Electron Microscopic Localization of Calcium in Vascular Smooth Muscle GAMIL DEBBAS, LOREN HOFFMAN, ERWIN J. LANDON AND LEON HURWITZ Departments of Pharmacology and Anatomy, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and Department of Pharmacology, University of New Mexico School of Medicine, Albuquerque, New Mexico 871 31

ABSTRACT Potassium pyroantimonate has been employed in this study to localize calcium in the vascular smooth muscle of the thoracic aorta of the rabbit. The pyroantimonate ion precipitates sodium, magnesium and calcium. Incubation of the isolated thoracic aorta in a high potassium bathing medium which does not contain sodium, magnesium or calcium depletes the tissue of sodium. Addition of 10.8 mM CaCl, to the incubation medium results in welllocalized depositions of reaction product, presumably that of calcium pyroantimonate, in mitochondria, sarcoplasmic reticulum, and at the plasma membrane. Some or all of these organelles may, therefore, play a vital role in the contractionrelaxation cycle of vascular smooth muscle.

The localization of calcium in muscle is a subject of considerable interest since the contraction-relaxation cycle of all types of mammalian muscle is controlled by calcium ions (Bohr, '64; Constantin et al., '65; Daniel, '65; Somlyo and Somlyo, '68; Bohr et al., '69; Hurwitz and Suria, '71). With the use of sodium oxalate as the precipitating agent, calcium has been visualized as electron opaque deposits of calcium oxalate in striated muscle from which the plasma membrane was removed (Constantin et al., '65; Podolsky et al., '70). Calcium has also been visualized at the subcellular level in intact cardiac (Legato and Langer, '69; Yarom and Braun, '71; Yarom et al., '72) and skeletal (Yarom and Meiri, '71) muscle fibers by employing potassium pyroantimonate to give an insoluble electron opaque deposit of calcium pyroantimonate. Somlyo and Somlyo ('71) have succeeded in localizing strontium in vascular smooth muscle. The accumulation of this ion in both mitochondria and sarcoplasmic reticulum, as well as the localization of barium in mitochondria reported earlier (Somlyo and Somlyo, '71; Devine et al., '73), has recently been confirmed by using electron probe analysis (Somlyo et al., '74). Moreover, x-ray microanalysis of mitochondria indicated the presence of calcium and ANAT. REC., 182: 447.472.

strontium (Somlyo et al., '74). As a result of these findings, Somlyo and his group suggested that both the sarcoplasmic reticulum and mitochondria are sites where calcium ion may be sequestered within the muscle fiber. Moreover, they postulated that these two organelles may play a vital role in the contraction-relaxation cycle of vascular smooth muscle (Somlyo and Somlyo, '71; Devine et al., '73; Somlyo et al., '74). The direct demonstration of the subcellular location of calcium in the smooth muscle of the rabbit aorta has not as yet been reported. In this study potassium pyroantimonate was employed to localize calcium in the vascular smooth muscle of the rabbit aorta. Rabbit aortic smooth muscle has a tightly bound pool of calcium that can be mobilized by excitatory agents (Hudgins and Weiss, '68; Van Breemen et al., '72; Deth and Van Breemen, '74) ; however, the intracellular location(s) of such a pool is a matter of speculation. This study, designed to demonstrate if and where intracellular depots of calcium may be located in such a muscle fiber, may, therefore, serve several useful purposes. First, it could verify or extend present notions Received Dec. 10, '74. Accepted Feb. 24, '75.

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G. DEBBAS, L. HOFFMAN, E. J. LANDON AND

L. HURWITZ

was stored overnight in the refrigerator. On the following day prior to its use, the fixative was passed through a millipore filter (0.22 LL, Millipore Corp.) under pressure to eliminate the presence of suspended pyroantimonate particles. The pH of the fixative was adjusted to 7.8 at 4°C with 0.05 N acetic acid, The cold fixative was again passed through a millipore filter a few minutes before it was used. Klein et al. ('72) recommended that the potassium pyroantimonate fixative be stored overnight in the refrigerator and centrifuged the next day prior to its use. We found our procedure of millipore filtration is an adequate substitute for the centrifugation step employed by Klein et al. ('72). During an MATERIALS AND METHODS experiment the circular segments of vasI. Methods cular tissue were immersed in the cold fixMale New Zealand white rabbits weigh- ative for a period of 20-25 minutes. The ing 1300-2300 gm were stunned by a blow segments were then cut into small strips, on the head and exsanguinated. A portion approximately 2 X 3 mm in size, using of the thoracic aorta, 1-3 cm above the several new stainless steel blades for each diaphragm and 2-3 cm below the aortic circular segment, and re-immersed in the arch, was gently but rapidly dissected free, fixative. Total fixation time was 90 mincut into circular segments approximately utes. It is important that fixation of tissues 5-7 mm in length and quickly blotted on be carried out in the cold (4°C) when pofilter paper to remove the blood that had tassium pyroantimonate is incorporated in adhered to the tissue. The circular seg- the fixative. Cold fixation minimizes damments were subjected to one of the follow- age to the tissue (Klein et al., '72; Debbas et al., '73; Debbas, '73). ing treatments : The pH of the fixative containing the A. Direct fixation in a potassium pyrotissue was 7.8 I 0 . 0 5 during fixation and antimonate-0s04 fixative after completion of fixation. It is essential One group of vascular tissues was im- that the pH of the fixative remain slightly mersed in a fixative immediately after it alkaline during fixation in order to avoid was isolated from the animal. The fixative the non-specific precipitation of the pyrowas prepared as follows: a 5% solution of antimonate anion from the fixing solution potassium pyroantimonate ( KnSbzOr.4Hz0, (Klein et al., '72). At the conclusion of fixFisher Scientific Co.) was made by boiling ation and prior to the first dehydration 5 grams of the reagent in 100 ml of dis- step, the tissue was rinsed twice during a tilled deionized water (Spicer et al., '68). 5-10 minute period in an aqueous alkaline The solution was left to cool to room tem- medium (0.05 M potassium acetate buffer, perature, restored to its original volume pH 7.8, at 4'C). This procedure has been and filtered (Whatman #5 filter paper) to shown to remove any unreacted potassium remove any pyroantimonate that precipi- pyroantimonate from the tissue and pretates on cooling. An appropriate aliquot vents non-specific precipitation of the reof the pyroantimonate solution was then agent by ethanol (Shiina et al., '70). mixed with 2% OsO, and 0.1 N acetic acid After rinsing the tissues in the potasso that the fixative consisted of 2% potas- sium acetate buffer, they were dehydrated sium pyroantimonate, 1% OsOl and 0.04 N in a graded series of cold ethanol, and acetic acid. The fixing solution was then finally, in propylene oxide. They were then placed in a stoppered glass container which embedded in araldite. The tissues were oriwas immersed in a bucket full of ice (4°C). ented under a dissecting microscope so The bucket of ice containing the fixative that the endothelial side would rest on

about the locations of intracellular calcium depots in this vascular smooth muscle deduced from indirect evidence (Baudouin et al., '72; Fitzpatrick et al., '72; Devine et al., '72; Somlyo, '72; Hurwitz et al., '73; Bohr, '73). Second, i t could, by revealing areas of high calcium concentration, pinpoint those intracellular loci or organelles that are most apt to contain active sequestration systems for removing free calcium ions from the cytoplasm. Third, it could pinpoint those intracellular loci and organelles that have the capacity to serve as possible internal reservoirs from which calcium ions may be mobilized for contraction.

