Heterogeneity of chondrocyte mitochondria

583

Biochimica et Biophysica Acta, 451 (1976) 583--591 © Elsevier/North-Holland Biomedical Press

BBA 28093

HETEROGENEITY OF CHONDROCYTE MITOCHONDRIA A STUDY OF THE Ca 2* CONCENTRATION AND DENSITY BANDING CHARACTERISTICS OF NORMAL AND RACHITIC CARTILAGE

I.M. SHAPIRO, A. BURKE and N.H. LEE

Department of Biochemistry and Center for Oral Health Research, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pa. 19174 (U.S.A.) (Received May 24th, 1976)

Summary Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca ~÷ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5--2.0 M) in the presence of inhibitors of Ca 2÷ transport (ruthenium red and 5,5'-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca 2. concentration (309 nmol/mg protein}. Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca 2÷ concentration (up to 5700 nmol/ mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca 2÷ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca 2÷ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca 2+ concentration necessary for initiation of the calcification mechanism was 4 pmol/mg protein.

Abbreviations: tissue zone RC-PC, resting-proliferating cartilage; PC-HTC, proliferating-hypertrophic cartilage; HTC-CC hypertrophic-calcifying cartilage; CC-PM, calcifying-cartilage zone of provisional mineralization. Nbs2, 5,5'-dithiobis-(2-nitrobenzoic acid).

584 Introduction It has been established that mitochondria of both hard and soft tissues accumulate Ca :÷ by processes that are dependent on the presence of extramitochondrial ATP or a respiratory substrate [1--8]. Recent studies suggest that by mediating Ca'* uptake, mitochondria can modify the intracellular ionic environment, and thereby regulate the activity of Ca~'÷-sensitive enzyme systems [ 1 , 9 - 1 3 ] . In soft tissues the intramitochondrial Ca 2÷ concentration is low. However, in hard tissues elevated levels of Ca 2* exist; most frequently, the accumulated ions are seen as electron-dense granules associated with mitochondria [14--16]. The importance of Ca 2. accumulation and granule formation in a calcifying tissue is poorly understood. There is some experimental data to indicate that these organelles accumulate Ca 2÷ as a way of protecting the cell f r o m high Ca 2÷ fluxes [1,9]. A second function attributed to these mitochon'dria is to prepare the cell for the mineralization process by accumulating high concentrations of Ca 2. and Pi [ 4 , 7 , 1 6 - 1 8 ] . It has been further suggested that subsequent transport of these ions into membrane-bound vesicles to the extracellular matrix initiates calcification [19]. In mineralizing tissues variations in mitochondrial Ca 2÷ levels have been shown to be related to differences in enzymatic activities [20]. Heterogeneity of mitochondria in hard tissues is further supported by the observations of Gay and Schraer [21] who showed that while intramitochondriai Ca 2÷ deposits could be seen in osteoblasts, the electron micrographs indicated that some, but not all the mitochondria were filled with granules. On the basis of their dehydrogenase activities, Meyer and Kunin [22] proposed that two types of mitochondria existed in cartilage and these workers speculated that some mitochondria were specialized for Ca2. accumulation. The objective of this investigation was to determine whether mitochondria of different Ca 2. concentrations exist in a mineralizing tissue and to relate the Ca :÷ status of the mitochondria to the degree of tissue mineralization. Employing density gradient centrifugation, the investigation revealed a heterogeneity of mitochondria in epiphyseal plate cartilage. The heterogeneity was greatest in the earliest stages of mineralization and in rickets, and decreased with progressive calcification of the cartilage plate. Materials and Methods Animals. White Rock chicks, aged 8 - 1 2 weeks, were used in all experiments. For studies of rachitic cartilage, 5-day-old chicks were maintained on a rachitogenic diet {Nutritional Biochemicals, Rachitogenic Diet) and deionized water. To initiate healing of the rachitic lesions, 8-week-old chicks were fed a normal diet for 10--14 days. The serum Ca 2. level of the rachitic chicks and the chicks recovering from rickets were 6--7.5 mg% and 9 mg%, respectively. Tissues. The chicks were killed by overdosing with pentobarbital. The epiphyseal plates of the tibiae and femuri were exposed and the tissuc was removed in thin slices as previously described [23]. The tissue zones were restingproliferating cartilage (RC-PC), proliferating-hypertrophic cartilage (PC-HTC), hypertrophic-calcifying cartilage (HTC-CC) and calcifying-cartilage zone of

