Phosphate Ion Targeted Colorimetric and Fluorescent Probe and Its Use to Monitor Endogeneous Phosphate Ion in a Hemichannel-Closed Cell

Abstract

Fluorescent probe 1, the first inorganic phosphate (Pi) targeted colorimetric and fluorescent probe to detect endogenous Pi in hemichannel-closed cells, has been developed. Probe 1 undergoes a unique Pi induced hydrolytic reaction in DMSO-HEPES (V/V = 9:1) buffered (0.02 M, pH 7.4) solutions that produces a colorimetric change associated with a 62 nm red-shift in the UV-vis absorption maximum and up to a 780-fold enhancement in the fluorescence intensity. The mechanistic proposal that these spectroscopic changes are associated with reaction Pi with 1 to form coumarin gains support from the results of theoretical calculations and mass spectrometry studies. Observations made in fluorescence imaging studies with HeLa cells and C. elegans show that 1 can be employed to monitor Pi production in vivo caused by apyrase-catalyzed ATP hydrolysis. Moreover, probe 1 was utilized to show that apoptosis of hemichannel-closed Sf9 cells is caused by Inx3 promoted dephosphorylation of Akt (RAC serine/threonine-protein kinase), leading to an elevation of the concentration of Pi. Overall, the study has produced the first fluorescent sensor 1 for endogenous inorganic phosphate. Moreover, the utility of 1 for measuring Pi release in vitro has been demonstrated and utilized to elucidate the mechanism of Inx3 action in hemichannel-closed Sf9 cells.

Black Phosphorus: Bioactive Nanomaterials with Inherent and Selective Chemotherapeutic Effects

“…As shown in Figure a and the Supporting Information, Figure S2, significantly enhanced cellular uptake and accelerated intracellular degradation were observed in the cancerous A549 and MCF‐7 cells compared to normal hBMSCs cells. At the same time, according to the phosphate anion probe, acute elevation of the intracellular phosphate concentration was observed in the cancer cells due to degradation of BPs, but such an increase was not detected in normal hBMSCs cells (Figure b). Faster biodegradation of BPs and more acute elevation of intracellular phosphate anions were observed in MCF‐7 cells than A549 cells.…”

TWO INNEXINS OF Spodoptera litura INFLUENCES HEMICHANNEL AND GAP JUNCTION FUNCTIONS IN CELLULAR IMMUNE RESPONSES

“…These two reports provide the initial evidence that steady‐state hemichannels are present on the hemocyte surface. According to our data, under immune challenge, polydnavirus‐mediated immunosuppression leads to down regulation of SpliInx2 and SpliInx3 expression during granulocyte apoptosis in S. litura hemocytes (Li et al., ), and recombinant baculovirus infection results in hemichannel‐closed Sf9 cells (Guo et al., ). These results suggested that steady‐state hemichannel could be disrupted during cellular immune responses and raise the question of what mechanisms controls steady‐state hemichannel formation?…”

Unexpected link between insect innexins and apoptosis of HeLa cells

“…Further, endogenous Inxs can be closed by overexpression of recombinant N‐terminally elongated Inxs using the insect Bac‐to‐Bac expression system and reduce baculovirus‐triggered apoptosis at an early stage (Chen et al., ). These reBac‐Nte‐Inxs‐infected cells with closed Inxs accumulate phosphate ions, which are known to stimulate apoptotic signaling and mediate the activation of caspase 3 dependent cell death via the loss of mitochondrial function (Adams, Mansfield, Perlot, & Shapiro, ; Guo et al., ; Zhang et al., ). Inxs are highly expressed in hemocytes and adipose tissue, which are the major tissues responsible for the innate immune response in the lepidopteran Spodoptera litura (Pang et al., ).…”