...why does NH4Cl acidify the cells?

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Hss
Hss's picture
...why does NH4Cl acidify the cells?

 Hi

In some experiments people use NH4Cl to decrease the ph of the cells; One question: why perfusing with NH4Cl the cells become acidific? The pH in the normal perfusion is about 7.4 and the pH in the NH4Cl solution is about 7.4 too, so I don´t understand why the change in pH of the cells (I suppose if there are more H+ in the NH4Cl solution then the pH should be more acid? 

Regards. 

The FFM
The FFM's picture
I believe that what you are

I believe that what you are asking about is the NH4+ prepulse acidification technique.

NH4+ in solution dissociates in NH3 and H+. In a HEPES buffered solution, replacing part of the Na+ concentration in the bath (e.g. 20 mM) with 20 mM NH4+ (NH4Cl) actually causes the intracellular pH (pHi) of the cell to increase (become more alkaline).  The increase is fast due to the rapid influx of NH3 (dissociated from the NH4+) into the cell and the subsequent combination of most of these NH3 molecules with intracellular H+.

After the fast alkalinization there is a slower "plateau-phase"  acidification caused by the slower entry of NH4+, a very small amount of which dissociates into NH3 and H+.

When the external NH4++ (NH4Cl) is removed, pHi rapidly falls (acidifies) because nearly all the internal NH4+ dissociates to NH3 (which rapidly leaves the cell) and H+ (which is trapped inside due to its charge).

If you observe that the pHi subsequently recovers from this acid loading step then this indicates the presence of an endogenous acid extrusion mechanism in the cell that you are studying.

Hss
Hss's picture
 ...yes, I think that was

 ...yes, I think that was exactly what I mean and what I needed to know.

Three things:

- So, in the perfusion solution the NH4+ don´t dissociates (and the solution don´t get acid)? It only get dissociates into the cell when external NH4+ is removed? 

- It happens too in HCO3- buffered solutions? I saw some papers in wich people used HCO3- for the same protocol of acidification with NH4Cl, so I was thinking I could used this too 

- And, in all this protocol, when we use 20mM of NH4Cl we used 20mM less NaCl to keep the osmolarity similar; so, the NH4Cl solution will have 20mM less Na+, oder? and I suppose it cann made some effect on the experiments too. 

Thanks  

The FFM
The FFM's picture
 For you to understand what

 For you to understand what is happening when you apply ammonium containing solutions, I refer you to the following paper and its associated references.

J Membr Biol. 2009 Mar;228(1):15-31. Epub 2009 Feb 26.
Concentration-dependent effects on intracellular and surface pH of exposing Xenopus oocytes to solutions containing NH3/NH4(+).
Musa-Aziz RJiang LChen LMBehar KLBoron WF.
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA. eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%72%61%69%66%2e%61%7a%69%7a%40%63%61%73%65%2e%65%64%75%22%3e%72%61%69%66%2e%61%7a%69%7a%40%63%61%73%65%2e%65%64%75%3c%2f%61%3e%27%29%3b'))

Abstract
Others have shown that exposing oocytes to high levels of NH(3)/NH(4)(+) (10-20 mM) causes a paradoxical fall in intracellular pH (pH(i)), whereas low levels (e.g., 0.5 mM) cause little pH(i) change. Here we monitored pH(i) and extracellular surface pH (pH(S)) while exposing oocytes to 5 or 0.5 mM NH(3)/NH(4)(+). We confirm that 5 mM NH(3)/NH(4)(+) causes a paradoxical pH(i) fall (-DeltapH(i) approximately equal 0.2), but also observe an abrupt pH(S) fall (-DeltapH(S) approximately equal 0.2)-indicative of NH(3) influx-followed by a slow decay. Reducing [NH(3)/NH(4)(+)] to 0.5 mM minimizes pH(i) changes but maintains pH(S) changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates -DeltapH(S) at both NH(3)/NH(4)(+) levels. During removal of 0.5 or 5 mM NH(3)/NH(4)(+), failure of pH(S) to markedly overshoot bulk extracellular pH implies little NH(3) efflux and, thus, little free cytosolic NH(3)/NH(4)(+). A new analysis of the effects of NH(3) vs. NH(4)(+) fluxes on pH(S) and pH(i) indicates that (a) NH(3) rather than NH(4)(+) fluxes dominate pH(i) and pH(S) changes and (b) oocytes dispose of most incoming NH(3). NMR studies of oocytes exposed to (15)N-labeled NH(3)/NH(4)(+) show no significant formation of glutamine but substantial NH(3)/NH(4)(+) accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH(i) and pH(S) demonstrate that NH(3) flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane-AmtB-enhances the flux of a gas across a biological membrane.

PMID: 19242745

Hss
Hss's picture
 ...thanks for the

 ...thanks for the information. I still would like to understand something in wich I don´t see so much clarification in papers.    So far I understand, in the bath solutiom, NH4Cl dissociates giving free H+; if so, why the pH of the solution doesn´t change (don´t get acid)?

Additionally this free H+ will activated some channels that made some alterations in the rest membrane potantial, for example.  

Regards.