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Intracellular pH
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{{Technical|date=March 2020}} [[File:Membrane_pH_gradient.jpg|thumb|pH gradient across a membrane, with protons traveling through a transporter embedded in the membrane.|alt=|200x200px]] '''Intracellular pH''' ('''pH<sub>i</sub>''') is the measure of the [[acidity]] or [[basicity]] (i.e., [[pH]]) of [[intracellular fluid]]. The pH<sub>i</sub> plays a critical role in membrane transport and other intracellular processes. In an environment with the improper pH<sub>i</sub>, biological cells may have compromised function.<ref>{{cite journal |last1=Harguindey |first1=S |last2=Stanciu |first2=D |last3=Devesa |first3=J |last4=Alfarouk |first4=K |last5=Cardone |first5=RA |last6=Polo Orozco |first6=JD |last7=Devesa |first7=P |last8=Rauch |first8=C |last9=Orive |first9=G |last10=Anitua |first10=E |last11=Roger |first11=S |last12=Reshkin |first12=SJ |title=Cellular acidification as a new approach to cancer treatment and to the understanding and therapeutics of neurodegenerative diseases. |journal=Seminars in Cancer Biology |date=April 2017 |volume=43 |pages=157β179 |doi=10.1016/j.semcancer.2017.02.003 |pmid=28193528|url=http://eprints.nottingham.ac.uk/43674/ }}</ref><ref>{{cite journal | vauthors = Flinck M, Kramer SH, Pedersen SF | title = Roles of pH in control of cell proliferation | journal = Acta Physiologica | volume = 223 | issue = 3 | pages = e13068 | date = July 2018 | pmid = 29575508 | doi = 10.1111/apha.13068 | s2cid = 4874638 }}</ref> Therefore, pH<sub>i</sub> is closely regulated in order to ensure proper cellular function, controlled cell growth, and normal cellular processes.<ref name=":0" /> The mechanisms that regulate pH<sub>i</sub> are usually considered to be [[plasma membrane]] transporters of which two main types exist β those that are dependent and those that are independent of the concentration of [[bicarbonate]] ({{chem|HCO|3|-}}). Physiologically normal intracellular pH is most commonly between 7.0 and 7.4, though there is variability between tissues (e.g., mammalian skeletal muscle tends to have a pH<sub>i</sub> of 6.8β7.1).<ref>{{cite web | first = Kerry | last = Brandis | name-list-style = vanc | work = Acid-Base Physiology | title = 2.6 Regulation of Intracellular Hydrogen Ion Concentration | url = https://www.anaesthesiamcq.com/AcidBaseBook/ab2_6.php | publisher = Anaesthesia Education Website }}</ref><ref>{{cite journal | vauthors = Madshus IH | title = Regulation of intracellular pH in eukaryotic cells | journal = The Biochemical Journal | volume = 250 | issue = 1 | pages = 1β8 | date = February 1988 | pmid = 2965576 | pmc = 1148806 | doi = 10.1042/bj2500001 }}</ref> There is also pH variation across different [[organelle]]s, which can span from around 4.5 to 8.0.<ref name=":2">{{cite journal | vauthors = Asokan A, Cho MJ | title = Exploitation of intracellular pH gradients in the cellular delivery of macromolecules | journal = Journal of Pharmaceutical Sciences | volume = 91 | issue = 4 | pages = 903β13 | date = April 2002 | pmid = 11948528 | doi = 10.1002/jps.10095 }}</ref><ref>{{cite journal | vauthors = Proksch E | title = pH in nature, humans and skin | journal = The Journal of Dermatology | volume = 45 | issue = 9 | pages = 1044β1052 | date = September 2018 | pmid = 29863755 | doi = 10.1111/1346-8138.14489 | doi-access = free }}</ref> pH<sub>i</sub> can be measured in a number of different ways.<ref name=":0">{{cite journal | vauthors = Boron WF | title = Regulation of intracellular pH | journal = Advances in Physiology Education | volume = 28 | issue = 1β4 | pages = 160β79 | date = December 2004 | pmid = 15545345 | doi = 10.1152/advan.00045.2004 | s2cid = 13242391 }} </ref><ref>{{cite journal | vauthors = Demuth C, Varonier J, Jossen V, Eibl R, Eibl D | title = Novel probes for pH and dissolved oxygen measurements in cultivations from millilitre to benchtop scale | journal = Applied Microbiology and Biotechnology | volume = 100 | issue = 9 | pages = 3853β63 | date = May 2016 | pmid = 26995606 | doi = 10.1007/s00253-016-7412-0 | s2cid = 8434413 | hdl = 11475/2259 | hdl-access = free }}</ref> == Homeostasis == Intracellular pH is typically lower than [[extracellular]] pH due to lower concentrations of HCO<sub>3</sub><sup>β</sup>.