CELLULAR BIOPHYSICS AND NEUROENDOCRINE TRANSPORT AND SECRETION 
Robert G. Johnson, Jr., M.D., Ph.D., Associate Investigator 
Dr. Johnson is interested in the application of 
biophysical and molecular techniques with physio- 
logical, spatial, and temporal resolution to the 
investigation of cellular regulation of biological 
membrane transport and release processes in endo- 
crine secretory cells. Specifically, the laboratory is 
investigating the cellular regulation and turnover 
of the Na^,K"*^ ATPase, the control and kinetics 
of voltage-dependent calcium channels, and the 
plasticity of chemical messengers in mature secre- 
tory vesicles. 
I. Cellular Regulation of the Na+,K+ ATPase. 
The Na"*" pump, w^hich appeared over 600 million 
years ago, functions to maintain an anisotropic dis- 
tribution of Na^ and across biological mem- 
branes by catalyzing the transport of influx and 
Na^ efflux against their respective gradients, using 
the high-energy phosphogens of ATP. Present in the 
plasma membranes of most eukaryotic cells, the 
Na^ pump has important functions in ion homeo- 
stasis, volume regulation, Na"*^ -coupled solute trans- 
port, and the excitability of certain cells. The regu- 
lation of the turnover of this ubiquitous pump has 
been thought to be primarily the ambient intracel- 
lular sodium concentration. However, a large body 
of phenomenological evidence suggests that, in cer- 
tain cells, hormones and intracellular messengers 
can independently and significantly increase the 
Na^ pump activity. It is difficult to measure accu- 
rately and quantitatively Na^ ATPase activity and 
the intracellular sodium concentration with reason- 
able kinetic time constants within intact cells. Un- 
derstanding the cellular regulation of the Na"*^ 
pump has important implications, given the dem- 
onstrated derangements in Na^ pump function in 
the pathologic states of hypertension and diabetes. 
Dr. Johnson and his colleagues have attempted to 
develop model systems and methodologies to mea- 
sure in a noninvasive and nondestructive manner 
the Na"*" pump activity in an intact cell or an intact 
tissue. The choice of an elasmobranch, Narcine 
brasiliensis, for this study has many advantages, in- 
cluding 1) the presence within the electrocyte of 
the highest density of Na"*" pump found within na- 
ture, 2) the homogeneous nature of the electric 
organ, 3) the simple composition with few proteins 
within the dorsal membrane, and 4) a primarily gly- 
colytic metabolic pathway. 
The noninvasive, nondestructive technique of 
nuclear magnetic resonance spectroscopy has been 
used to measure the ATPase activity of the Na"*^ 
pump in a time-dependent fashion; simultaneously 
the transmembrane sodium gradient has been mea- 
sured through the use of custom triple-tuned sur- 
face coils. The resting ATPase activity is quite low, 
but with stimulation the activity increases over 
three orders of magnitude, despite only a small in- 
crease in the internal sodium concentration. Other 
noninvasive techniques, including saturation trans- 
fer measurements, have confirmed this finding. 
These measurements represent the first evidence 
that the Na^ pump activity in an excitable tissue 
can be significantly regulated by a mechanism inde- 
pendent of intracellular sodium. Ongoing studies 
are focused on the biochemical signals that regu- 
late the activation and deactivation of the Na"*" 
pump activity. 
n. Plasticity of Neuroendocrine Chemical 
Messengers. 
One significant observation during the past de- 
cade has been that many neurotransmitters and 
neuromodulators are colocalized to the same secre- 
tory vesicle and that the amount of each compound 
can be regulated independently. The chromaffin 
granule from the adrenal medulla has provided a 
unique opportunity to study the plasticity of chemi- 
cal messengers, because of the amount and diver- 
sity of the compounds contained there (including 
catecholamines, enkephalins, and ATP). The en- 
kephalins are present primarily in the high-molecu- 
lar-weight precursor form, but under the appropri- 
ate conditions the degree of processing can be 
increased significantly. Extensive analysis has indi- 
cated that enkephalin content and enkephalin pro- 
cessing can increase when catecholamine content 
decreases. Radiochemical labeling studies support 
the conclusion that although enkephalin synthesis 
and processing increase in newly formed granules, 
there is a significant increase in processing in ma- 
ture granules. These findings indicate that a trans- 
membrane signal across the secretory vesicle mem- 
brane must activate enzymes responsible for this 
increased processing. The ability to influence pro- 
cessing of peptides within a mature granule has im- 
portant implications for secretory vesicles within 
a nerve terminal, where processing could conceiv- 
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