of the Ca⁺⁺ channel in sevoflurane induced vasodilation. Separate experiments examined whether verapamil, which blocks the sarcolemmal voltage-operated Ca⁺⁺ channel (Series 3), SK&F 96365 an inhibitor of sarcolemmal voltage-independent Ca⁺⁺ channels (Series 4A+B), or ryanodine an inhibitor of the sarcoplasmic reticulum Ca⁺⁺ channel (Series 5A+B), modulated sevoflurane-mediated vasodilation.

Results

Sevoflurane produced dose dependent vasodilatation of chorionic plate arterial rings in all studies. Prior blockade of the K_Ca++ and K_ATP channels augmented the vasodilator effects of sevoflurane. Furthermore, exposure of rings to sevoflurane in advance of TEA occluded the effects of TEA. Taken together, these findings suggest that sevoflurane blocks K⁺ channels. Blockade of the voltage-operated Ca⁺⁺channels inhibited the vasodilator effects of sevoflurane. In contrast, blockade of the voltage-independent and sarcoplasmic reticulum Ca⁺⁺channels did not alter sevoflurane vasodilation.

Conclusion

Sevoflurane appears to block chorionic arterial K_Ca++ and K_ATP channels. Sevoflurane also blocks voltage-operated calcium channels, and exerts a net vasodilatory effect in the in vitro foeto-placental circulation.     

Differential superoxide dismutase expression in ryegrass cultivars in response to short term aluminium stress

Background and aims

Saline soils limit plant production worldwide through osmotic stress, specific-ion toxicities, and nutritional imbalances.

Methods

The ability of Ca²⁺ and K⁺ to alleviate toxicities of Na⁺ and Mg²⁺ was examined using 89 treatments in short-term (48 h) solution culture studies for cowpea (Vigna unguiculata (L.) Walp.) roots. Root elongation was related to ionic activities at the outer surface of the root plasma membrane.

Results

The addition of K⁺ was found to alleviate the toxic effects of Na⁺, and supplemental Ca²⁺ improved growth further in these partially-alleviated solutions where K⁺ was present. Therefore, Na⁺ appears to interfere with K⁺ metabolism, and Ca²⁺ reduces this interference. Interestingly, the ability of Ca²⁺ to improve K-alleviation of Na⁺ toxicity is non-specific, with Mg²⁺ having a similar effect. In contrast, the addition of Ca²⁺ to Na-toxic solutions in the absence of K⁺ did not improve growth, suggesting that Ca²⁺ does not directly reduce Na⁺ toxicity in these short-term studies (for example, by reducing Na⁺ uptake) when supplied at non-deficient levels. Finally, K⁺ did not alleviate Mg²⁺ toxicity, suggesting that Mg²⁺ is toxic by a different mechanism to Na⁺.

Conclusions

Examination of how the toxic effects of salinity are alleviated provides clues as to the underlying mechanisms by which growth is reduced.     

Root-induced processes controlling phosphate availability in soils with contrasted P-fertilized treatments

Aims

In this study we identified the nature of the root-induced chemical processes controlling changes in phosphate (P) availability in a soil with two P loadings resulting from long-term fertilization treatments.

Methods

We used a set of mechanistic adsorption models (surface complexation and ion exchange) within the framework of the component additive approach to simulate the effect of durum wheat roots on P availability. We had to consider the influence of adsorption of other ions to ensure the goodness-of-fit of the simulations.

Results

We found that Ca²⁺ uptake, in addition to P uptake and root-induced alkalization, controlled P availability in the rhizosphere regardless of the fertilization level. The relative influence of these three processes depends primarily on the extractant used to estimate P availability. Calcium uptake was the most significant process in water extracts, whereas P uptake was the dominant root-induced chemical process in CaCl₂ extracts. Under low Ca concentrations, Ca²⁺ uptake decreased the promoting influence of Ca²⁺ adsorption on P adsorption.

Conclusions

In addition to confirming the validity of our approach to model P availability, the present investigation indicated that root-induced processes markedly affect P availability irrespective of the fertilization level.     

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background

Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44^mapk) and ERK2 (p42^mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca²⁺ and Ca²⁺-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F_2α.

Results

Pretreatment of the cells with the Ca²⁺ chelators BAPTA-AM or EGTA, as well as the Ca²⁺ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca²⁺/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF_2α.

Conclusion

The present data indicate that Ca²⁺ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.     

Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons

Background

Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP₃-gated thapsigargin sensitive intracellular calcium (Ca²⁺) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn²⁺) that has been reported to play a role in cell signaling.