LOCALIZATION OF CALCIUM IN SMOOTH MUSCLE

the bottom of the flat embedding mold. After hardening, the blocks of tissue were trimmed in order to remove the fibrous adventitia from the segments of aorta. This step facilitated subsequent sectioning (Pease and Paule, '60). Sectioning was done on a Sorvall, Porter-Blum ultramicrotome MTII using a diamond knife. Ultrathin sections obtained from tissues of procedures A, B and C (described below) were examined with a Hitachi HU-11B electronmicroscope a s follows : ( 1) Unstained, ( 2 ) Stained with a 6% solution of uranyl acetate in 50% ethanol for periods of 1-5 minutes, ( 3 ) Stained only with lead citrate for 1-5 minutes, ( 4 ) Stained with uranyl acetate and then with lead citrate for 1-5 minutes in each of the staining solutions. Both uranyl acetate and lead citrate were passed through a millipore filter under pressure (0.05 y , Millipore Corp. ) to eliminate the possible presence of suspended particles which could otherwise randomly precipitate on the sections. The distribution of reactions product (within the aortic tissue) in serially cut sections which were unstained was compared with those that were stained a s mentioned above in ( 2 ) , ( 3 ) and ( 4 ) to determine if either uranyl, lead, or both altered the distribution of reaction product within the aortic smooth muscle fiber. Since no differences in distribution were found, all electron micrographs presented in this report were obtained from sections stained with uranyl acetate for 3-5 minutes and then with lead citrate for 2-2.5 minutes. B. Incubation in a depolarizing solution containing CaCL (1) A second group of circular segments of aorta, before being fixed, were incubated in vials containing a depolarizing solution for a total of 50 minutes. The depolarizing solution had the following composition: tris buffer (tris (hydroxymethyl) aminomethane, Sigma Chemical Co.), 23 mM; KC1, 127 mM; glucose, 11 mM; and CaCL, 10.8 mM. The pH of the solution was adjusted to 7.5 with 6 N HC1 and its temperature was maintained at 25°C C 1 ° C or at 34°C 1°C in a water bath. Just prior to use all solutions were aerated with 100% oxygen for 15-20 minutes. Every ten minutes the vials containing the tis-

*

449

sues were drained and replenished with fresh oxygenated depolarizing solution, the temperature of which was identical to that drained from the vial. At the end of the incubation period the circular segments were blotted on filter paper and immersed at once in the cold fixative. They were then processed as described under procedure A. ( 2 ) A third group of circular segments was incubated in the depolarizing solutions as i n ( 1 ) . The period of incubation was 45 minutes. The temperature of the solution was 25°C 1°C. Following its incubation in the depolarizing solution, the third group was divided into three subgroups, a, b, and c. Each subgroup was then subjected to one of the following treatments prior to fixation in the potassium pyroantimonate-0s04 fixative: ( a ) The first subgroup of tissues was washed for 15 minutes i n the depolarizing solution from which CaCL was omitted (calcium-depleted depolarizing solution). The solution in which the tissues were immersed was exchanged three times for fresh solution during this 15 minute period. ( b ) The second subgroup of tissues was washed for 10 minutes in calcium-depleted depolarizing solution that contained M EGTA (ethylene-guanidine-tetraacetate) . It was then washed for another five minutes in the calcium depleted depolarizing solution. During the ten minute and five minute washes each solution was exchanged three times for fresh solution. (c) The third subgroup of tissues was washed for five minutes in calcium-depleted depolarizing solution that contained M EGTA. It was then washed for an additional five minutes in the calcium-depleted depolarizing solution. During each five minute wash, the solution was exchanged three times for fresh solution. Finally, the tissues were immersed for five minutes in a potassium pyroantimonate solution a t 4°C. This solution had the same composition and pH as the potassium pyroantimonate-0s04fixative except that the OsOl was omitted. Tissues from subgroups a , b and c were then blotted on filter paper, immersed at once in the cold fixative and subsequently processed as described under procedure A. As indicated above, the tissues subjected to procedures b and c were washed in a calcium-depleted depolarizing solution that

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G. DEBBAS, L. HOFFMAN, E. J. LANDON AND L. HURWITZ

contained EGTA. They were then washed reports it was indicated that potassium three times in calcium-depleted depolariz- pyroantimonate can precipitate a number ing solution to remove unreacted EGTA of substances to various degrees (Komnick and Ca-EGTA complex present in the ex- and Komnick, '63; Bulger, '69; Shiina et tracellular space. The removal of Ca-EGTA al., '70; Klein et al., %), that it forms comis essential because EGTA and pyroanti- plexes with others (Clark and Ackerman, monate will compete for calcium ions '71b), that its reaction with certain cations (Klein et al., '72). Moreover, if EGTA is may be influenced by the buffer (Torack present in the tissues or is incorporated in and LaValle, '70) or fixative employed the potassium pyroantimonate fixative, it (Clark and Ackerman, '71a; Garfield et al., could lead to diffusion artifacts and poor '72), that the reagent itself may precipitate osmium fixation (Klein et al., '72). from solution at low pH (Tani et al., '69; Klein et al., '72) or be precipitated withC . Incubation in a depolarizing solution in the tissue during dehydration as mencontaining n o added CaCL tioned earlier (Shiina et al., '70). In contrast to procedure B in which tisThe precipitation of cations such as SOsues were incubated for 45 or 50 minutes dium, calcium and magnesium by pyroin a depolarizing solution containing cal- antimonate ion in various tissues has been cium, tissues in a fourth group were incu- confirmed by electron microprobe analysis bated for 50 minutes at 25°C 2 1°C in (Tandler et al., '70; Kierszenbaum et al., a calcium-depleted depolarizing solution. '71). These studies (Tandler et al., '70; Every ten minutes the vials containing the Kierszenbaum et al., '71) showed that potissues were drained and replenished with tassium pyroantimonate, when used withfresh oxygenated calcium-depleted depolar- out the addition of a conventional fixative, izing solution, the temperature of which produces intracellular precipitates of calwas identical to that drained from the vial. cium, sodium and magnesium pyroantimoAt the end of the incubation period the nates in a variety of tissues. Klein et al. circular segments were blotted on filter ('72), using atomic absorption analysis, paper and immersed at once in the cold found that a 2% solution of potassium fixative. They were then processed as de- pyroantimonate could precipitate calcium, scribed under procedure A. Table 1 out- magnesium and sodium ions when these lines the steps taken in the performance ions were present in a concentration as of procedures A, B (1) and ( 2 ) and C. The low as 0.001 mM, 0.01 mM, and 7-8 mM, number of animals studied in each of the respectively. This finding indicates that at above mentioned procedures and the num- least under in vitro conditions this method ber of blocks examined are given in table 2. is more sensitive to calcium than to the other two cations. Moreover, these investi11. Rationale for the pyroantimonategators (Klein et al., '72) proposed several reaction in this study important guidelines for the preparation Komnick ('62) introduced the reagent and proper use of the potassium pyroantipotassium pyroantimonate to localize so- monate reagent in conjunction with elecdium ions in cells and tissues. Recently, tronmicroscopy. Such guidelines, in our however, this reagent has gained popular- opinion, are of extreme importance for the ity to localize calcium ions at the subcellu- proper use of the pyroantimonate reagent lar level (Legato and Langer, '69; Yarom in any electronmicroscopic study and most and Braun, '71; Yarom and Meiri, '71; Ya- of their guidelines have been used in this rom et al., '72; Brighton and Hunt, '74; study. Hales et al., '74) in spite of reports conThe fact that potassium pyroantimonate cerning its polyvalency, and the complex- can form precipitates with sodium, calcium ities (pitfalls) that arise when using this and magnesium makes it a useful tool for reagent in cytochemical studies (Komnick examining the subcellular distribution of and Komnick, '63; Bulger, '69; Tani et al., these inorganic cations. Thus, a number of '69; Shiina et al., '70; Torack and LaValle, investigators have utilized the pyroantimo'70; Clark and Ackerman, '71a,b; Klein et nate anion to localize sodium ion (Spicer al., '72; Garfield et al., '72). In the latter et al., '68; Zadunaisky et al., '68; Shiina et

