The solution to bicarbonate

Letter to the EditorThe solution to bicarbonateEbbe Boedtkjer and Christian AalkjaerEbbe BoedtkjerDepartment of Biomedicine, Aarhus University, Aarhus, Denmark and Christian AalkjaerDepartment of Biomedicine, Aarhus University, Aarhus, DenmarkDepartment of Biomedical Sciences, University of Copenhagen, Copenhagen, DenmarkPublished Online:24 Mar 2022https://doi.org/10.1152/ajpheart.00057.2022MoreSectionsPDF (204 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat to the editor: A foundation of ex vivo experimental studies is the use of appropriately composed physiological salt solutions. Tight control of the chemical environment is one key strength of well-performed ex vivo investigations allowing for dissection of specific cellular contributions and molecular mechanisms under simulated physiological and pathophysiological conditions.Experiments relying on rigorous evidence-based designs rather than variable laboratory traditions should improve reproducibility. In a recent issue of the American Journal of Physiology-Heart and Circulatory Physiology, we therefore commend the recent efforts by Wenceslau et al. (1) to provide scientific guidelines, including composition of salt solutions, for studies of isolated blood vessels. We particularly appreciate their argument that the composition of traditional salt solutions should be adjusted to reflect physiological in vivo levels, for instance, regarding ionized Ca2+ and glucose, which have classically been present in supraphysiological concentrations.In line with this, we also recommend that the traditional supraphysiological levels of 5.4–6 mM K+ suggested by Wenceslau et al. (1) be reduced to a physiological level around 4 mM. This is particularly relevant for studies evaluating extracellular K+ accumulation as an activity-related signal for vasodilation.Considering the intent of the guideline authors to reproduce in vivo concentrations of circulating ions, we were surprised by their suggestion to use 14.9 or 19 mM HCO3−, which is well below typical plasma concentrations in humans and rodents. We disagree with the authors that [HCO3−] can be lowered without influencing contractile responses (1).We would like to raise two important points in particular:First, if the proposed salt solution is aerated with a gas mixture containing 5% CO2—as suggested in the guidelines (1)—according to the Henderson–Hasselbalch equation, 14.9 mM HCO3− produces a pH of 7.24. This level of acidity will affect tone in many vascular beds. If, alternatively, the salt solution at 5% CO2 is adjusted with NaOH to pH 7.40—as described in the original paper cited for the absence of HCO3−-dependent vascular effects (2)—equilibration of the CO2/HCO3− buffer system during titration will inevitably generate HCO3− until, at pH 7.40, the solution contains 22 mM HCO3−, as predicted by the Henderson–Hasselbalch equation. The 5% CO2 gas mixtures used by almost everyone and readily commercially available correspond in a humidified atmosphere to roughly 36 mmHg. If a more physiological gas mixture with 40 mmHg CO2 is used, the corresponding [HCO3−] is 24 mM at pH 7.40. In any case, the conclusion that low [HCO3−] does not influence vascular tone is erroneous, because the final pH-adjusted salt solutions used for the cited control and "reduced HCO3−" experiments (2) in reality all contained 22 mM HCO3−. It is obviously no surprise that identical solutions resulted in identical vascular tone development.Second, we strongly recommend using a physiological [HCO3−] of 22–24 mM because HCO3− plays at least three important roles that are concentration-dependent: it constitutes with CO2 a mobile physiological buffer system that stabilizes pH and minimizes spatial pH gradients (3–5), it serves as a substrate for Na+, HCO3− cotransporters, and Cl–/HCO3− exchangers (6–11), and it can be sensed in both the intracellular (12) and surrounding extracellular (13, 14) space of endothelial cells to modify arterial function and structure.Multiple studies show that H+-sensitive targets are important in most vascular beds, and more recent investigations reveal that HCO3− also plays a direct signaling role in the control of vascular tone (15). HCO3− transporters are critically important for setting the intracellular pH of vascular smooth muscle and endothelial cells (6–9), and omission of CO2/HCO3− can modify, for instance, vascular smooth muscle Ca2+ sensitivity (7, 8), endothelium-dependent hyperpolarizations (6), and NO production (8). Intracellular HCO3− sensing has been ascribed to the soluble adenylyl cyclase (12), whereas extracellular sensing requires the receptor protein tyrosine phosphatase (RPTP)γ (13, 14).These abovementioned considerations are important for all types of studies on isolated blood vessels. Specifically, when evaluating physiological and pathophysiological roles of HCO3−, two experimental approaches have been employed:First, due to the chemical equilibrium between CO2, HCO3−, and H+, it is not possible under standard experimental conditions to vary only one of the parameters