Results

In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn²⁺ indicators, we showed that intracellular regions of Zn²⁺ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn²⁺ chelator TPEN, indicating that the local Zn²⁺ fluorescence represented free Zn²⁺ localized to the ER in the basal condition. Blockade of the ER Ca²⁺ pump by thapsigargin produced a steady increase of intracellular Zn²⁺. Furthermore, we determined that the thapsigargin-induced Zn²⁺ increase was not dependent on extracellular Ca²⁺ or extracellular Zn²⁺, suggesting that it was of intracellular origin. The applications of caged IP₃ or IP₃-3Kinase inhibitor (to increase available IP₃) produced a significant increase in intracellular Zn²⁺.

Conclusions

Taken together, these results suggest that Zn²⁺ is sequestered into thapsigargin/IP₃-sensitive stores and is released upon agonist stimulation.     

Pepper mildew resistance locus O interacts with pepper calmodulin and suppresses Xanthomonas AvrBsT-triggered cell death and defense responses

Main conclusion

Pepper CaMLO2 specifically interacts with CaCaM1 and translocates cytoplasmic CaCaM1 to the plasma membrane, leading to the suppression of Xanthomonas AvrBsT-triggered Ca ²⁺ influx, hypersensitive cell death and defense responses.

Abstract

Pathogen-induced cell death is closely linked with disease susceptibility and resistance in plants. Pepper (Capsicum annuum) mildew resistance locus O (CaMLO2) and calmodulin (CaCaM1) genes are required for disease-associated cell death and hypersensitive cell death, respectively. Here, we demonstrate that pathogen-responsive CaMLO2 interacts with CaCaM1 in yeast and in planta. Bimolecular fluorescence complementation and co-immunoprecipitation analyses confirm a specific interaction between CaMLO2 and CaCaM1 at the plasma membrane (PM) in plant cells. Subcellular localization analyses of CaCaM1 fused to green fluorescent protein reveals that treatment with Ca²⁺ and co-expression with CaMLO2 induce translocation of cytosolic CaCaM1 to the PM where CaMLO2 is localized. Transient CaMLO2 expression negatively regulates CaCaM1 accumulation in Nicotiana benthamiana. Xanthomonas avrBsT-triggered Ca²⁺ influx and hypersensitive cell death are disrupted by CaCaM1 and/or CaMLO2 expression. CaMLO2 silencing in pepper significantly enhances reactive oxygen species burst, cell death, and resistance responses to Xanthomonas campestris pv. vesicatoria Ds1 and Ds1 (avrBsT), which is accompanied by enhanced induction of CaCaM1, CaPR1 (PR-1), and CaPO2 (peroxidase). These results suggest that CaMLO2 interacts with CaCaM1 and suppresses AvrBsT-triggered cell death and defense responses.     

Alteration in mitochondrial Ca<Superscript>2+</Superscript> uptake disrupts insulin signaling in hypertrophic cardiomyocytes

Background

Cardiac hypertrophy is characterized by alterations in both cardiac bioenergetics and insulin sensitivity. Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca²⁺ release from the endoplasmic reticulum, however, how this translates to changes in mitochondrial metabolism in either healthy or hypertrophic cardiomyocytes is not fully understood.

Results

In the present study we investigated insulin-dependent mitochondrial Ca²⁺ signaling in normal and norepinephrine or insulin like growth factor-1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca²⁺-fluorescent probes we showed that insulin increases mitochondrial Ca²⁺ levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca²⁺ uniporter, as well as by siRNA-dependent mitochondrial Ca²⁺ uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor-1-stimulated cells. This resulted in a reduction in mitochondrial Ca²⁺ uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca²⁺ uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling.

Conclusions

Mitochondrial Ca²⁺ uptake is a key event in insulin signaling and metabolism in cardiomyocytes.

     

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

Background

Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP₃/Ca²⁺ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca²⁺ signaling and InsP₃-induced Ca²⁺ release from the endoplasmic reticulum (ER).

Results

Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca²⁺]_i changes into a sustained increase. Intraluminal Ca²⁺ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP₃-induced Ca²⁺ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP₃ receptor Ca²⁺ channel for InsP₃. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP₃-induced Ca²⁺ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP₃/Ca²⁺ signaling pathway for 5-HT, because IBMX potentiated Ca²⁺-dependent Cl^- transport activated by a threshold 5-HT concentration.

Conclusion

This report shows, for the first time for an insect system, that cAMP can potentiate InsP₃-induced Ca²⁺ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP₃/Ca²⁺ signaling pathways enhances transepithelial electrolyte transport.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.