ited

Fixed in potassium pyroantimonate-0s04

Not incL

Procedure A

\

Fixed as

CaCL

Incubated for 50 min. at 25°C or 34°C i n depolarizing solution

TABLE 1

Fixed as A

Washed for 15 min. in calcium depleted deuolarizing solutio

Fixed as A

Washed for 5 min. in calcium depleted depolarizing solution

Washed for 5 min. in calcium dedeted depolai :ing solution

Fixed as A

i

in a 2% solution of potassium pyroantimonate - no OsOr present

Washed for 5 min. in calcium depleted depolarizing solution containing 10-3 M EGTA

Washed for 10 min. in calcium depleted depolarizing solution containing M EGTA

Incubated for 45 min. at 25°C in depolarizing solution containing 10.8 mM CaCI2

Procedure B (2)

Fresh aortic circular segments

Steps performed in procedures A, B. C

Fixc

as A

Incubated for 50 min. at 25°C in depolarizing solution containing no added CaCL (control tissue)

452

G. DEBBAS, L. HOFFMAN, E. J. LANDON AND L. HURWITZ TABLE 2 Number of blocks examined i n each treatment group Number of animals studied

A

B1

BZ < a )

Bz (b)

B2 ( r )

2

C

~~

3 3 2

18 20

10

2

1 1 3

14

9

9 19

~~~

1 Temperature of incubation 34°C. 2 A minimum of 2 4 grids were examined from each block of tissue. Thin sections were usually obtained from two different levels within each block by penetrating (trimming) deeper into the tissue.

al., '70) and to determine, in a nonspecific manner, the subcellular distribution of cations (Tandler et al., '70;,Kierszenbaum et al., '71; Tandler and Kierszenbaum, '71; Tisher et al., '72; Yarom and Meiri, '72; Herman et al., '73). Others have extended the usefulness of the pyroantimonate method to disclose specific subcellular sites where calcium and/or magnesium are localized (Legato and Langer, '69; Klein et al., '70; Klein-Thureson and Klein, '71; Yarom and Braun, '71; Yarom and Meiri, '71; Yarom et al., '72; Brighton and Hunt, '74; Hales et al., '74). In some of the latter studies electron probe analysis (Yarom et al., '72) or x-ray microanalysis (Hales et al., '74) were used to prove that calcium was the predominant cation in the electron opaque precipitate which had been visualized by the electron microscope. These latter analyses thus support the applicability of the pyroantimonate method to localize calcium in mammalian cells and tissues. In view of what has been discussed above concerning the pyroantimonate method, our experimental planning took into consideration the following: (1) The recommendations of Klein et al. ('72) and personal communication (R. L. Klein), and Shiina et al. ('70) for the proper use of the pyroantimonate reagent (see METHODS); ( 2 ) Most of the limitations of the pyroantimonate method as discussed above, especially its polyvalency concerning sodium and magnesium; ( 3 ) The physiologic and pharmacologic properties of the rabbit aortic smooth muscle; ( 4 ) The effect of OsOl (Krames and Page, '68) on translocation of extracellular calcium into muscle fibers; and ( 5 ) The effect of stains, uranyl and/or lead on the distribution of reaction product (see METHODS). The following rationale as

discussed below underlies the experimental plan chosen as a consequence of the considerations, i.e., ( 2 ) , ( 3 ) and ( 4 ) mentioned above. The tissues that were fixed without prior incubation (procedure A ) served as a measure of the best fixation that can be achieved with the fixative employed in this study. In addition, since the concentration of sodium is high relative to the concentrations of calcium and magnesium in the rabbit aorta (Altura and Altura, '71), much or most of the precipitate is likely to be sodium pyroantimonate in such a tissue. The results of procedure A would, therefore, reflect the distribution of sodium, as well as that of magnesium and calcium in the aortic smooth muscle. Due to the very rapid efflux of sodium from vascular smooth muscle (for review, Somlyo and Somlyo, '68) incubation of the tissues in the absence of extracellular sodium, calcium and magnesium (procedure C) washes out a substantial fraction of intracellular sodium. A major reduction in the sodium content of the rabbit aorta following incubation in a sodium-free medium has been reported by several investigators (Dodd and Daniel, '60; Jones et al., '73). Some calcium and magnesium may also be washed out. As described under RESULTS, comparison by electron microscopy of tissues incubated for 50 minutes in calcium depleted depolarizing solution (procedure C ) with those that were not incubated prior to fixation (procedure A ) supports the contention that depletion of cations does indeed occur. Such a depleted tissue (procedure C ) may be used as a control for tissues incubated for 50 or 45 minutes in depolarizing solution in which sodium and magnesium are lacking but

LOCALIZATION OF CALCIUM IN SMOOTH MUSCLE

453

10.8 mM CaCl, is present (procedures B lular calcium ion in solution was removed by rinsing the tissue in a calcium-depleted ( 1 ) and B ( 2 ) , respectively). By incubating tissues for 50 minutes in depolarizing solution that contained a depolarizing medium that contains CaCl, M EGTA, then in a calcium-depleted de(procedure B ( 1 ) ) the smooth muscle fi- polarizing solution in which EGTA was bers can be loaded with calcium and, at omitted, and finally in a solution of potasthe same time, depleted of their sodium sium pyroantimonate that was free of and magnesium. Thus procedure B ( 1 ) oso4.Our aim i n exposing the tissue to will delineate sites where the storage or potassium pyroantimonate prior to fixation, binding of calcium occurs in the depolar- was ( 1 ) to precipitate any remaining exized fiber without undue interference from tracellular calcium ion that may not have sodium and magnesium precipitates. As been chelated or washed out in the predescribed above, tissues subjected to pro- ceding steps and ( 2 ) to insure the prescedure B ( 1 ) were incubated in a calcium ence of the pyroantimonate ion in the containing depolarizing solution and then extracellular space of the tissue before inipromptly fixed in a potassium pyroantimo- tiating the fixation procedure. The latter nate-Os04 solution. As a result, a high con- condition was assumed to enhance the centration of calcium was present i n the likelihood that the pyroantimonate reagent extracellular space when the tissues were would move ahead of the OsO, into the immersed in the fixative. Under these ex- intracellular areas of the smooth muscle perimental conditions it is quite possible fiber. This, i n turn, might provide a better that extracellular calcium may enter the opportunity for the pyroantimonate ion to smooth muscle fiber during fixation. The reach and react with the calcium ions lopossibility is enhanced by evidence sug- cated in these areas before the OsOr could gesting that oso4makes the plasma mem- get there and allegedly modify the internal brane more permeable to calcium ions distribution of this cation. The above rea(Krames and Page, '68). Furthermore, the soning is based on results of previous calcium that moves into the smooth muscle studies which have indicated that potascell during fixation may attach itself to sium pyroantimonate, in the absence of a intracellular sites and may subsequently fixative can penetrate intact mammalian be precipitated at these sites by the pyro- cells (Tandler et al., '70; Kierszenbaum et antimonate ion. If the latter sequence of al., '71). events were to occur, reaction product seen RESULTS at specific sites inside the smooth muscle Figures 1 and 2 represent the results fiber would be a n artifact of OsO4fixation, rather