585 provisional mineralization (CC-PM). No a t t e m p t was made to isolate tissue zones from rachitic chicks and from chicks recovering from the rachitic condition; instead the whole epiphyseal plate was utilized. Preparation of mitochondria. Mitochondria were prepared from the tissue zones in the presence of 0.05 mM Nbs2 (5,5'-dithiobis-(2-nitro benzoic acid)) and/or ruthenium red (5 mg/100 ml). These inhibitors were added to the isolation medium to decrease exogenous Ca 2÷ uptake and redistribution [24--26]. Mitochondria were isolated from the cartilage using a modification of a previously described technique [23]. Thus, following homogenization in a Polytron (Brinkmann Instruments, Inc.) and centrifugation at 14000 X g, a mitochondrial pellet was collected. The pellet was washed, gently homogenized and then re-centrifuged at 300 X g for 2 rain. This washing procedure was repeated twice. The supernatants were collected, combined and centrifuged at 14000 X g for 10 rain and a mitochondrial fraction was obtained. Mitochondria from rachitic cartilage and from cartilage of recovering rachitic chicks were isolated using this procedure. To isolate mitochondria from tissue zone CC-PM, the tissue was first washed extensively with saline to remove blood. The saline was then replaced with an isolation medium [23] that contained both 0.5 mM Nbs: and 0.1 mM ruthenium red. The tissue was homogenized with the Polytron, centrifuged at 900 X g for 5 min and the supernatants collected. The sediment was washed, centrifuged and the supernatants combined. The supernatant was then centrifuged at 14000 X g for 10 min. The pellet was collected, washed and gently homogenized for the density centrifugation step. Using this procedure, a 4to 6-fold increase in the relative specific activity of the cytochrome oxidase was obtained. Density centrifugation. Mitochondria were obtained from cartilage of normal and rachitic chicks and from chicks recovering from rickets. The organelles were isolated in the presence of 0.1 mM ruthenium red and 0.5 mM Nbs:. They were gently homogenized and resuspended in 0.5 ml of an isolation medium containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 10 mM MgCl: and adjusted to isotonicity with 0.25 M sucrose. This was subjected to continuous gradient centrifugation in a gradient made up of 1.5 and 2.0 M sucrose. The mitochondria were centrifuged in a S.W. 29 rotor at 25000 Xg for 90 min. Chemical analysis. The Ca 2÷ concent of the mitochondria was measured by atomic absorption spectroscopy (Perkin-Elmer, Model 360) in the presence of 0.1% lanthanum chloride. The method of Lowry et al. [27] was used to determine the protein concentration of the pellets. Cytochrome oxidase activity was measured by observing the oxidation of cytochrome c at 550 nm [28]. Results and Discussion This study revealed that mitochondria of cells of mineralizing cartilage could be separated into different density fractions using continuous gradient centrifugation. In the earliest of the mineralizing zones (RC-PC) two bands are seen; the major fraction (band 2) exists as a distinct band, or as a broad suspended zone (Fig. 1). In tissue zone PC-HTC the major fraction (band 2b) is denser than