<ref>{{cite journal |vauthors=Flinck M, Kramer SH, Pedersen SF |title=Roles of pH in control of cell proliferation |journal=Acta Physiol (Oxf) |volume=223 |issue=3 |pages=e13068 |date=July 2018 |pmid=29575508 |doi=10.1111/apha.13068 |s2cid=4874638 }} {{verify source |date=September 2019 |reason=This ref was deleted Special:Diff/901265489 by a bug in VisualEditor and later restored by a bot from the original cite located at Special:Permalink/901264743 cite #5 - verify the cite is accurate and delete this template. [[User:GreenC bot/Job 18]]}}</ref> A rise of extracellular (e.g., [[Serum (blood)|serum]]) [[partial pressure]] of [[carbon dioxide]] ([[PCO2|pCO<sub>2</sub>]]) above 45 [[mmHg]] leads to formation of [[carbonic acid]], which causes a decrease of pH<sub>i</sub> as it [[Dissociation (chemistry)|dissociates]]:<ref>Flinck M, Kramer SH, Pedersen SF (July 2018). "Roles of pH in control of cell proliferation". ''Acta Physiol (Oxf)''. '''223''' (3): e13068. [[Digital object identifier|doi]]:10.1111/apha.13068. [[PubMed Identifier|PMID]] 29575508.</ref> : H<sub>2</sub>O + CO<sub>2</sub> {{eqm}} H<sub>2</sub>CO<sub>3</sub> {{eqm}} H<sup>+</sup> + HCO<sub>3</sub><sup>β</sup> Since biological cells contain fluid that can act as a buffer, pH<sub>i</sub> can be maintained fairly well within a certain range.<ref>{{cite journal | vauthors = Slonczewski JL, Fujisawa M, Dopson M, Krulwich TA | title = Cytoplasmic pH measurement and homeostasis in bacteria and archaea | journal = Advances in Microbial Physiology | volume = 55 | pages = 1β79, 317 | date = 2009 | pmid = 19573695 | doi = 10.1016/S0065-2911(09)05501-5 | isbn = 9780123747907 }}</ref> Cells adjust their pH<sub>i</sub> accordingly upon an increase in acidity or basicity, usually with the help of CO<sub>2</sub> or HCO<sub>3</sub><sup>β</sup> sensors present in the membrane of the cell.<ref name=":0" /> These sensors can permit H+ to pass through the cell membrane accordingly, allowing for pH<sub>i</sub> to be interrelated with extracellular pH in this respect.<ref>{{cite journal | vauthors = Jensen FB | title = Red blood cell pH, the Bohr effect, and other oxygenation-linked phenomena in blood O2 and CO2 transport | journal = Acta Physiologica Scandinavica | volume = 182 | issue = 3 | pages = 215β27 | date = November 2004 | pmid = 15491402 | doi = 10.1111/j.1365-201X.2004.01361.x }}</ref> Major intracellular buffer systems include those involving proteins or phosphates. Since the proteins have acidic and basic regions, they can serve as both proton donors or acceptors in order to maintain a relatively stable intracellular pH. In the case of a phosphate buffer, substantial quantities of weak acid and conjugate weak base (H<sub>2</sub>PO<sub>4</sub><sup>β</sup> and HPO<sub>4</sub><sup>2β</sup>) can accept or donate protons accordingly in order to conserve intracellular pH:<ref>{{cite web | first = Michael J | last = Bookallil | name-list-style = vanc |url= http://www.anaesthesia.med.usyd.edu.au/resources/lectures/acidbase_mjb/control.html#co2control|title= Acid-Base: pH of the Blood - 3 - Control mechanisms | publisher = The University of Sydney Nuffield Department of Anaesthetics | work = Lectures and Study Notes Listing |access-date=2019-05-28}}</ref><ref>{{cite journal | vauthors = Burton RF | title = Intracellular buffering | journal = Respiration Physiology | volume = 33 | issue = 1 | pages = 51β8 | date = April 1978 | pmid = 27854 | doi = 10.1016/0034-5687(78)90083-X }}</ref> :OH<sup>β</sup> + H<sub>2</sub>PO<sub>4</sub><sup>β</sup> {{eqm}} H<sub>2</sub>O + HPO<sub>4</sub><sup>2β</sup> :H<sup>+</sup> + HPO<sub>4</sub><sup>2β</sup> {{eqm}} H<sub>2</sub>PO<sub>4</sub><sup>β</sup> == In organelles == [[File:PH_of_organelles.jpg|alt=|thumb|207x207px|Approximate pHs of various organelles within a cell.<ref name=":2" />]] The pH within a particular organelle is tailored for its specific function. For example, lysosomes have a relatively low pH of 4.5.<ref name=":2" /> Additionally, fluorescence microscopy techniques have indicated that phagosomes also have a relatively low internal pH.