than the accumulation of calcium obtained using procedure A. The reaction generated by physiological processes prior product was randomly distributed throughto fixation. out the smooth muscle cells. Nuclei and The above considerations led to a n at- nucleoli were observed to have a n abuntempt to wash out and/or precipitate the dance of reaction product compared with calcium i n the extracellular space before that seen in the cytoplasm and its organexposing the tissue to the potassium pyro- elles (fig. 1). Mitochondria had a modantimonate-OsOc fixative (procedures B erate-to-dense accumulation of reaction ( 2 ) a, b and c ) . I n procedure B ( 2 ) a, product (fig. 1 ) although, in a few inextracellular calcium ion was washed out stances, mitochondria with very little reof the tissue, prior to fixation, by rinsing action product were encountered (fig. 2 ) . the tissue for 15 minutes i n a depolarizing On occasion reaction product was also solution that did not contain any added found at the inner surface of the plasma CaC1,. I n procedure B ( 2 ) b, as a n added membrane. Its distribution appeared to be precaution, the tissues were first rinsed in erratic (figs. 1, 2 ) . The quantity of rea calcium-depleted depolarizing solution action product found in the sarcoplasmic reticulum was inconsistent, varying from M EGTA before washthat contained ing the tissues in the calcium-depleted de- a slight amount to none (fig. 1 and Debpolarizing solution that did not contain bas, '73). This near random distribution EGTA. In procedure B ( 2 ) c, the extracel- of reaction product is expected, since one

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G . DEBBAS, L. HOFFMAN, E. J. LANDON AND L. HURWITZ

would predict that any unbound sodium would be randomly precipitated. In view of these results, it was deemed essential that an attempt be made to wash sodium ions out of the tissue. By reducing or removing the formation of sodium precipitates, the selective sequestration of calcium ions by organelles could be more clearly delineated in electron micrographs. The distribution of reaction product observed in aortic smooth muscle that was incubated for 50 minutes at 25°C in a calcium-depleted depolarizing solution (procedure C) is shown in figure 3. The amount of non-specific cytoplasmic reaction product present in the tissues following their incubation in a sodium-free medium was far less than that observed in fresh, unincubated tissues (figs. 1, 2). This reduction in reaction product within the smooth muscle is presumed to reflect the loss of unbound sodium as well as some of its calcium and magnesium. The nuclei and nucleoli appeared to be the only two organelles where reaction product was prominent. The mitochondria contained a very slight amount of reaction product or had none at all. The plasma membrane had a sparse amount of reaction product, while the sarcoplasmic reticulum usually had none (fig. 3). Figures 4 and 5 represent the results obtained using procedure B ( 1) , The presence of a relatively high concentration of calcium in the incubating medium led to the appearance of new sites of reaction product deposition. As in procedure C, the cytoplasm had little non-specific randomly distributed reaction product. Nuclei were observed to contain a sparse amount of reaction product (fig. 4); i.e., less than control; however, nucleoli (not shown in this report, but elsewhere (Debbas, '73) ) retained a concentration of reaction product that was similar to that seen in nucleoli of control tissues (i.e., procedure C ) . Mitochondria appeared to have accumulated large amounts of reaction product (fig. 4), a finding not encountered in the control tissues. Reaction product deposition was almost uniform along the entire inner surface of the plasma membrane of the smooth muscle cell (figs. 4, 5). It was also attached to the membrane of the sarcoplasmic reticulum (figs. 4, 5). The ac-

cumulation of reaction product at this site varied from slight to dense (figs. 4, 5). The results obtained with a variation of procedure B (1) in which tissues were incubated at a higher temperature, i.e., at 34°C (to approach a physiological temperature) were essentially identical to those obtained at 25°C (figs. 4, 5). The mitochondria, plasma membrane, and sarcoplasmic reticulum accumulated reaction product to about the same degree at both temperatures (Debbas, '73). With procedures A, B and C extracellular reaction product was mainly confined to collagen rather than elastic tissue (Debbas, '73). Figures 6-9 show the results that were obtained when steps were taken to wash out and/or precipitate the extracellular calcium at the conclusion of the incubation period just prior to fixation (procedure B ( 2 ) a, b and c ) . It may be seen that the effort made to remove extracellular calcium ions in solution by washing the tissues in calcium-depleted depolarizing SOlution without EGTA (procedure B ( 2 ) a, fig. 6 ) or in calcium-depleted depolarizing solution with EGTA (procedure B (2) b, figs. 7, 8) did not alter the distribution of reaction product that was encountered in the unwashed vascular tissues (procedure B ( l ) , figs. 4, 5). Similarly, when extracellular calcium ion was exposed to the calcium chelator, EGTA and subsequently to the calcium precipitating agent pyroantimonate anion (procedure B (2) c, fig. 9 ) , the results were the same as those encountered with procedure B ( 1), B ( 2 ) a, and B (2) b (compare figs. 4, 5 to 6-9). The nuclei under these conditions were observed to contain a slight amount of reaction product (figs. 8, 9) and the plasma membrane bound an appreciable amount of reaction product to its inner surface (figs. 6, 8, 9 ) . The sarcoplasmic reticulum (fig. 7) also accumulated reaction product that was similar in quantity and distribution to that seen in the sarcoplasmic reticulum of tissues subjected to procedure B (1). In figure 7, it can be seen that reaction product is mainly attached to both sides of the membrane of the sarcoplasmic reticulum, i.e., the side facing the cytoplasm and/or the side facing the lumen. In addition to its presence in mitochondria, plasma membrane, and sarcoplasmic re-

LOCALIZATION OF CALCIUM IN SMOOTH MUSCLE

455

ticulum, reaction product was occasionally would be intracellular. The sodium content seen adhering to the membrane of some of under these conditions decreased to less the surface vesicles (procedure B ( 1) and than 5 meq/liter cell water. B ( 2 ) a, b, c, fig. 6 and (Debbas, '73)). DISCUSSION Since most membrane systems in the For general information regarding the smooth muscle fiber appeared to have the capacity to bind appreciable amounts of ultrastructure of the rabbit aorta the reader reaction product, the observation that the is referred to previous reports (Bierring nuclear envelope rarely had such deposits and Kobayasi, '63; Seifert, '63; Stein et al., suggests that the pyroantimonate precipi- '69). The quality of fixation of the aortic tate did not adhere to cellular membranes smooth muscle fibers as afforded by the potassium pyroantimonate-OsOc fixative is in a non-specific manner. The distribution of reaction product in adequate for the purpose of this study. The aortic tissues visualized in unstained sec- majority of the smooth muscle fibers and tions (not shown in the present report) their organelles were intact, even when the was compared with those that were stained tissues were incubated in various bathing (see METHODS). The use of uranyl acetate, media for 50-60 minutes prior to fixation. lead citrate or staining by