Resistance to swelling of mitochondria from new-born rat liver

BIOCHIMICA ET BIOPHYSICA ACTA

411

Preliminary Notes Resistance to swelling of mitochondria from new-born rat liver T h e ability of certain agents to induce swelling of isolated mitochondria has been extensively studied in preparations of adult-rat liver 1. In view of the metabolic differences known to exist between adult and new-born tissues, it appeared of interest to compare the effect of swelling agents on mitochondria from liver of very young rats with those from liver of adult rats. Liver of rats from single litters, 2-48 h of age, were pooled and homogenized gently for approx. 30 sec in IO vol. 0.33 M sucrose. Adult-rat liver was similarly homogenized for 2 min. Mitochondria were isolated using 0.33 M sucrose as the suspending medium throughout and final suspensions of mitochondria were prepared to contain 30-4 ° mg protein/ml. Suspensions of equal protein concentration from both sources had approximately equal absorption after similar dilution. Mitochondria prepared in this way from new-born rat liver had the same microscopic appearance as those from adult liver and were capable of oxidizing ~-hydroxybutyrate, fumarate and octanoate. Mitochondrial swelling was measured by the change in absorbancy at 520 m/~ of a suspension in 0. 3 M sucrose-o.o2 M Tris buffer (pH 7.4). The swelling agents studied were L-thyroxine, calcium, phosphate, fumarate and reduced glutathione. Mitochondria prepared from liver of new-born rats showed resistance to the swelling action of thyroxine in concentrations below IO-~ M (Fig. I), while swelling of mitochondria from adult-rat liver was marked with lO -5 M thyroxine and occurred with as little as lO -7 M thyroxine. Swelling induced b y thyroxine was somewhat increased in the new-born preparations when the mitochondria were suspended in o.125 M KCl-o.o2 M Tris rather than sucrose-Tris, but the marked difference in swelling between adult and new-born preparations persisted. Minimal swelling likewise occurred with lO -3 M CaCI~ and lO -4 M potassium fumarate (Fig. 2), both of which caused rapid swelling of mitochondria from adult rats. 0.02 M potassium phosphate buffer (pH 7.4) caused swelling to occur in the new-born preparations but at a considerably slower rate and to a lesser degree than in the adult. The addition of 0.005 M reduced glutathione to the test system caused swelling of the mitochondria from new-born animals only after a prolonged lag period, usually 30-60 min following exposure, but the absorbancy then fell nearly to zero as also occurred with mitochondria from adult liver. In the latter, however, the lag period was usually approx. IO min. No consistent difference in swelling reaction was found in mitochondria from rats 2-48 h of age, nor was there any observable difference between mitochondria from animals which had been suckled and those which Were taken from the mother directly after birth. Extreme fragility of mitochondria from foetal tissue has been reported ~. The Abbreviation: Tris, tris(hydroxymethyl)aminomethane.

Biochim. Biophys. Acta, 47 (1961) 411-412

412

PRELIMINARY NOTES

possibility that the observed resistance to swelling in these mitochondria from newborn rats might be a reflection of "pre-swelling" during preparation appears improbable in view of the constant relationship of absorbancy to protein concentration in suspensions derived from both adult and new-born sources. The observed oxidative ability of the mitochondria also suggests that significant damage had not occurred. 07

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Fig. I. T h y r o x i n e - i n d u c e d swelling of m i t o c h o n d r i a f r o m livers of a d u l t a n d n e w - b o r n rats. T e s t s y s t e m c o n t a i n e d o.o 5 m l of m i t o c h o n d r i a l s u s p e n s i o n in 3.o m l of 0. 3 M sucrose-o.o2 M Tris (pH 7.4), c o n t a i n i n g L - t h y r o x i n e in conc e n t r a t i o n s s h o w n . T e m p e r a t u r e , 2o °. N e w b o r n , t--I ; adult, O - - O .

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Fig. 2. R e s p o n s e of m i t o c h o n d r i a f r o m newb o r n a n d a d u l t - r a t livers to swelling a g e n t s . T e s t c o n d i t i o n s as in Fig. i, w i t h a d d i t i o n s of lO -3 M CaC12, IO - t M p o t a s s i u m f u m a r a t e a n d o.oi M p o t a s s i u m p h o s p h a t e (pH 7.4).

It is possible that lack of availability of oxidizable substrate to the liver mitochondria of new-born rats might cause resistance to swelling4, 5, although added fumarate did not induce swelling. The failure of several unrelated agents to produce swelling at concentrations effective with adult mitochondria indicates a non-specific mechanism which may reflect a metabolic or structural difference between mitochondria from new-born and adult animals. This work was supported in part by Research Grant No. A-776 from the National Institutes of Health, U.S. Public Health Service.

Departments of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pa., and Washington University School of Medicine, St. Louis, Mo. (U.S.A.)

MARGARET L. WILLIAMS

1 D. F. TAPLI~Y, J. BioL Chem., 222 (I956) 325 • 2 N. H . CARI~Y AND G. [D. GREVILLE, Bioehem. J., 71 (1959) 161. s F. E. HUNTER, JR., J. F. LEVY, J. F1NK, F. GUERRA AND A. HURWITZ, J. Biol. Chem., 234 (1959) 2176. 4 j . B. CHAPPELL AND G. D. GREVILLE, Nature, 182 (I958) 813.

Received December 24th, 196o Biochim. Biophys. Acta, 47 (1961) 4 i t - 4 I ~