586

CONTINUOUS GRADIENT

ZONE

~ODISTRIBUTION BAND

UU i

RC-PC

ii

i

PC-HTC

HTC-CC

CC-PM

ii

UU U

U

i

ii

20 20 80 80

i

ii

10 10 10

1 2a

80 90

2b

5

1

95

2

1 99

Fig. 1. C o n t i n u o u s gradient centrifugation characteristics of c h o n d r o c y t e m i t o c h o n d r i a . M i t o c h o n d r i a were c e n t r i f u g e d in a c o n t i n u o u s density gradient ( 1 . 5 - - 2 . 0 M sucrose) at 2 5 0 0 0 r e v . / m i n for 9 0 rain. In b o t h z o n e s RC-PC a n d PC-HTC t w o t y p e s of banding p a t t e r n s w e r e o b s e r v e d (i a n d ii). Z o n e CC-PM r e p r e s e n t s calcified cartilage, t h e z o n e of provisional m i n e r a l i z a t i o n .

2.0 M sucrose. Traces of the suspended fraction (band 2a) are frequently seen and a small low density fraction is also present (band 1). While never occupying more than 10% of the total mitochondrial protein, variations in the banding pattern of tissue zones RC-PC and PC-HTC probably represented zonal contamination. For example, band 2a would represent contamination of tissue zone PC-HTC with mitochondria of the contiguous RC-PC zone. This type of contamination is unavoidable in the chick due to the anatomical interdigitation of the tissue zones of the growth plate. In all zones of the plate, at least two bands are seen. These comprise a major high density band (band 2) and lighter suspended bands (band 1). With progressive mineralization, the lighter suspended mitochondria gain Ca ~÷ and thereby produce the characteristic banding of tissue PC-HTC, HTC-CC and CC-PM. Changes in banding patterns as a function of Ca 2. loading have been previously demonstrated by other workers [29,30]. For example, using liver mitochondria that normally form a single low density band, Greenawalt et al. [29] showed

* OF MITOCHONDRIA

ISOLATED

214 525

73--464 287--699

OXIDASE ACTIVITY

* OF MITOCHONDRIA

541 2025

ISOLATED

395-- 821 1100--2720

Range

0.015 0.028

0.010--0.019 0.020--0.037

Range

• * Mean of four determinations. • ** M e a n o f f i v e d e t e r m i n a t i o n s . T Mean of six d e t e r m i n a t i o n s .

• nmol/g protein.

Band 1 Band 2

Mean * *

RC-PC

0.028 0.036

Mean ***

PC-HTC

1530 4178

Mean * *

HTC-CC

0.025 0.056

Mean T

HTC-CC

0.020--0.057 0.044-0.094

Range

CARTILAGE

0.015 0.087

Mean T

CC-PM

730 5691

Mean * *

CC-PM

0.007--0.030 0.069--O.119

Range

585--1031 2914--9450

Range

sucrose density gradient (1.5--2.0 M sucrose).

836--2360 1738--7500

Range

FROM CHICK EPIPHYSEAL

0.010--O.064 0.016---0.047

Range

M i t o c h o n d r i a w e r e i s o l a t e d u s i n g t h e s a m e c o n d i t i o n s as d e s c r i b e d i n T a b l e I.

CYTOCIIROME

T A B L E II

* nmol/g protein. ** M e a n o f six d e t e r m i n a t i o n ~ *** Mean of five determinations.

Band 1 Band 2

Mean * * *

Mean * *

Range

PC-HTC

RC-PC

PLATE

red; final purification was on a continuous

FROM CHICK EPIPHYSEAL

Mitochondria were isolated in the presence of Nbs 2 and ruthenium

C a 2÷ C O N C E N T R A T I O N

TABLE I

¢j1 GO

588

band

% distribution

RACHITIC

~

1

10

CARTILAGE

U

20

]0

2b

80

~-'-I

]

10

U

2

9o

HEALING CARTILAGE

Fig. 2. Continuous gradient eentrifugation characteristics of mitochondria of raehitie cartilage and cartilage of raehitie chicks t r e a t e d w i t h v i t a m i n D a n d Ca 2+.

