Parathyroid Hormone-induced Mitochondrial Swelling

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  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
                                                                                  by pyruvate-malate.
                                                                                     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
                                                                                  nearly correspond.
                                                                                      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
                                                         -I 100
                                                                                  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
                                                        -----i the
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



                                                                                                                                -    400

 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.
        I                                                       Al

                                                                     -    400


                                                                     -   100

   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
between the sites of action of 2,4-dinitrophenol,                 Dianemycin,
and Nigericin        and that of aurovertin and oligomycin.                   This     1. CHAPPELL,        J. B., AND CROFTS,             A. R., Biochem.        J., 95, 378
proposed site of hormone action also appears to be utilized for                              (1965).
                                                                                       2. UTSUMI,      K.: Acta Med. Okayama, 18, 189 (1964).
the hormone-stimulated           respiration     response which occurs in                                                               G. D., AND BICKNELL,
                                                                                       3. CHAPPELL,        J. B., GREVILLE,                                            K. E.,
the absence of Mgff (11). The hormone must therefore lower                                  Biochem:      J., 84; 61~ (1962):
the steady state level of the nonphosphorylated                  intermediates         4. RASMUSSEN,          H., FISHER,        J., AND ARNAUD,          C., Proc.      Natl.
of oxidative       phosphorylation.        This in itself may result in                     Acad. Sci. U. S., 52, 1198 (1964a).
                                                                                       5. UTSUMI,      K., Acta Med. Okayama,               17, 259 (1963).
swelling, or it may decrease the supply of energy for the contrac-
                                                                                       6. MOORE.       C.. AND PRESSMAN.               B. C.. Biochem.       Biophw. -
                                                                                                                                                                  _       Res.
tile mechanism.          To what extent the hormone participates                 in          Cornhun.;       15, 562 (1964).        ’         ’
the latter mechanism is a question which cannot be answered                            7. SALLIS, J. D., DELUCA,             H. F., AND RASMUSSEN,              H., J. Biol.
until much more definitive data on ion transport under a wide                                Chem., 238, 4098 (1963).
variety of conditions are available.                                                   8. RASMUSSEN,          H., SALLIS,       J. D., FANG, M., DELUCA,               H. F.,
                                                                                            AND YOUNG,           R., Endocrinology,         74, 388 (1964b).
    Rasmussen, Fisher, and Arnaud (4) have also reported para-                         9. SALLIS, J. D., DELUCA,              H. F., AND MARTIN,           D. L., J. Biol.
thyroid hormone-induced            mitochondrial       swelling, which they                 Chem., 240, 2229 (1965).
attribute     specifically    to K+ accumulation.              In our hands,          10. FANG, M., RASMUSSEN,               H., DELUCA,          H. F., AND YOUNG,          R.,
however, parathyroid          hormone-induced        swelling has not been                  Biochem.       Biophys.     Res. Commun.,           10, 260 (1963).
                                                                                      11. SALLIS, J. D., AND DELUCA,                  H. F., J. Biol. Chem., 241, 1122
restricted to conditions         of K+ transport.          In fact, the most                 (1966).
rapid and extensive swelling change takes place in a medium in                        12. SALLIS, J. D., AND DELUCA,                  H. F., J. Biol.    Chem., 239, 4304
which Tris is the sole cation (see Fig. 4). At no time have we                               (1964).
been able to demonstrate a K+ dependence or enhancement of                            13. UTSUMI,      K., AND YAMAMOTO,              G., Acta Med. Okayama,           18, 111
parathyroid        hormone-induced         swelling.     Therefore,      we are              (1964).
                                                                                      14. GFLEENBAUM,          A. L., AND DICKER,             S. E., Biochem.       Biophys.
forced to conclude that, under the present circumstances, K+                                Res. Commun.,           12, 402 (1963).
accumulation        alone cannot account for the hormone-induced                      15. HUNTER,      F. E., JR., LEVY, J. F., FINK, J., SCHUTZ, B., GUERRA,
swelling.                                                                                   F., AND HURWITZ,            A., J. Biol.       Chem., 234, 2176 (1959).
    It has been observed by others that oxytocin and vasopressin                      16. LEHNINGER,         A. L., Physiol.        Rev., 42, 467 (1962).
also induce mitochondrial            swelling     (14, 17, 18). Recently,             17. LEHNINGER,         A. L., AND NEUBERT,             D., Proc. Natl.      Acad. Xci.
                                                                                             U. S., 47, 1929 (1961).
the specificity of this phenomenon has been questioned and has                        18. GREENBAUM,           A. L., AND DICKER,             S. E., Biochim.       Biophys.
been attributed        to contaminating        metal ions in the hormonal                   Acta, 74, 519 (1963).
preparations      (19). This point is of obvious importance to the                    19. CASH, W. D., GARDY, M., AMEND,                     W. J. C., JR., AND EVANS,
results reported here for the parathyroid               hormone.      However,              F. O., JR., Biochem.          Biophys.       Res. Commun.,      17, 655 (1964).