Pco2, [HCO3−], and pH. However, because of the relatively slow spontaneous hydration of CO2—requiring several hundred milliseconds for equilibration—separate control of Pco2, [HCO3−], and pH can be achieved using out-of-equilibrium CO2/HCO3− solutions based on rapid mixing and laminar propulsion of solutions with dissimilar CO2/HCO3−/H+ composition (16). This technique applied to cerebral and mesenteric arteries from rats and mice has provided new insights into the physiology of HCO3−-dependent proteins and their vascular consequences (5, 13).Second, as a simpler approach, investigators have been comparing vascular responses in the presence and absence of CO2/HCO3−. To ensure appropriate buffering capacity, it is common in these cases to replace NaHCO3 with an artificial buffer such as HEPES. In these types of experiments, we suggest adding a similar (osmotically compensated) concentration of HEPES to the CO2/HCO3− containing solutions to distinguish the influence of removing CO2/HCO3− from the potential effect of adding HEPES.In conclusion, we strongly recommend physiological concentrations of 22–24 mM HCO3− in experimental salt solutions for ex vivo tissue studies. In combination with a gas mixture containing 5% or (better) 40 mmHg CO2, this establishes a pH of 7.40, which is appropriate for studies on arteries under physiological conditions. Obviously, other combinations of Pco2, [HCO3−], and pH can be relevant if evaluating veins, disease circumstances, or blood vessels from other animal species (e.g., marine organisms).DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.AUTHOR CONTRIBUTIONSE.B. drafted manuscript; E.B. and C.A. edited and revised manuscript; E.B. and C.A. approved final version of manuscript.REFERENCES1. 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Extracellular acidosis and very low [Na+] inhibit NBCn1- and NHE1-mediated net acid extrusion from mouse vascular smooth muscle cells. Acta Physiol (Oxf) 221: 129–141, 2017. doi:10.1111/apha.12877.Crossref | PubMed | ISI | Google Scholar12. Schmitz B, Nedele J, Guske K, Maase M, Lenders M, Schelleckes M, Kusche-Vihrog K, Brand S-M, Brand E. Soluble adenylyl cyclase in vascular endothelium: gene expression control of epithelial sodium channel-α, Na+/K+-ATPase-α/β, and mineralocorticoid receptor. Hypertension 63: 753–761, 2014. doi:10.1161/HYPERTENSIONAHA.113.02061. Crossref | PubMed | ISI | Google Scholar13. Boedtkjer E, Hansen KB, Boedtkjer DM, Aalkjaer C, Boron WF. Extracellular HCO3– is sensed by mouse cerebral arteries: regulation of tone by receptor protein tyrosine phosphatase γ. J Cereb Blood Flow Metab 36: 965–980, 2016. doi:10.1177/0271678X15610787. Crossref | PubMed | ISI | Google Scholar14. Hansen KB, Staehr C, Rohde PD, Homilius C, Kim S, Nyegaard M, Matchkov VV, Boedtkjer E. PTPRG is an ischemia risk locus essential for HCO3–-dependent regulation of endothelial function and tissue perfusion. eLife 9: e57553, 2020. doi:10.7554/eLife.57553. Crossref | PubMed | ISI | Google Scholar15. Boedtkjer E. Acid-base regulation and sensing: accelerators and brakes in metabolic regulation of cerebrovascular tone. J Cereb Blood Flow Metab 38: 588–602, 2018. doi:10.1177/0271678X17733868. Crossref | PubMed | ISI | Google Scholar16. Zhao J, Hogan EM, Bevensee MO, Boron WF. Out-of-equilibrium CO2/HCO3– solutions and their use in characterizing a new K/HCO3 cotransporter. Nature 374: 636–639, 1995. doi:10.1038/374636a0. Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESCorrespondence: E. Boedtkjer ([email protected]au.dk). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationRelated articlesGuidelines for the measurement of vascular function and structure in isolated arteries and veins 25 Jun 2021American Journal of Physiology-Heart and Circulatory PhysiologyReply to Boedtkjer and Aalkjaer 24 Mar 2022American Journal of Physiology-Heart and Circulatory PhysiologyCited ByReply to De Mey et al.Camilla F. Wenceslau, Cameron G. McCarthy, Scott Earley, Sarah K. England, Jessica A. Filosa, Styliani Goulopoulou, David D. Gutterman, Brant E. Isakson, Nancy L. Kanagy, Luis A. Martinez-Lemus, Swapnil K. Sonkusare, Pratish Thakore, Aaron J. Trask, Stephanie W. Watts, and R. Clinton Webb24 March 2022 | American Journal of Physiology-Heart and Circulatory Physiology, Vol. 322, No. 4Reply to Boedtkjer and AalkjaerCamilla F. Wenceslau, Cameron G. McCarthy, Scott Earley, Sarah K. England, Jessica A. Filosa, Styliani Goulopoulou, David D. Gutterman, Brant E. Isakson, Nancy L. Kanagy, Luis A. Martinez-Lemus, Swapnil K. Sonkusare, Pratish Thakore, Aaron J. Trask, Stephanie W. Watts, and R. Clinton Webb24 March 2022 | American Journal of Physiology-Heart and Circulatory Physiology, Vol. 322, No. 4 More from this issue > Volume 322Issue 4April 2022Pages H685-H686 Crossmark Copyright & PermissionsCopyright © 2022 the American Physiological Society.https://doi.org/10.1152/ajpheart.00057.2022PubMed35324335History Received 1 February 2022 Accepted 7 February 2022 Published online 24 March 2022 Published in print 1 April 2022 KeywordsarteriesbicarbonatemyographysolutionspH Metrics