<ref name=":4">{{cite journal | vauthors = Nunes P, Guido D, Demaurex N | title = Measuring Phagosome pH by Ratiometric Fluorescence Microscopy | journal = Journal of Visualized Experiments | issue = 106 | pages = e53402 | date = December 2015 | pmid = 26710109 | pmc = 4692782| doi = 10.3791/53402 }}</ref> Since these are both degradative organelles that engulf and break down other substances, they require high internal acidity in order to successfully perform their intended function.<ref name=":4" /> In contrast to the relatively low pH inside lysosomes and phagosomes, the mitochondrial matrix has an internal pH of around 8.0, which is approximately 0.9 pH units higher than that of inside intermembrane space.<ref name=":2" /><ref>{{cite journal | vauthors = Porcelli AM, Ghelli A, Zanna C, Pinton P, Rizzuto R, Rugolo M | title = pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant | journal = Biochemical and Biophysical Research Communications | volume = 326 | issue = 4 | pages = 799β804 | date = January 2005 | pmid = 15607740 | doi = 10.1016/j.bbrc.2004.11.105 }}</ref> Since oxidative phosphorylation must occur inside the mitochondria, this pH discrepancy is necessary to create a gradient across the membrane. This membrane potential is ultimately what allows for the mitochondria to generate large quantities of ATP.<ref>{{cite book | vauthors = Alberts B, Johnson A, Lewis J, etal | title = Molecular Biology of the Cell | edition = 4th | location = New York | publisher = Garland Science | date = 2002 | chapter = The Mitochondrion | chapter-url = https://www.ncbi.nlm.nih.gov/books/NBK26894/ }}</ref> [[File:Mitochondria Intermembrane pH.jpg|thumb|Protons being pumped from the mitochondrial matrix into the intermembrane space as the electron transport chain runs, lowering the pH of the intermembrane space.]] == Measurement == There are several common ways in which intracellular pH (pH<sub>i</sub>) can be measured including with a microelectrode, dye that is sensitive to pH, or with nuclear magnetic resonance techniques.<ref name=":3">{{cite journal | vauthors = Roos A, Boron WF | title = Intracellular pH | journal = Physiological Reviews | volume = 61 | issue = 2 | pages = 296β434 | date = April 1981 | pmid = 7012859 | doi = 10.1152/physrev.1981.61.2.296 }}</ref><ref name=":1" /> For measuring pH inside of organelles, a technique utilizing pH-sensitive green fluorescent proteins (GFPs) may be used.<ref name=":5">{{cite journal | vauthors = Roberts TM, Rudolf F, Meyer A, Pellaux R, Whitehead E, Panke S, Held M | title = Corrigendum: Identification and Characterisation of a pH-stable GFP | journal = Scientific Reports | volume = 8 | pages = 46976 | date = May 2018 | pmid = 29769631 | pmc = 5956236 | doi = 10.1038/srep46976 | bibcode = 2018NatSR...846976R }}</ref> Overall, all three methods have their own advantages and disadvantages. Using dyes is perhaps the easiest and fairly precise, while NMR presents the challenge of being relatively less precise.<ref name=":3" /> Furthermore, using a microelectrode may be challenging in situations where the cells are too small, or the intactness of the cell membrane should remain undisturbed.<ref name=":1" /> GFPs are unique in that they provide a noninvasive way of determining pH inside different organelles, yet this method is not the most quantitatively precise way of determining pH.<ref name=":6" /> === Microelectrode === The microelectrode method for measuring pH<sub>i</sub> consists of placing a very small electrode into the cellβs cytosol by making a very small hole in the plasma membrane of the cell.<ref name=":1" /> Since the microelectrode has fluid with a high H+ concentration inside, relative to the outside of the electrode, there is a potential created due to the pH discrepancy between the inside and outside of the electrode.<ref name=":3" /><ref name=":1" /> From this voltage difference, and a predetermined pH for the fluid inside the electrode, one can determine the intracellular pH (pH<sub>i</sub>) of the cell of interest.