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 0.26mMI 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 1-2nmoles.mg"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
