Kraut Jeffrey A, Cheetham-Wilkinson Izaak J, Swan Laura E, Stagi Massimiliano, Kurtz Ira
Medical and Research Services VHAGLA Healthcare System, UCLA Membrane Biology Laboratory, and Division of Nephrology VHAGLA Healthcare System and David Geffen School of Medicine Los Angeles California USA.
Department of Cellular and Molecular Physiology, Institute of Systems, Molecular and Integrative Biology University of Liverpool Liverpool UK.
FASEB Bioadv. 2023 Mar 15;5(4):149-155. doi: 10.1096/fba.2022-00062. eCollection 2023 Apr.
Acidification of the cellular lysosome is an important factor in infection of mammalian cells by SARS-CoV-2. Therefore, raising the pH of the lysosome would theoretically be beneficial in prevention or treatment of SARS-CoV-2 infection. Sodium bicarbonate, carbicarb, and THAM are buffers that can be used clinically to provide base to patients. To examine whether these bases could raise lysosomal pH and therefore be a primary or adjunctive treatment of SARS-CoV-2 infection, we measured lysosomal and intracellular pH of mammalian cells after exposure to each of these bases. Mammalian HEK293 cells expressing RpH-LAMP1-3xFLAG, a ratiometric sensor of lysosomal luminal pH, were first exposed to Hepes which was then switched to sodium bicarbonate, carbicarb, or THAM and lysosomal pH measured. In bicarbonate buffer the mean lysosomal pH was 4.3 ± 0.1 ( 20); NS versus Hepes ( 20). The mean lysosomal pH in bicarbonate/carbonate was 4.3 ± 0.1 ( 21) versus Hepes ( 21), NS. In THAM buffer the mean lysosomal pH was 4.7 ± 0.07 ( 20) versus Hepes (4.6 ± 0.1, 20), NS. In addition, there was no statistical difference between pH in bicarbonate, carbicarb or THAM solutions. Using the membrane permeable base NHCl (5 mM), lysosomal pH increased significantly to 5.9 ± 0.1 ( 21) compared to Hepes (4.5 ± 0.07, 21); < 0.0001. Similarly, exposure to 1 mM hydroxychloroquine significantly increased the lysosomal pH to (5.9 ± 0.06, 20) versus Hepes (4.3 ± 0.1, 20), < 0.0001. Separately steady-state pHi was measured in HEK293 cells bathed in various buffers. In bicarbonate pH was 7.29 ± 0.02 ( 12) versus Hepes (7.45 ± 0.03, [ 12]), < 0.001. In cells bathed in carbicarb pH was 7.27 ± 0.02 ( 5) versus Hepes (7.43 ± 0.04, [ 5]), < 0.01. Cells bathed in THAM had a pH of 7.25 ± 0.03 ( 12) versus Hepes (7.44 ± 0.03 [ 12]), < 0.001. In addition, there was no statistical difference in pH in bicarbonate, carbicarb or THAM solutions. The results of these studies indicate that none of the buffers designed to provide base to patients alters lysosomal pH at the concentrations used in this study and therefore would be predicted to be of no value in the treatment of SARS-CoV-2 infection. If the goal is to raise lysosomal pH to decrease the infectivity of SARS-CoV-2, utilizing lysosomal permeable buffers at the appropriate dose that is non-toxic appears to be a useful approach to explore.
细胞溶酶体的酸化是严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染哺乳动物细胞的一个重要因素。因此，从理论上讲，提高溶酶体的pH值将有利于预防或治疗SARS-CoV-2感染。碳酸氢钠、碳酸氢碳酸钠缓冲液和三羟甲基氨基甲烷（THAM）是临床上可用于为患者提供碱的缓冲剂。为了研究这些碱是否能提高溶酶体pH值，从而成为SARS-CoV-2感染的主要或辅助治疗方法，我们在将这些碱分别作用于哺乳动物细胞后，测量了其溶酶体和细胞内的pH值。首先将表达溶酶体腔pH值的比率传感器RpH-LAMP1-3xFLAG的哺乳动物人胚肾293（HEK293）细胞暴露于羟乙基哌嗪乙磺酸（Hepes）中，然后换成碳酸氢钠、碳酸氢碳酸钠缓冲液或THAM，并测量溶酶体pH值。在碳酸氢盐缓冲液中，溶酶体平均pH值为4.3±0.1（n = 20）；与Hepes相比无统计学差异（n = 20）。在碳酸氢盐/碳酸盐缓冲液中，溶酶体平均pH值为4.3±0.1（n = 21），与Hepes相比（n = 21），无统计学差异。在THAM缓冲液中，溶酶体平均pH值为4.7±0.07（n = 20），与Hepes（4.6±0.1，n = 20）相比，无统计学差异。此外，碳酸氢钠、碳酸氢碳酸钠缓冲液或THAM溶液中的pH值之间无统计学差异。使用可透过膜的碱氯化铵（5 mM），与Hepes（4.5±0.07，n = 21）相比，溶酶体pH值显著升高至5.9±0.1（n = 21）；P<0.0001。同样，暴露于1 mM羟氯喹啉可使溶酶体pH值显著升高至（5.9±0.06，n = 20），而Hepes组为（4.3±0.1，n = 20），P<0.0001。另外，在不同缓冲液中培养的HEK293细胞中测量了稳态细胞内pH值（pHi）。在碳酸氢盐缓冲液中，pH值为7.29±0.02（n = 12），而Hepes组为（7.45±0.03，[n = 12]），P<0.001。在碳酸氢碳酸钠缓冲液中培养的细胞，pH值为7.27±0.02（n = 5），而Hepes组为（7.43±0.04，[n = 5]），P<0.01。在THAM缓冲液中培养的细胞pH值为7.25±0.03（n = 12），而Hepes组为（7.44±0.03 [n = 12]），P<0.001。此外，碳酸氢钠、碳酸氢碳酸钠缓冲液或THAM溶液中的pH值无统计学差异。这些研究结果表明，在本研究中使用的浓度下，设计用于为患者提供碱的缓冲剂均未改变溶酶体pH值，因此预计对SARS-CoV-2感染的治疗没有价值。如果目标是提高溶酶体pH值以降低SARS-CoV-2的感染性，那么使用适当剂量的无毒的可透过溶酶体的缓冲剂似乎是一种值得探索的有用方法。