both of these However, the sarcoplasmic reticulum was reagents did not alter the localization of poorly preserved as evidenced by its dilareaction product within the aortic smooth tion. Previous studies on vascular smooth muscle have shown that the sarcoplasmic muscle fibers. A Baird Atomic flame photometer was reticulum will appear swollen after priemployed to measure sodium and potas- mary fixation with OsOc and especially sium levels in fresh unincubated tissues when the tissue is incubated in a high KCl and in tissues that were incubated for 50 solution prior to fixation (Jones et al., '73). minutes at 25°C in the calcium containing Tissues that were fixed without first beand calcium-depleted depolarizing solu- ing incubated for a period of time in a tions. Tissues were assayed for their so- bathing medium consistently showed an dium content as described by Bowman and abundance of reaction product in the nuLandon ('67). These flame photometric clear compartment. Smooth muscle nuclei measurements were not extensively pur- of the rabbit aorta, therefore, appear to sued using only the thoracic aortas of two serve as a sink for cations. Presumably, a rabbits for each of the fresh or incubated large portion of this reaction product is tissues. These experiments showed that sodium pyroantimonate. Electron microduring the incubation period in the sodium- scopic cytochemistry on a variety of tissues free depolarizing solution more than 90% does indeed support the view that the nuof the initial sodium ion content of the cell clear compartment sequesters sodium (Spiwater was lost. Our estimate of the intra- cer et al., '68; Zadunaisky et al., '68; Klein cellular sodium content of fresh unincu- et al., '70; Klein-Thureson and Klein, '71). bated tissues was based on the approxima- The cytoplasm and its organelles showed tion that the extracellular space of the a random distribution of reaction product. rabbit aorta is 35% of the total tissue Such a result is not unexpected since an space (Altura and Altura, '71). On this intracellular concentration of 7 to 8 mM basis we obtained a sodium level of 55 sodium may produce a detectable quantity meq/liter cell water. This value appears to of reaction product (Klein et al., '72). The be high compared to that previously re- presence of magnesium and calcium may ported for aortic smooth muscle (for review further add to the precipitate. In contrast see Somlyo and Somlyo, '68); and it is to unincubated tissues, those that were inprobable that our figure includes an extra- cubated for 50 minutes in a medium that cellular bound fraction of sodium. In esti- did not contain any added sodium, calcium mating the intracellular sodium content of or magnesium displayed much less reacthe aortic smooth muscle after it was in- tion product in the cytoplasm and in cubated for 50 minutes in depolarizing so- all other organelles excluding the nuclear lution (with or without added calcium) we compartment. This observation correlates assumed that all the sodium measured with our flame photometric measurements,

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G . DEBBAS, L. HOFFMAN, E. J. LANDON AND L. HURWITZ

which indicated a major loss of sodium from the latter tissue. Rabbit aorta loses approximately 65% of its original sodium content during a 20-minute incubation period in a Ringer's solution in which choline has been substituted for sodium (Dodd and Daniel, '60). Moreover, it has been shown that the smooth muscle of rabbit aorta loses more than 90% of its intracellular sodium when incubated for thirty minutes in a high potassium, sodium-free medium (Jones et al., '73). It also loses 40% of its magnesium content when incubated in a magnesium-free Kreb's Ringer medium for one hour (Altura and Altura, '71). When the rabbit aorta was incubated in a medium containing 10.8 mM CaCL with no added sodium or magnesium, reaction product was increased at a number of specific sites by comparison with control tissues. First, there was an appreciable increase in reaction product in the mitochondria and at the plasma membrane. Second, the sarcoplasmic reticulum, which was devoid of reaction product in the control tissues, now appeared to have accumulated it. Only one site, the nucleus, showed a diminution of reaction product by comparison with nuclei of control tissues. Although the underlying basis for this observation is obscure, it would appear that the nucleus does not directly participate in the calcium regulation associated with contraction. In any cytochemical study there is a possibility that artifacts in the localization of reaction product can occur. As mentioned earlier, one such artifact could have resulted from the translocation of extracellular calcium into the smooth muscle cell during fixation and its subsequent precipitation by pyroantimonate anion at various intracellular sites. The possibility of experiencing this artifact stems from the fact that extracellular calcium ion in some of our experiments was not removed from the calcium loaded aortic tissue prior to fixation (procedure B (1)). However, results obtained in experiments in which extracellular calcium ion was chelated, precipitated and rinsed away from the calcium loaded aortic tissue prior to fixation serve to negate this possibility (procedures B ( 2 ) a, b, c ) . Our results showed that the localization of reaction product (in mito-

chondria, sarcoplasmic reticulum and at the plasma membrane) after taking steps to remove extracellular calcium was similar to that encountered with procedure B ( 1 ) (compare figs. 4, 5 to 6-9). A second artifact that may have occurred during fixation or dehydration in procedures B and C could have been induced by the diffusion of precipitated reaction product from its original site(s) of sequestration to a new site(s), e.g., diffusion of reaction product from the sarcoplasmic reticulum to the plasma membrane or the reverse. If diffusion of reaction product had occurred one would expect a random distribution of reaction product in the cytoplasm as well as in the vicinity of specific organelles. The lack of excessive reaction product in the cytoplasm would argue against the occurrence of such artifactual diffusion of reaction product in this study. A third artifact that may have occurred could have arisen as a consequence of using uranyl and lead as stains. It is conceivable that such heavy metals will form precipitates with the pyroantimonate ion or that the staining process will change the distribution of the pyroantimonate precipitate. Comparison of stained sections with those that were not stained (as mentioned earlier) negate the occurrence of such an artifact. Based on the above observation, we assume that the increase in reaction product seen in tissues that were exposed for 50 minutes to an extracellular concentration of 10.8 mM CaCl, over that seen in tissues that were exposed to a calcium-depleted environment was due to the precipitation of calcium as calcium pyroantimonate. The increased quantity of reaction product appeared to be concentrated at mitochondria, sarcoplasmic reticulum and the inner surface of the plasma membrane. It would appear, therefore, that these organelles are capable of accumulating or binding calcium ions that gain entrance into the cytoplasm from the external medium. The following considerations lead us to believe that our assumptions are correct: ( I ) Treatment of control tissues and of experimental tissues was identical except for the addition of 10.8 mM CaCL to the bathing medium of the experimental tis-

