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Title: Ca2⁺ and Na⁺ transport by chromaffin granules of the adrenal medulla
Author: Haigh, Julian R.
ISNI:       0000 0001 3524 0782
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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Chromaffin granules, the secretory organelles of the adrenal medulla, store and secrete catecholamines, proteins and nucleotides. Their very high internal catecholamine concentrations are achieved by a chemiosmotic coupling between an inwardly directed, electrogenic H+-translocating adenosine triphosphatase (H+- ATPase) and a separate catecholamine/H+ exchanger. Ca2+ is also a major component of the granule matrix, its nominal concentration being 20-30mM. The mechanism of Ca2+ uptake has hitherto only been partially characterised. Ca2+ uptake via electroneutral Ca2+/Na+ exchange has been demonstrated in resealed membrane "ghosts" and intact granules (Phillips (1981), Biochem. J. 200: 99-107; Krieger-Brauer & Gratzl (1982), Biochem. Biophys. Acta. 691: 61-70). To sustain Ca2+ uptake it is therefore necessary for granules to have an independent mechanism of Na+ uptake. In this thesis I show that chromaffin granules possess a novel amiloridesensitive Na+/H+ antiporter in their membranes. This explains why Na+ is mildly inhibitory to catecholamine transport (which is driven principally by the transmembrane pH difference generated by the ATPase). Na+/H+ antiport activity has been assayed using a variety of techniques: direct assay of 22Na+ accumulation in response to a transmembrane pH difference (ApH) generated by a rapid increase in external pH, or by ATP hydrolysis; generation of a ApH (measured with a fluorescent probe) in response to an imposed Na+ gradient; loss of a pH gradient due to Na+ uptake; and Na+/Na+ exchange. The antiporter has a relatively high Michaelis constant (Km) for extragranular Na+ (4.7mM at pH 7.0 determined from fluorescence experiments) and is inhibited competitively by the diuretic drug amiloride, a well known inhibitor of plasma membrane Na+/H+ antiporters, with an apparent inhibition constant (K|) of 0.26mM I have also measured the total and free concentrations of the inorganic cations Na+ and K+ inside the granule matrix. The total concentrations were found to vary depending on the ionic composition of the isolation buffer used to prepare the granules, with the lowest values being obtained with nominally Na+ and K+-free buffered sucrose solutions. The activity coefficients of both ions were found to be about 0.8, indicating that most of the Na+ and K+ is free within the matrix. The free concentration of Ca2+, however, was found to be approximately 5^M, a value markedly lower than the previously measured total concentration of 20mM (Phillips et al., (1977), Neuroscience, 2: 147-152). In other words, its free concentration is only 0.03% of the total. Because monovalent ion redistribution occurs during granule isolation, the concentrations of Na+, K+ and Ca2+ measured in vitro cannot be extrapolated to the intact cell in situ. Using Na+-loaded resealed membrane "ghosts", and using Ca2+ buffers to achieve various extravesicular free Ca2+ concentrations, I have determined that the apparent Km for Ca2+ uptake during Ca2+/Na+ exchange is about 1|iM, with maximal rates of Ca2+ uptake of the order of"1.sec"1. In "ghosts", the activity of the Na+/H+ antiporter described above can be used to couple Ca2+ uptake via Ca2+/Na+ exchange to electrogenic proton translocation via the granule membrane ATPase. Therefore, Ca2+ uptake can be indirectly linked to the proton pump. However, under conditions designed to mimic the environment of a granule in the cytosol of a chromaffin cell, only very low amounts of Na+ could be accumulated within the granule matrix. Consequently, measured rates of Ca2+ accumulation are also low. Under such circumstances, the granules seem unlikely to play a major role in calcium homeostasis in the intact cell.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry