THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 241, No. 5, Issue of March 10, 1966
Printed in U.S.A.
Parathyroid Hormone-induced Mitochondrial Swelling*
(Received for publication, July 19, 196.5)
Kozo UTZUMI, JOHN D. SALLIS, AND H. F. DELUCA
From the Department of Biochemistry, Cancer Institute of Okayama University Medical School, Okayama,
Japan, and the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 5.9’06
SUMMARY EXPERIMENTAL PROCEDURE
In addition to stimulating ion transport and respiration, it 2=ncubalion-Rat liver mitochondria were isolated from male
is now established that the parathyroid hormone induces albino rats (150 to 200 g) as previously described (5). Mito-
rapid and extensive mitochondrial swelling. The swelling chondria equivalent to 0.5 mg of nitrogen were incubated at 25’
process takes place in the absence of Mg++ in a medium in a medium containing 100 mM sucrose, 3.3 mM Tris-HCl buffer
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composed of sucrose, substrate, and tris(hydroxymethyl)- (pH 7.2), and 10 mM substrate in a total volume of 2.0 ml.
aminomethane. If Mgff is present, the swelling change is Further addition of 13.3 mM Pi or 6.7 mM MgClz was made
greatly minimized. The swelling is accompanied by a where indicated in the text. An apparatus for the simultaneous
simultaneous increase in pyridlne nucleotide oxidation and measurement of fluorescent emission of pyridine nucleotide, 90”
respiration, and is neither dependent upon nor enhanced by light scattering, and oxygen consumption of mitochondria was
K+ or Na+. Inorganic phosphate, acetate, and, to a lesser used as previously reported (13), and incubations were carried
extent, arsenate stimulate the rapidity and extent of the out directly in the cuvette. Following a 1-min thermoequilibra-
parathyroid hormone-induced swelling. The results suggest tion period, 100 pg of purified parathyroid hormone2 contained
that parathyroid hormone-induced swelling is closely related in 0.001 M acetic acid were added to the cuvette. Controls
to the ion transport and respiration responses. received the appropriate amount of acetic acid.
Measurement-(u) Swelling: The 90” light scattering change
at 650 rnp was recorded as a percentage of the initial scattering
(defined as 100%) observed after the addition of a reagent as
recorded on the chart (13). (b) Respiration: Oxygen consump-
tion was measured polarographically by means of a rotating
The recent interest in ion transport by mitochondria has re-
platinum electrode (13). (c) Pyridine nucleotide oxidation:
opened many questions relating to mitochondrial swelling.
The reduced pyridine nucleotides of mitochondria were excited
Investigations by several groups have already revealed that
at the 365 rnp line of a mercury lamp and the fluorescence was
under appropriate conditions extensive mitochondrial swelling
monitored at 450 m,u and positioned at 90” to the exciting light.
can be associated with the transport of Ca+C (1, 2), Mnff (3),
The relative intensity is recorded on the chart (13).
K+ (4), inorganic orthophosphate (5), and many other ions.
Explanation of TracesTrace A (Fig. 1, A to C) represents
Our further interest in the relationship between these effects
respiration, a downward deflection indicating an increase in
has been stimulated by the knowledge that the antibiotic pep-
oxygen consumption. Trace B represents swelling, an increase
tides valinomycin and gramicidin, both well known swelling
being noted by a downward deflection. Trace C represents
agents, are also capable of stimulating ion transport (6).’
fluorescent emission of pyridine nucleotides, a downward deflec-
Recent studies on the mechanism of action of parathyroid
tion representing oxidation.
hormone have shown that this peptide, too, is capable of induc-
ing ion transport in isolated mitochondria (7-9). In addition, RESULTS
this hormone has been shown to stimulate nonphosphorylative Hormolze- and
and Pi-induced Swelling in Presence Absence of
oxidation under a variety of conditions (10, 11) and its site of Mg++--Fig. 1A clearly indicates that, in the absence of Mg++,
action is well defined (7, 10-12). It therefore appeared fruitful extensive swelling is produced by both Pi and parathyroid
to study the action of the hormone on mitochondrial swelling hormone. Although respiration is slightly increased by Pi, a
and, if possible, to correlate the effect with ion transport and much more dramatic change appears upon the further addition
respiration. This study was made feasible through the availa- of parathyroid hormone. Both the parathyroid hormone and
bility of a device for the simultaneous measurement of swelling Pi appear necessary for these alterations in swelling and respira-
(90’ light scattering), respiration, and pyridine nucleotide oxi- tion, as illustrated in Fig. lC, where the reverse order of addition
dation (13). These experiments form the basis of the present of these reagents was examined. The respiratory activity was
report. found proportional to the magnitude of swelling. It should
* Published with the approval of the Director of the Wisconsin also be noted (in Fig. 1C) that the addition of parathyroid
Agricultural Experiment Station. Supported by Grant AM hormone in the absence of Pi does result in an increased pyridine
O-5800-04 NTN from the National Institutes of Health.
1 Personal communication. * R. B. Sanders, J. D. Sallis, and H. F. DeLuca, in preparation.
Issue of March 10, 1966 Utzunai, Sallis, and DeLuca 1129
nucleotide oxidation. When Mg++ was initially present (Fig.
1B) and both Pi and parathyroid hormone were subsequently
added, the magnitude of the swelling by these reagents was
greatly depressed, and only a slight response in respiration was
observed. Identical patterns have been observed in further
experiments (not shown here) in which succinate was replaced
Bnion Injhence in Relation to Swelling and Hormone-induced
Respiration-Parathyroid hormone has been shown previously
to induce to a varying degree the transport of a number of other
anions besides Pi (14). It was of interest, therefore, to examine
the effects of these anions on the swelling and respiration phe-
nomenon. These data are recorded in Fig. 2, A and B. The
magnitude of swelling appeared in the order Pi > acetate >
arsenate. With the exception of sulfate, the presence of all of
cl MINi I these anions promoted varying degrees of swelling which were
greatly heightened upon the addition of hormone.
10 A respiration response was also induced by the anions, and
again a marked stimulation was evident in the presence of
B hormone. Greatest response appeared with arsenate followed
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by Pi, acetate, and sulfate, respectively. However, it will be
noted that the arsenate control value is much greater than the
- 400 response induced by the other anions, so that the true order of
magnitude is probably Pi > acetate > arsenate > sulfate.
Thus, the patterns for respiration and swelling would more
Cation Influence on Hormone-stimulated Respiration and Xwell-
&g-The importance of Na+ or K+ to the reaction was investi-
gated by measuring responses in the presence of Na+ or Kf
alone or in the absence of either of these cations by replacing
them with Tris. The previous data were recorded in a medium
containing only Na+, but Fig. 3 shows the effect of substituting
Na+ with K+. By comparison with Fig. 14, it is obvious that
the responses are identical. Of singular importance, however,
are the results obtained in the absence of either of these cations
(Fig. 4), where, again, hormone-induced swelling and respiration
have been demonstrated.
The present report establishes unequivocally that the para-
thyroid hormone induces extensive and rapid mitochondrial
swelling. It is tempting to reflect that swelling of mitochondria
is a consequence of osmotic pressure determined by ion accumu-
lation in response to parathyroid hormone. At this time, how-
ever, there is insufficient evidence to support this claim. In
the absence of Mg+f, a situation in which the parathyroid hor-
mone induces the most dramatic swelling changes, ion accumula-
tion in response to hormone has not yet been demonstrated
satisfactorily (11). This important point obviously needs
further intensive investigation and would require more sophisti-
cated techniques than have been used currently.
That the swelling phenomenon reported here is in some way
related to ion transport is suggested by the importance of the
anions in the medium. Although the hormone-induced swelling
I I I I I I I 0 occurs in a medium containing only sucrose, Tris, and substrate
(see Fig. 4), this response is greatly enhanced by the addition of
FIG. 1. Parathyroid hormone (PTH)- and Pi-induced swelling a suitable anion. Pi, itself a well known swelling agent (E),
in the presence or absence of Mg++. Conditions were as described was found most effective in this capacity, followed in effective-
in the text. Sodium succinate (10 mM) was used as substrate, ness by acetate and arsenate. It is interesting to note that with
and 13.3 mM sodium phosphate, 50 rg per ml of parathyroid hor-
mone, and 6.7 mM MgC& were added where indicated. the single exception of acetate, a similar order of effectiveness
control value obtained in the absence of parathyroid hormone. has been demonstrated with regard to parathyroid hormone-
1130 Pardhyroid Hormone-induced Mitochrmdrial Swelling Vol. 241, Yo. 5
1 I I I I , >PHOSPHATE
0 I I I I I I I I 0
FIQ. 2. Relationship of anions to parathyroid hormone (PTH)-induced respiration and swelling. Conditions were as described in
the text. Sodium succinate (10 rn@ was used as substrate and the separate addition of the sodium salt of the respective anion (13.3
mM) was made at the point indicated.
