Abstract
Intracellular pH (pHi) and Ca2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca2+. The sources of the Ca2+ increase are from the endoplasmic reticulum (ER) Ca2+ pools as well as from Ca2+ influx. The store-mobilization component of the Ca2+ increase induced by the pHi rise was not sensitive to antagonists for either IP3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), leading to depletion of the ER Ca2+ store. We further showed that the physiological consequence of depletion of the ER Ca2+ store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca2+ leak from ER pools followed by Ca2+ influx via SOCs.
Highlights
The activity of virtually all proteins and macromolecules can be modulated by protons; intracellular pH is rigorously regulated for survival [1,2,3]
In the process of synthesizing cell permeant analogs of the Ca2+ releasing messangers IP3, nicotinic acid adenine dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR), we found the hydrobromide salt of diisopropylethyl amine (DIEA.HBr), an organic base commonly used in the organic chemistry, can induce cytosolic Ca2+ increases, similar to that of NH4Cl, in a dose dependent manner, whereas sodium acetate, a weak acid, failed to change Ca2+ (Figure 1A)
Previous studies on intracellular alkalinization induced Ca2+ release from intracellular stores suggested that the endoplasmic reticulum (ER) Ca2+ pools are the main target, yet involvement of IP3 was a topic of debate [19,23,24]
Summary
The activity of virtually all proteins and macromolecules can be modulated by protons; intracellular pH (pHi) is rigorously regulated for survival [1,2,3]. Subtle and transient pHi changes occur under many physiological conditions. Activity-dependent membrane depolarization elevates pHi in astrocytes of rat cortex [4]. Likewise, both capacitation of spermatozoa [5] and fertilization of eggs [6], induce intracellular alkalinization. Cells passively stabilize pHi by the buffering capacity of a variety of intracellular weak acids and bases, especially HCO32, generated by CO2 hydration and subsequent deprotonation of carbonic acid. These intrinsic buffering systems can be overpowered during continued extra- and intracellular stress or stimulation. Cells have evolved a complicated proton transporting system to regulate cytosolic pH as well as the pH in other cellular compartments [1]
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