LOCALIZATION OF CALCIUM I N SMOOTH MUSCLE

~

sues, It should also be noted that the augmentation of reaction product in the experimental tissues was not modified when steps were taken to remove calcium ions from the extracellular space of the tissues following their incubation in the calcium containing depolarizing solution and just prior to their fixation, ( 2 ) Marked enhancement of calcium entry into the smooth muscle and the accumulation or binding of calcium ions by organelles would be expected when a high concentration of CaC12 is present in the incubating medium. This expectation is based on results of previous reports which showed that potassium-induced contractures in the rat tail artery (Hinke, '65) and the rabbit aorta (Hudgins and Weiss, '68; Van Breemen, '69; Van Breemen et al.;'72) utilize a n extracellular or loosely-bound pool of calcium and further, that these contractures are accompanied by a n increased influx of calcium ions (Briggs, '62; Van Breemen et al., '72) and a n associated increase in tissue calcium content (Van Breemen et al., '72); ( 3 ) the intracellular concentration of sodium in tissues which were incubated in the high-calcium medium was reduced to a level below that which would be precipitated by the pyroantimonate anion in vitro (Klein et al., '72). Moreover, a fraction of intracellular magnesium is also presumed to be lost because of the absence of magnesium in the bathing fluid. A s a result, reaction product in excess of that visualized under control conditions is not likely to be due to precipitated sodium or magnesium in the aforementioned organelles; ( 4 ) Pyroantimonate is more sensitive to precipitation by calcium ion than it is to magnesium or sodium ion in vitro (Klein et al., '72). ( 5 ) Direct electron microscope microprobe or x-ray microanalysis as mentioned earlier (Tandler et al., '70; Kierszenbaum et al., '71; Yarom et al., '72; Hales et al., '74) verifies the presence of calcium in the electron opaque precipitate seen when tissues are treated with the pyroantimonate reagent. The intracellular localization of calcium found in this electron microscopic study of vascular smooth muscle is supported by numerous physiological studies. These physiological studies have suggested that

457

vascular smooth muscle contains a store of intracellularly bound calcium that is available for muscle contraction. Evidence for this intracellular store of calcium rests on the observation that the contractile process is not completely inhibited in the absence of a n extracellular or a loosely bound pool of calcium. With the perfused rat tail artery, contractures induced by norepinephrine are sustained for twenty minutes in the absence of extracellular calcium. Under similar conditions, contractures induced by potassium decay in four minutes (Hinke, '65). The isolated rabbit aorta or main pulmonary artery incubated in the absence of calcium for a prolonged period of time responds like the rat tail artery Norepinephrine produces a diminished but significant contractile response, while the response to high potassium is markedly reduced or abolished (Hudgins and Weiss, '68; Devine et al., '72; Van Breemen et al., '72). It appears that norepinephrine can utilize intracellular pools of calcium but may also cause a delayed increase in calcium influx (for review see Somlyo, '72). On the other hand, a high potassium depolarizing medium mobilizes extracellular or loosely bound calcium (Hinke, '65; Hudgins and Weiss, '68; Van Breemen, '69; Van Breemen et al., '72). However, a more recent study has shown that a high potassium medium may also release some calcium from the intracellular pool (Goodman et al., '72). I n a n earlier study using strontium Somlyo and Somlyo ('71) showed that this cation localizes in the lumen of the sarcoplasmic reticulum and in mitochondria of vascular smooth muscle. Strontium is a n electron-opaque divalent cation postulated to behave like calcium (Somlyo and Somlyo, '71 ). Localization of calcium pyroantimonate in the mitochondria and sarcoplasmic reticulum in this study is in agreement with the strontium study. However, i n contrast to that found i n the strontium study, the point of concentration of the calcium pyroantimonate in our study was a t the membrane of the sarcoplasmic reticulum. Reaction product was rarely seen in the sarcoplasmic cisterns. The reason for this difference in the two studies is not obvious. It is possible that calcium ion has a significantly higher affinity for

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G . DEBBAS, L. HOFFMAN, E. J. LANDON AND L. HURWITZ

anionic sites in the membrane of the sarcoplasmic reticulum than does strontium ion. Consequently, only the calcium at the membrane would be present in a high enough concentration to form visible reaction product. Alternatively, one could conceive of the possibility that cation precipitates of pyroantimonate have a tendency to adhere to membranes. If this were, indeed, the case, it would be in conflict with our findings that the cation precipitates of pyroantimonate were randomly dispersed throughout the cell after employing procedure A and that the nuclear membrane was conspicuously devoid of any such precipitate at its surface after employing procedures B (1) and ( 2 ) . It should also be noted that in our study, as opposed to the strontium study, the sarcoplasmic reticulum had swelled considerably. The introduction of a normal Kreb's solution following the exposure of the tissues to a high potassium medium would have reduced the swelling (Jones et al., '73). However, this procedure would have introduced a sodium pyroantimonate precipitate that could serve to obscure our results. The effect that a swollen sarcoplasmic reticulum has on the distribution of reaction product between the lumen and the membrane is, of course, impossible to assess at this point in time. One additional factor that may account for the difference in the location of the calcium and strontium in the sarcoplasmic reticulum is one involving a difference in procedure between the two studies. In our study the aortic tissues were fixed immediately after being exposed to a high potassium medium. In the strontium study the tissues were fixed immediately after being exposed to a normal Kreb's solution. Since a high potassium medium, as opposed to a high sodium medium, stimulates, to some extent, the release of intracellular stores of calcium (Goodman et al., ' 7 2 ) , the absence of a high concentration of this cation in the lumen of the sarcoplasmic reticulum in our study may have resulted from a difference in the dynamic state of the calcium exchange processes that exist in the presence of these two different kinds of bathing media. Evidence demonstrating the capacity of mitochondria (Fitzpatrick et al., '72) and