t h a t mitochondrial Ca 2. accumulation resulted in the formation of 1--3 bands of increasing Ca 2* concentration and density. In soft tissues, undergoing dystrophic change, there is an increase in high density m i t ochondri a and a conc o m i t a n t increase in the Ca 2÷ concentration of the high density fraction [30]. The Ca 2÷ concentration of the m i t ochondri a isolated by continuous gradient centrifugation is shown in Table I and Table III. Even in the rachitic state, mitochondrial Ca 2÷ levels are very high. Compared to liver, c h o n d r o c y t e mitochondria o f rachitic and normal chicks show a 20- to 100-fold and 350-fold increase in Ca ~-÷ concentration respectively [1]. The Ca 2. levels reported here are o f a similar magnitude to the values described by Chen et al. [7] for the m i t o ch o n d r ia o f the blue crab hepatopancreas where mitochondrial Ca 2÷ mobilization is associated with the moulting cycle. With mineralization of the chick epiphyseal growth plate, there is a progressive increase in the Ca 2÷ concentration of m i t o c h o n d r i a isolated from tissue zones RC-PC, PC-HTC, ttTCCC and CC-PM. With the exception of the very small low density band of tissue zone CC-PM, this increase in Ca ~÷ concentration is exhibited by both the high and low density mitochondria. The mitochondrial Ca 2÷ concentrations noted ill this study are higher than previously reported [20]. This is probably due to the differences in the centrifugation procedure employed. With reference to the c y t o c h r o m e oxidase activities of the mitochondrial fractions, Table II shows that the activities of the high density mitochondria are considerably greater than the low density band. Fur t he rm ore, as Arsenis 120] reported, there appears to be an increase in the activity of this e n z y m e with tissue mineralization. The banding pattern of m i t oc hondr i a from rachitic cartilage and cartilage of recovering rachitic chicks is shown in Fig. 2. The sedimentation patterns are similar to tissue zone PC-HTC, i.e., a dense band comprising about 80% of the total mito ch o n d ri a (band 2b) and a less dense fraction which is seen as a single band (band 1), or doubl e t (bands 1 and 2a). Table III shows that the Ca 2+ concentration of the rachitic m i t oc hondr i a is low and comparable with tissue zone PC-HTC. In addition, the c y t o c h r o m e oxidase activities of tissue zone PC-HTC

ISOLATED

FROM

RACHITIC

262 1605

55-- 430 1047--2181

Range

• Mean o f five d e t e r m i n a t i o n s . • * Mean o f t h r e e d e t e r m i n a t i o n s .

Band 1 Band 2

Mean *

(nmol/mg protein)

Ca 2+

Rachitic

0.031 0.027

Mean * 0.016---0.055 0.015---0.034

Range

Cytoehrome oxidase (retool/rain per mg protein)

1265 4171

M e a n **

1100--1503 3413--4817

Range

Ca2+ (nmol/mg protein)

"HeaLing"

CHICK CARTILAGE

0.016 0.051

Mean * *

0.012---0.020 0.037---0.061

Range

Cytoehrome oxidase (mmol/min per mg protein)

M i t o e h o n d r i a w e r e i s o l a t e d f r o m t h e w h o l e e p i p h y s e a l g r o w t h p l a t e . T h e m e t h o d o f i s o l a t i o n w a s t h e s a m e as t h a t d e s c r i b e d in Fig. 3.

Ca 2 . C O N C E N T R A T I O N AND CYTOCHROME OXIDASE ACTIVITY OF MITOCHONDRIA CARTILAGE OF CHICKS RECOVERING FROM THE RACHITIC CONDITION ("HEALING")

