Intracellular ion activity

Fig. 6. Double-barrel micropipet ISE for measuring transient intracellular ion activities, (From 11, with permission)...

Microelectrodes can be made sufficiently small to measure intracellular ion activities, as shown schematically in Fig. 5 The micro ion-selective electrode is inserted into a single cell by means of a micromanipulator. The intracellular activity of the... 

NMR can help to monitor energization, see, e.g. [121, 273], especially the levels of 31P-containing metabolites, e.g. [45, 366], enzyme kinetics, compartmentalized intracellular ion activities, the fate of 3H-, 2H-, 13C-, 15N-, or 19F-labeled tracers, e.g. [108,109], 02 tension, compartmentalized redox potential, membrane potential, cell number or cell volume, see [133], and even pH. Major drawbacks are the cost of the equipment, the low intrinsic sensitivity and the interpretation of spectra [430]. 

Once is known, Eq. (8) can be used to evaluate the intracellular ion activity. Usually, E is measured several times in many cells, and an average value is used to calculate the unknown ion activity. If the microelectrode is highly selective for the analyte ion, little attention need be given to the selectivity coefiBcient term of Eq. (8). However, when less selective electrodes are employed, selectivity coefficient values and estimates as to the interferent ion activities within the cells must be made. Sometimes, actual measurement of these interferent ion activities can be made first with other micro-ISEs, and the resulting values used in the calculations. 

The purpose of the reference barrel is to allow for the simultaneous measurement of the cell membrane potential while measuring the intracellular ion activities with the ISE portion of the device. Tims, in practice, a second single-barrel reference micropipet electrode is placed in the bathing solution outside the cell so that the potential between the two KCl-filled electrodes can always be monitored to obtain the instantaneous cell membrane potential (E ). The potential of the liquid membrane ISE barrel can also be monitored versus the external reference electrode. In this manner, potential changes due to variations in the cell membrane potential can be taken into account when calculating the intracellular ion activities. Alternatively, only the potential difference between both barrels of the electrode could be monitored. This potential should only be dependent on the intracellular activity of the analyte ion (not affected by the cell membrane potential). For certain ion measurements, e.g., K using a valinomycin based liquid micropipet electrode, leakage of K+ from the reference barrel could present a problem. In such cases, the reference barrel and the outer reference pipet should be filled with a solution other than 3 M KCl. 

Applications of pH and ion-selective microelectrodes are described in microanalysis (Wright), measurement of intracellular ion activity and calculation of equilibrium potentials (Brown and Kunze), and then studies of the kidney (Wright, Malnic et al., and Khuri), brain (Zeuthen et al., Morris and Krnjevic), frog heart (Walker and Ladle), and human skeletal muscle (Filler and Das). 

In these bio-related fields, it is primarily the pH, pNa, pK, pCa, pCl or pF electrodes which are employed. Depending upon whether extra- or intracellular ion activities are to be determined, different electrode constructions are utilized. Measurements in extracellular fluids (whole blood, serum, etc.) are usually carried out with the conventional macroelectrodes. Anaerobic conditions are harder to maintain with this method.is expedient to thermostat the entire sample beaker and electrode pair set-up. For more accurate pH and pCa determinations the air space above the sample must also be equilibrated with an O2-CO2 mixture, to establish the same partial pressure of CO2 as in the original sample environment. This expense can be spared by working with microprobes under anaerobic conditions with small surface areas (low CO2 loss). In such cases the commercially available flow-thru cells or capillary cells are used. For ion activity measurements within the cell special microelectrodes with tip diameters about one micron are employed, so that the cell membrane is not damaged too much. To date, such electrodes are available only for pH and pNa measurements (Transidyne, U.S.A.). 

The Ag/AgCl micropipette reference electrodes usually employed in physiological measurements of cell membrane potentials can be used as reference electrodes for intracellular ion activity measurements as well. The salt bridge electrolyte must be chosen with care so that the intracellular measured ion concentration is not influenced. 

Figure 11. A summary cartoon illustrating the relationships between the first messengers and the release of Ca ions from the SR, the various pathways that influence the intracellular ion concentration, and the activation of MLCK, which leads to contraction.

Ion-selective microelectrodes [18, 70,71, 164] are chiefly used for measurement of ion activities in individual cells and in intracellular liquid. They were developed from micropipettes, which are miniature liquid bridges used for measurement of cell membrane potentials [94]. Micropipettes and ion-selective microelectrodes are made using commercial drawing devices. Ion-selective... 

Certain drugs affect the cell by binding to a receptor that is linked to a Gs protein. The activated receptor activates the Gs protein, which in turn activates the effector system that opens an ion channel or activates a specific enzyme. Conversely, a drug that binds to a receptor that is linked to a G protein, inhibits channel opening or intracellular enzyme activity. 

Miniature liquid ion-exchanger electrodes have been made of such small size that ion activities in intracellular fluid can be measured. 

Cardiac muscle cells are normally depolarised by the fast inward flow of sodium ions, following which there is a slow inward flow of calcium ions through the L-type calcium channels (phase 2, in Fig. 24.1) the consequent rise in free intracellular calcium ions activates the contractile mechanism. 

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