sarcoplasmic reticulum (Baudouin et al., '72) of aortic smooth muscle to sequester calcium has also been obtained in studies on subcellular fractions. In these studies both mitochondrial and microsomal fractions were isolated from the aorta of the rabbit. Active uptake of calcium was found to occur in both these subcellular fractions. In subsequent experiments in which the microsomal preparation was further divided on a sucrose density gradient column, and correlations between the activities of various enzyme markers and an energy-dependent calcium pump were examined, the data strongly suggested that an appreciable degree of calcium pump activity was located in microsomal vesicles that were derived from the sarcoplasmic reticulum (Hunvitz et al., '73). Energy dependent uptake of calcium by microsomal and mitochondrial fractions obtained from bovine aorta has also been recently reported (Hess and Ford, '74). If the sarcoplasmic reticulum were not functional in the regulation of activator calcium, it seems unlikely that the mitochondria would serve as an intracellular store of calcium from which the divalent ion can be mobilized for muscle contraction. This is evidenced by the observation that the longitudinal muscle of the guinea pig ileum, which appears to contain ATPdependent calcium pumps primarily in its mitochondria (Hurwitz, unpublished data) and its plasma membrane (Hurwitz et al., '73) does not contract in the absence of external or loosely bound calcium (Hurwitz and Joiner, '70). Furthermore, Devine et al. ('72) have demonstrated that the capacity of smooth muscles to contract in the absence of external calcium appears to correlate with the visible quantity of sarcoplasmic reticulum found in the fiber. In the present study, localization of calcium was seen not only in the mitochondria and the sarcoplasmic reticulum but also at the inner surface of the plasma membrane. Deposition of calcium at these sites may have been brought about by the activity of an energy dependent calcium binding process and, possibly, a calcium transport system in the plasma membrane. Results of other studies have also suggested that transport or binding sites for calcium may be present in the plasma

LOCALIZATION OF CALCIUM IN SMOOTH MUSCLE

membrane of smooth muscle fibers (Van Breemen, '69; Hurwitz and Joiner, '70; Goodford and Wolowyk, '72; Devynck et al., '73). The large quantity of reaction product that was observed at the inner surface of the plasma membrane constitutes an area of localization of calcium that was not detected by Somlyo and Somlyo ('71) in their study of the intracellular distribution of strontium in the main pulmonary artery and the mesenteric vein. This difference in the localization of the two cations was apparent despite the effort made by us to remove extracellular calcium prior to fixation and the steps taken in one experiment by Somlyo and Somlyo to maintain a high level of extracellular strontium during fixation. Thus, the possibility exists that calcium ion may have a much different affinity for specific sites on the plasma membrane of the aortic smooth muscle than does strontium for plasma membrane sites in the other vascular smooth muscles. In addition, previous studies on vascular smooth muscle have shown that both magnesium (Altura and Altura, '71) and sodium ions (Bohr et al., '69; Sitrin and Bohr, '71) compete with calcium ions for cellular sites. As a result, the concentration of magnesium and sodium ions in both the external and internal environments of the cell could modify the degree to which calcium or strontium will be bound or taken up by the various organelles in the smooth muscle cell. Differences in the types of bathing media employed in our study and in the strontium study could, therefore, be another possible cause for the differences observed in the localization of the two cations. It should be emphasized that the inward movement and intracellular distribution of calcium was investigated in the presence of a high potassium depolarizing medium that contained 10.8 mM CaCl,. Although in this bathing solution aortic smooth muscle is physiologically functional (i.e., can be stimulated to undergo changes in contractile state by appropriate stimuli), the environment for such muscle in vivo is different. The limitation of the pyroantimonate method (Klein et al., '72) and the relative impermeability of the polarized plasma membrane of tonic vascular smooth

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muscles to strontium ion (Somlyo and Somlyo, '71) and to calcium ion (present study) necessitated the use of a high KC1, sodium-free and magnesium-free medium to accomplish the objective of this study. The observation that much smaller quantities of reaction product were found in the sarcoplasmic reticulum and at the plasma membrane of tissues that were fixed by procedure A would indicate that these organelles may sequester less calcium under normal physiological conditions than under the conditions defined by procedure B. Thus, our study reveals the intracellular organelles of the aortic smooth muscle which possess the capacity to sequester calcium, but does not indicate the amount of calcium which may be stored at each of these sites in an in vivo environment. In conclusion, several organelles in the aortic smooth muscle of the rabbit are capable of accumulating or binding a reaction product, presumed to be mostly calcium pyroantimonate under the influence of a high potassium depolarizing medium. These organelles are the plasma membrane, the mitochondria and the sarcoplasmic reticulum. Our results are interpreted to indicate that these organelles are capable of sequestering calcium ion and that some of them may constitute intracellular storage sites from which the divalent ion may be mobilized for contraction. ACKNOWLEDGMENTS

We wish to acknowledge the valuable technical assistance of Janice Carolyn, Sally Little, and Donald Swagler. Thanks are due to Dr. Jack Davies, Chairman of the Department of Anatomy, for providing the electron microscopic facilities and to Brenda Lair for secretarial assistance. This study represents a portion of the dissertation research carried out by the first author in partial fulfillment of requirements for the Ph.D. degree in the Department of Pharmacology, Vanderbilt University School of Medicine. Supported by U.S.P.H.S. Training Grant GM-00058-14, U.S.P.H.S. Grant AM-04703, National Science Foundation Grant GB361059, and HEW Grant HL16179. LITERATURE CITED Altura, B. M., and B. T . Altura 1971 Influence of magnesium on drug induced contractions

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chondria as cation accumulation sites in and ion content in rabbit aorta. Am. J. Physiol., smooth muscle. Phil. Trans. R. SOC. Lond., 220: 938-944. B265: 17-23. Baudouin, M., P. Meyer, S. Fermandjian and J. L. Devynck, M. A., M. G. Pernollet, P. Meyer, S. Morgat 1972 Calcium release induced by inFermandjian and P. Fromageot 1973 Angioteraction of angiotensin with its receptors in tensin receptors in smooth muscle cell memsmooth muscle cell microsomes. Nature, 235: branes. Nature New Biol., 245: 55-58. 336-338. Dodd, W, A., and E. E. Daniel 1960 ElectroBierring, F., and T. Kobayasi 1963 Electronlytes and arterial muscle contractility. Circ. microscopy of the normal rabbit aorta. Acta Res., 8: 451-463. Pathol. Microbiol. Scand., 57: 154-168. Fitzpatrick, D. F., E. J. Landon, G. Debbas and Briggs, A. H. 1962 Calcium movements during L. Hurwitz 1972 A calcium pump in vascupotassium contracture in isolated rabbit aortic lar smooth muscle. Science, 176: 305-306. strips. Am. J. Physiol., 203: 849-852. Garfield, R. E., R. M. Henderson and E. E. Daniel Brighton, C. T., and R. M. Hunt 1974 Mito1972 Evaluation of the pyroantimonate techchondrial calcium and its role in calcification: nique for the localization of tissue sodium. TisHistochemical localization of calcium in elecsue and Cell, 4: 575-589. tron micrographs of the epiphyseal growth plate with K-pyroantimonate. Clin. Orthopaed., 100: Goodford, P. J., and M. W. Wolowyk 1972 Localization of cation interactions in the smooth 406-416. muscle of the guinea-pig taenia coli. J. Physiol., Bohr, D. F. 1964 Electrolytes and smooth mus224: 521-535. cle contraction. Pharmacol. Rev., 16: 85-111. Goodman, F. R., G. B. Weiss, M. N. Weinberg and 1973 