Fisetin-Mediated Perturbations of Membrane Permeability and Intracellular pH in Candida albicans

Younhee Kim The Korean Society for Microbiology and Biotechnol 2024 Journal of microbiology and biotechnology Vol.34 No.4

The antifungal activity of fisetin against Candida albicans is explored, elucidating a mechanism centered on membrane permeabilization and ensuing disruption of pH homeostasis. The Minimum Inhibitory Concentration (MIC) of fisetin, indicative of its interaction with the fungal membrane, increases in the presence of ergosterol. Hoechst 33342 and propidium-iodide staining reveal substantial propidium-iodide accumulation in fisetin-treated C. albicans cells at their MIC, with crystal violet uptake assays confirming fisetin-induced membrane permeabilization. Leakage analysis demonstrates a significant release of DNA and proteins in fisetin-treated cells compared to controls, underscoring the antifungal effect through membrane disruption. Green fluorescence, evident in both the cytoplasm and vacuoles of fisetin-treated cells under BCECF, AM staining, stands in contrast to controls where only acidic vacuoles exhibit staining. Ratiometric pH measurements using BCECF, AM reveal a noteworthy reduction in intracellular pH in fisetin-treated cells, emphasizing its impact on pH homeostasis. DiBAC4(3) uptake assays demonstrate membrane hyperpolarization in fisetintreated cells, suggesting potential disruptions in ion flux and cellular homeostasis. These results provide comprehensive insights into the antifungal mechanisms of fisetin, positioning it as a promising therapeutic agent against Candida infections.

Na+/H+ exchanger와 HCO-₃Transporter에 의한 흰쥐 타액선 선세포내 pH 조절

서정택,박동범,손흥규,이종갑 大韓小兒齒科學會 1998 大韓小兒齒科學會誌 Vol.25 No.2

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the activity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is Na+/H+ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of H+ extrusion through Na+/H+ exchanger before and during muscarinic stimulation and to investigate the possible existence of HCO-₃ transporter which is responsible for the continuous supply of HCO-₃ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7' -bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluormeter. 1. By switching the perfusate from HCO-₃-free to HCO-₃-buffered solution, pHi decreased by 0.39±0.02 pH units followed by a slow increase at an initial rate of 0.04±0.007 pH units/min. the rate of pHi increase was reduced to 0.01±0.002 pH units/min by the simultaneous addition of 1 mM amilorede and 100μM DIDS. 2. An addition and removal of NH+₄ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in HCO-₃-free perfusate which implied that the pHi increase was entired dependent on the activation of Na+/H+ exchanger in HCO-₃=free condition. 3. An addition of 10μM carbachol increased the initial rate of pHi recovery from 0.16±0.01 pH units/min to 0.28±0.03pH units/min. 4. The initial rate of pHi decrease induced by 1mM amilorede was also increased by the exposure of the acinar cells 10μM carbachol(0.06±0.008pH unit/min) compared with that obtained before carbachol sitmulation (0.03±0.004pH unit/min). 5. The intracellular buffering capacity β1 was 14.31±1.82 at pHi 7.2-7.4 and β1 increased as pHi decreased. 6. The rate of H+ extrusion through Na+/H+ exchanger was greatly enhanced by the stimulation of the cells with 10μM carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of HCO-₃ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-ford increased in Na+/H+ exchanger by the stimulation of the acinar cells with 10μM carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported HCO-₃ into the cells.

$Na^+/H^+$ exchanger와 $HCO_3^-$ transporter에 의한 흰쥐 타액선 선세포내 pH 조절

박동범,서정택,손흥규,이종갑,Park, Dong-Bum,Seo, Jeong-Taeg,Sohn, Heung-Kyu,Lee, Jong-Gap 대한소아치과학회 1998 大韓小兒齒科學會誌 Vol.25 No.2

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is $Na^+/H^+$ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of $H^+$ extrusion through $Na^+/H^+$ exchanger before and during muscarinic stimulation and to investigate the possible existence of $HCO_3^-$ transporter which is responsible for the continuous supply of $HCO_3^-$ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from $HCO_3^-$-free to $HCO_3^-$-buffered solution, pHi decreased by $0.39{\pm}0.02$ pH units followed by a slow increase at an initial rate of $0.04{\pm}0.007$ pH units/min. The rate of pHi increase was reduced to $0.01{\pm}0.002$ pH units/min by the simultaneous addition of 1 mM amiloride and $100{\mu}M$ DIDS. 2. An addition and removal of $NH_4^+$ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in $HCO_3^-$-free perfusate which implied that the pHi increase was entired dependent on the activation of $Na^+/H^+$ exchanger in $HCO_3^-$-free condition. 3. An addition of $10{\mu}M$ carbachol increased the initial rate of pHi recovery from $0.16{\pm}0.01$ pH units/min to $0.28{\pm}0.03pH$ units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to $10{\mu}M$ carbachol ($0.06{\pm}0.008pH$ unit/min) compared with that obtained before carbachol stimulation ($0.03{\pm}0.004pH$ unit/min). 5. The intracellular buffering capacity ${\beta}1$ was $14.31{\pm}1.82$ at pHi 7.2-7.4 and ${\beta}1$ increased as pHi decreased. 6. The rate of $H^+$ extrusion through $Na^+/H^+$ exchanger was greatly enhanced by the stimulation of the cells with $10{\mu}M$ carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of $HCO_3^-$ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in $Na^+/H^+$ exchanger by the stimulation of the acinar cells with $10{\mu}M$ carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported $HCO_3^-$ into the cells.