<ref name=":1">{{cite book | vauthors = Loiselle FB, Casey JR | title = Membrane Transporters in Drug Discovery and Development | chapter = Measurement of Intracellular pH | volume = 637 | pages = 311β31 | date = 2010 | pmid = 20419443 | doi = 10.1007/978-1-60761-700-6_17 | isbn = 978-1-60761-699-3 | series = Methods in Molecular Biology }} </ref> === Fluorescence spectroscopy === Another way to measure Intracellular pH (pH<sub>i</sub>) is with dyes that are sensitive to pH, and fluoresce differently at various pH values.<ref name=":4" /><ref>{{cite journal | vauthors = Specht EA, Braselmann E, Palmer AE | title = A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging | journal = Annual Review of Physiology | volume = 79 | pages = 93β117 | date = February 2017 | pmid = 27860833 | doi = 10.1146/annurev-physiol-022516-034055 | pmc = 12034319 }}</ref> This technique, which makes use of fluorescence spectroscopy, consists of adding this special dye to the cytosol of a cell.<ref name=":3" /><ref name=":1" /> By exciting the dye in the cell with energy from light, and measuring the wavelength of light released by the photon as it returns to its native energy state, one can determine the type of dye present, and relate that to the intracellular pH of the given cell.<ref name=":3" /><ref name=":1" /> === Nuclear magnetic resonance === In addition to using pH-sensitive electrodes and dyes to measure pH<sub>i</sub>, Nuclear Magnetic Resonance (NMR) spectroscopy can also be used to quantify pH<sub>i</sub>.<ref name=":1" /> NMR, typically speaking, reveals information about the inside of a cell by placing the cell in an environment with a potent magnetic field.<ref name=":3" /><ref name=":1" /> Based on the ratio between the concentrations of protonated, compared to deprotonated, forms of phosphate compounds in a given cell, the internal pH of the cell can be determined.<ref name=":3" /> Additionally, NMR may also be used to reveal the presence of intracellular sodium, which can also provide information about the pH<sub>i</sub>.<ref>{{cite journal | vauthors = Eliav U, Navon G | title = Sodium NMR/MRI for anisotropic systems | journal = NMR in Biomedicine | volume = 29 | issue = 2 | pages = 144β52 | date = February 2016 | pmid = 26105084 | doi = 10.1002/nbm.3331 | s2cid = 29964258 }}</ref> Using NMR Spectroscopy, it has been determined that [[lymphocyte]]s maintain a constant internal pH of 7.17Β± 0.06, though, like all cells, the intracellular pH changes in the same direction as extracellular pH.<ref>{{cite journal | vauthors = Deutsch C, Taylor JS, Wilson DF | title = Regulation of intracellular pH by human peripheral blood lymphocytes as measured by 19F NMR | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 79 | issue = 24 | pages = 7944β8 | date = December 1982 | pmid = 6961462 | pmc = 347466 | doi = 10.1073/pnas.79.24.7944 | bibcode = 1982PNAS...79.7944D | doi-access = free }}</ref> === pH-sensitive GFPs === To determine the pH inside organelles, pH-sensitive GFPs are often used as part of a noninvasive and effective technique.<ref name=":5" /> By using cDNA as a template along with the appropriate primers, the GFP gene can be expressed in the cytosol, and the proteins produced can target specific regions within the cell, such as the mitochondria, golgi apparatus, cytoplasm, and endoplasmic reticulum.<ref name=":6">{{cite journal | vauthors = Kneen M, Farinas J, Li Y, Verkman AS | title = Green fluorescent protein as a noninvasive intracellular pH indicator | journal = Biophysical Journal | volume = 74 | issue = 3 | pages = 1591β9 | date = March 1998 | pmid = 9512054 | pmc = 1299504 | doi = 10.1016/S0006-3495(98)77870-1 | bibcode = 1998BpJ....74.1591K }}</ref> If certain GFP mutants that are highly sensitive to pH in intracellular environments are used in these experiments, the relative amount of resulting fluorescence can reveal the approximate surrounding pH.<ref name=":6" /><ref>{{cite journal | vauthors = Rizzuto R, Brini M, Pizzo P, Murgia M, Pozzan T | title = Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells | journal = Current Biology | volume = 5 | issue = 6 | pages = 635β42 | date = June 1995 | pmid = 7552174 | doi = 10.1016/s0960-9822(95)00128-x | s2cid = 13970185 | doi-access = free | bibcode = 1995CBio....5..635R }}</ref> == References == {{Reflist}} [[Category:Cell biology]]
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