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I I I I I I ’ 0
FIG. 3. Parathyroid hormone (PTZZ)-induced respiration and swelling in a potassium medium. Conditions were as described in the
text. Potassium succinute (10 mM) was used as substrate and potassium phosphate (13.3 mAr) was added at the point indicated.
FIG. 4. Parathyroid hormone (PTH)-induced respiration and swelling in the absence of Na+ or K+. Conditions were as described
in the text. Tris-succinate (10 mM) was used as substrate and Tris-phosphate was added at the point indicated.
Issue of March 10, 1966 Utzumi, Sallis, and DeLuca 1131
stimulated anion uptake in the presence of Mg++. Unfortu- in experiments not shown here, either ashing of the hormone
nately, acetate transport in response to hormone has not yet preparations as described by Cash et al. (19) or simply oxidative
been demonstrated, although it is known that acetate is necessary destruction of the hormone with peracetic acid completely elimi:
to provide optimum conditions for parathyroid hormone-stimu- nates the mitochondrial responses reported in both the present
lated mitochondrial ATPase (12). When magnesium is present, and previous reports. Thus, the parathyroid hormone response
there is no question that ion accumulation does occur (7), but cannot be attributed to contaminating metal ions.
swelling is minimal under these conditions (see Fig. 1C). The The respiratory response appears to be closely related to, and
lack of swelling under these circumstances is probably due to a occurs simultaneously with, the appearance of swelling. Two
precipitation of the transported ions (for example, Mgs(POJ2) criteria were employed to measure the respiratory response, (a)
inside the mitochondria, thus greatly reducing their effectiveness polarographic measurement of oxygen consumption and (b)
in increasing osmotic pressure. fluorometric measurement of reduced pyridine nucleotides. The
Aside from osmotic considerations, there is abundant evidence latter appeared to be the more sensitive index as the response
(16) to indicate that other mechanisms are involved in mito- was always evident earlier than that demonstrated by the plati-
chondrial swelling. One of the most popular ideas involves a num electrode trace. The oxidation of the pyridine nucleotides
contractile or mechanoprotein mechanism, and still another always occurred simultaneously with the initiation of swelling.
involves conformational changes as a result of the level of high Clearly, therefore, the respiratory response is not an aftermath
energy intermediates generated by oxidative phosphorylation. of swelling damage, but, rather, both probably result from a
No doubt all these mechanisms can operate in varying degrees more primary action of the hormone. Further support for this
under divergent circumstances, with the result that the data view is offered by the observation that Mg++ inhibits both the
are often conflicting, confusing, and difficult to interpret. Al- respiratory and the swelling response.
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though it seems certain that the osmotic mechanism must Regardless of the mechanisms by which it is induced, para-
inevitably be involved in the hormone-induced swelling, the thyroid hormone-dependent mitochondrial swelling is an im-
mere fact that parathyroid hormone-stimulated respiration portant response which is unquestionably linked to the already
occurs in a medium of only sucrose, Tris, and substrate demands well established actions of the hormone in vitro. This phenome-
that other mechanisms which have been proposed to account non alone, however, would seem to be of little physiological
for the swelling cannot be ignored at the present time. importance. Nevertheless, when considered together with the
It would seem of interest, therefore, to consider the swelling ion transport and respiration responses of the mitochondria,
changes in the light of our knowledge concerning the site of inter- it may be of great value in determining the molecular mecha-
action of parathyroid hormone with the oxidative phosphoryla- nism of parathyroid hormone action.
tive chain. Our previous studies (7, 10) on ion transport indi-
cated that the parathyroid hormone interacted somewhere REFERENCES
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