T A B L E IlI AND

Ln e,D

590 and rachitic mitochondrial fractions are very similar. On the basis of these analyses it can be concluded that the effects of withholding vitamin D and Ca 2. from a chick produces an intracellular environment in which the mitochondrial Ca :+ concentration and cytochrome oxidase activity are characteristic of an early stage in the mineralization process. During recovery from a vitamin D deficiency these parameters radically change and the profile of the whole plate resembles the advancing mineralization edge of the normal epiphyseal plate. Thus, the mitochondrial Ca 2÷ concentration and the oxidase activity dramatically increase while the percentage of light mitochondria drops. It is clear from these analyses of mitochondria of rachitic and normai cartilage that the mitochondrial cation concentration can be used as a qualitative indicator of a process that is customarily described in morphological terms. While a detailed study of re-mineralization of cartilage following the rachitic conditions was not undertaken, the data shown in Table III indicate that in the rachitic state mitochondria contain a third less Ca 2÷ than when vitamin D and Ca 2. are restored to the diet. When recovery commences, there is a rise in the serum Ca :+ concentration and a concomitant increase in cellular Ca :+. Whether the cellular changes characteristic of a mineralizing system occur as a result of a rise in cytosol Ca:+ or are secondary to a change in mitochondrial function is unknown. However, recent work suggests that the mitochondria load with Ca :+ until a critical concentration is reached and then there is interference with coupled oxidative phosphorylation [1,31]. If there is now insufficient ATP being generated by the mitochondria to maintain the accumulated Ca 2+, then efflux could occur and this could initiate the calcification process. The critical mitochondrial cation levels necessary for Ca :+ efflux have ~ot been determined, but the values found in band 2 of cartilage of rachitic chicks treated with vitamin D suggest that this is in the region of 4 pmol/mg protein. Acknowledgements The critical comments of Dr. Neils Haugaard are gratefully acknowledged. This investigation was supported by National Institute of Dental Research grant DE-02623. References 1 L e h n i n g e r , A.L., Carafoli, E. a n d Rossi. C.S. ( 1 9 6 7 ) Adv. E n z y m o l . 2 9 , 2 5 9 - - 3 1 9 2 Tager, J.M., Papa, S., Quagliariello, E. a n d Slater, E.C. ( 1 9 6 6 ) in R e g u l a t i o n of M e t a b o l i c Processes in M i t o c h o n d r i a , p. 559, Elsevier Publishing Co., A m s t e r d a m 3 V a s i n g t o n , F.D. a n d M u r p h y , J.V. ( 1 9 6 2 ) J. Biol. C h e m . 2 3 7 , 2 6 7 0 - - 2 6 7 7 4 L e h n i n g e r , A.L. ( 1 9 7 0 ) B i o c h e m . J. 119, 1 2 9 - - 1 3 8 5 C h a n c e , B. ( 1 9 6 5 ) J. Biol. C h e m . 240, 2 7 2 9 - 2 7 4 8 6 C h a n c e , B., Azzi, A. a n d Mela, L. ( 1 9 6 9 ) in T h e Molecular Basis of M e m b r a n e F u n c t i o n (D.C. Tosteson. ed.), p. 561, P r i n c e t o n Hall, New Jersey 7 Chert, C . H . , G r e e n a w a l t , J.W. a n d L e h n i n g e r , A.L. ( 1 9 7 4 ) J. Cell Biol. 6 1 , 3 0 1 - - 3 1 5 8 Shapiro, I.M. a n d Lee. N.H. ( 1 9 7 5 ) Clin. O r t h o p a e d . 1 0 6 , 3 2 3 - - 3 2 9 9 Borle, A.B. ( 1 9 7 5 ) J. M e m b r a n e Biol. 21, 1 2 5 - - 1 4 6 10 R a s m u s s e n , H. a n d Bordter, P. ( 1 9 7 4 ) in T h e Physiological a n d Cellular Basis of Metabolic Bone Disease, pp. 1 2 8 - - 2 0 5 , Williams & Wiikins Co. 11 R a s m u s s e n , H., G o o d m a n , D.B.P. a n d T ~ n e n h o u s e , A. ( 1 9 7 2 ) in Critical R e v i e w s in B i o c h e m i s t r y , pp. 95-..148 12 Meli, J. a n d Bygrave, F.L. ( 1 9 7 2 ) B i o c h e m . J. 128, 4 1 5 - - 4 2 0

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