Cellular respiration energises all biological activities. An undesirable waste by-product of respiration is acid (H⁺-ions or their equivalent). Specialised mechanisms must be implemented to prevent acid accumulation inside cells and a fall in intracellular pH (pH_i; an inverse measure of acidity). This task is challenging as typical rates of acid-production can double resting H⁺ levels in 1/100^th of a second, if not regulated. Virtually all biochemical reactions are sensitive to pH_i. Even a modest fall in pH_i can have a major effect on cell function, growth and development, and may even trigger cell death. Highly controlled regulatory mechanisms are of utmost importance to keep pH_i within a narrow, physiological range. In addition, favourable pH_i must be unified across a tissue to ensure coordinated tissue growth and development.

There are two challenges to performing pH_i regulation in tissue. Firstly, H⁺-ions have a high charge-density and therefore cannot cross cell membranes passively at any significant rate. For this reason, cells have developed transporter proteins embedded in the cell-surface to transfer acid between the intra- and extracellular milieu. Secondly, H⁺-ions are highly reactive and undergo binding reactions with chaperone molecules, called pH buffers. A majority of these buffers are large molecules such as proteins. When bound to these buffers, H⁺-ion diffusion is severely reduced, typically by 2-3 orders of magnitude. Therefore, although H⁺-ions are the smallest of cations, they diffuse very slowly in biological systems.

Healthy tissues perform pH_i regulation by transporting acid across the surface membrane using specialised H⁺, OH^- or HCO₃^- carrying proteins, controlled by pH_i. Extracellularly-deposited acid is then removed in the blood. This arrangement can work efficiently, provided the degree of blood perfusion matches respiratory output. If blood perfusion fails, acid will accumulate around cells and slow the rate of acid-removal across the membrane. In light of low H⁺-ion mobility, cell-capillary distances are therefore critical for coordinated spatial pH_i regulation in tissue.

There are important disease states where blood perfusion may not suffice, leaving the tissue prone to acid accumulation. One such condition is ischaemia during which blood perfusion fails and as a result of which, cells can die. One organ susceptible to this is the heart. Tumours are characterised by a very high respiratory rate, coupled with poor perfusion and large cell-capillary distances. Despite the propensity to acidosis, tumours can thrive under conditions that would be untenable for other tissues. This observation suggests that tumours may express specialised mechanisms for regulating pHi to compensate for poor perfusion.

Most studies on pH_i regulation have been performed on single, isolated cell preparations. These investigations have contributed a wealth of information on regulatory processes operating at single-cell level. A complete understanding of tissue-level pH_i regulation must take into account diffusion in the extracellular milieu and cell-cell communication (through channels called gap junctions). These can only be studied in multi-cellular structures that mimic tissue. The aim of our research is to identify the mechanisms that regulate pH_i spatially in spherical 3-D multi-cellular clusters of cells (called ‘spheroids’) grown from tumour cells such as colon or renal bladder carcinomas. Diffusible buffer molecules, the enzyme carbonic anhydrase, and gap junctions are likely to play a role in spatial pH_i regulation. The role of these molecules will be investigated in spheroids using gene expression and pharmacological inhibition. Experimental results will be married with computational simulations and analysis. The selective expression of this enzyme in tumours may underlie the survival advantage of cancer. Inhibition of these mechanisms may form the basis of anti-cancer therapy. The spheroid model will also shed light on our general understanding of the fundamental processes that control pH_i in tissue.

Further information can be found at: http://www.physiol.ox.ac.uk/~ps/

See also Vaughan-Jones Research

Pawel Swietach