Vascular smooth muscle updated. S. D. Pomarantz 1972 Effects of added or Circ. Res., 32: 665-672. substituted potassium ion on 45Ca movements Bohr, D. F., C. Seidel and J. Sobieski 1969 POSin rabbit aortic smooth muscle. Circ. Res., 31: sible role of sodium-calcium pumps in tension 672-681. development of vascular smooth muscle. MicroHales, C. N., J. P. Luzio, J. A. Chandler and L. vasc. Res., I : 335-343. Herman 1974 Localization of calcium in the Bowman, F.J., and E. J. Landon 1967 Organic smooth endoplasmic reticulum of rat isolated Mercurials and net movements of potassium in fat cells. J. Cell Sci., 15: 1-15. rat kidney slices. Am. J. Physiol., 213: 1209Herman, L., T. Sato and C. N. Hales 1973 The 1217. electron microscopic localization of cations to Bulger, R. E. 1969 Use of potassium pyroantipancreatic Islets of Langerhans and their posmonate in the localization of sodium ions i n sible role in insulin secretion. J. Ultrastruct. rat kidney tissue. J. Cell Biol., 40: 79-94. Res., 42: 298-311. Clark, M. A., and G. A. Ackerman 1971a AlterHess, M. L., and G. D. Ford 1974 Calcium acation of nuclear and nucleolar pyroantimonatecumulation by subcellular fractions from vasosmium reactivity by glutaraldehyde fixation. cular smooth muscle. J. Molec. Cell. Card., 6: J. Histochem. Cytochem., 19: 388-390. 275-282. 1971b A histochemical evaluation of the pyroantimonate-osmium reaction. J. Histo- Hinke, J. A. M. 1965 Calcium requirements for noradrenaline and high potassium ion contracchem. Cytochem., 19: 727-737. tion in arterial smooth muscle. In: Muscle. Constantin, L. L., C. Franzini-Armstrong and R. J. W. M. Paul et al., eds. Pergamon, New York, Podolsky 1965 Localization of calcium-accumulating structures in striated muscle fibers. pp. 269-285. Hudgins, P. M., and G. B. Weiss 1968 DifferenScience, 147: 158-160. tial effects of calcium removal upon vascular Daniel, E. E. 1965 Attempted synthesis of data regarding divalent ions in muscle function. In: smooth muscle contraction induced by norepiMuscle. W. M. Paul et al., eds. Pergamon, New nephrine, histamine, and potassium. J. Pharmacol. Exptl. Therap., 159: 91-97. York, PP. 295-313. Hurwitz, L., D. F. Fitzpatrick, G. Debbas and E. J. Debbas, G. 1973 Electron microscopic localization of calcium in vascular smooth muscle. Landon 1973 Localization of calcium pump Ph.D. thesis. Department of Pharmacology, Vanactivity in smooth muscle. Science, 179: 384derbilt University, Nashville, Tennessee 37232. 386. Available through University Microfilms, 300 Hurwitz, L., and P. D. Joiner 1970 Mobilization of cellular calcium for contraction in intesNorth Zeeb Road, Ann Arbor, Michigan 48106. tinal smooth muscle. Am. J. Physiol., 218: 12Debbas, G., L. Hoffman, E. J. Landon and L. Hurwitz 1973 Electron microscopic localization 19. of calcium i n vascular smooth muscle. Fed. Hurwitz, L., and A. Suria 1971 The link beProc., 32: 767. tween agonist action and response in smooth muscle. Ann. Rev. Pharmacol., I 1: 303-326. Deth, R., and C. Van Breemen 1974 Relative contributions of calcium influx and cellular Jones, A. W., A. P. Somlyo and A. V. Somlyo 1973 calcium release during drug induced activation Potassium accumulation in smooth muscle and of the rabbit aorta. Pflugers Arch., 348: 13-22. associated ultrastructural changes. J. Physiol., 232: 247-273. Devine, C. E., A. V. Somlyo, and A. P. Somlyo 1972 Sarcoplasmic reticulum and excitation- Kierszenbaum, A. L., C. M. Libanati and C. J. contraction coupling i n mammalian smooth Tandler 1971 The distribution of inorganic muscles. J . Cell Biol., 52: 690-718. cations in mouse testis. J. Cell Biol., 48: 3141973 Sarcoplasmic reticulum and mito323.

LOCALIZATION O F CALCIUM IN SMOOTH MUSCLE Klein, R. L., C. R. Horton and A. Thureson-Klein 1970 Studies on nuclear amino acid transport and cation content in embryonic myocardium of the chick. Am. J. Card., 25: 300-310. Klein, R. L., S. S. Yen and A. Thureson-Klein 1972 Critique on the K-pyroantimonate method for semiquantitative estimation of cations in conjunction with electron microscopy. J. Histochem. Cytochem., 20: 65-78. Klein-Thureson, A., and R. L. Klein 1971 Cation distribution and cardiac jelly in early embryonic heart: A histochemical and electron microscopic study. J. Molec. Cell. Card., 2: 3140. Komnick, H. 1962 Electronenmikroskopische Lokalisation von N a + und C1- in Zellen und Geweben. Protoplasma., 55: 414-418. Komnick, H., and U. Komnick 1963 Elektronenmikroskopische Untersuchungen zur funktionellen Morphologie des Ionentransportes i n der Salzdriise von Larus argentatus. 2. Zellforsch. Mikrosk. Anat., 60: 163-203. Krames, B., and E. Page 1968 Effects of electron microscopic fixatives on cell membranes of the perfused rat heart. Biochim. Biophys. Acta, 150: 24-31. Legato, M. J., and G. A. Langer 1969 The subcellular localization of calcium ion in mammalian myocardium. J. Cell Biol., 41: 401-423. Pease, D. C., and W. J. Paule 1960 Electron microscopy of elastic arteries; the thoracic aorta of the rat. J. Ultrastruct. Res., 3: 469483. Podolsky, R. J., T. Hall and S. L. Hatchett 1970 Identification of oxalate precipitates in striated muscle fibers. J. Cell Biol., 44: 699-702. Seifert, K. 1963 Electronenmikroskopische untersuchungen der Aorta des Kaninchens. 2. Zellforsch. Mikrosk. Anat., 60: 293-312. Shiina, %-I., V. Mizuhira, T. Amakawa and Y. Futaesaku 1970 A n analysis of the histochemical procedure for sodium ion detection. J. Histochem. Cytochem., 18: 644-649. Sitrin, M. D., and D. F. Bohr 1971 Ca and Na interaction in vascular smooth muscle contraction. Am. J. Physiol., 220: 1124-1128. Somlyo, A. P. 1972 Excitation-contraction coupling i n vertebrate smooth muscle: Correlation of ultrastructure with function. The Physiologist, 15: 338-348. Somlyo, A. P., and A. V. Somlyo 1968 Vascular smooth muscle. I. Normal structure, pathology, biochemistry, and biophysics. Pharmacol. Rev., 20: 197-272. Somlyo, A. P., A. V. Somlyo, C. E. Devine, P. D. Peters and T. A. Hall 1974 Electron microscopy and electron probe analysis of mitochondrial cation accumulation in smooth muscle. J. Cell Biol., 61: 723-742.

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Somlyo, A. V., and A. P. Somlyo 1971 Strontium accumulation by sarcoplasmic reticulum and mitochondria in vascular smooth muscle. Science, 174: 955-958. Spicer, S. S.,J. H. Hardin and W. B. Greene 1968 Nuclear precipitates in pyroantimonate-osmium tetroxide-fixed tissues. J. Cell Biol., 39: 216221. Stein, O., S. Eisenberg and Y. Stein 1969 Aging of aortic smooth muscle cells in rats and rabbits. A morphologic and biochemical study. Lab. Invest., 21: 386-397. Tandler, C. J., and A. L. Kierszenbaum 1971 Inorganic cations in rat kidney. Localization with potassium pyroantimonate-perfusion fixation. J. Cell Biol., 50: 830-839. Tandler, C. J., C. M. Libanati and C. A. Sanchis 1970 The intracellular localization of inorganic cations with potassium pyroantimonate. J. Cell Biol., 45: 355-366. Tani, E., T. Ametani and H. Handa 1969 Sodium localization in the adult brain. I. Normal brain tissue. Acta neuropath., 14: 137-150. Tisher, C. C., B. A. Weavers and W. J. Cirksena 1972 X-ray microanalysis of pyroantimonate complexes in rat kidney. Am. J. Pathol., 69: 255-270. Torack, R. M., and M. LaValle 1970 The specificity of the pyroantimonate technique to demonstrate sodium. J. Histochem. Cytochem., 18: 635-643. Van Breemen, C. 1969 Blockade of membrane calcium fluxes by lanthanum in relation to vascular smooth muscle contractility. Arch. Int. Physiol. Biochim., 77: 710-716. Van Breemen, C., B. R. Farinas, P. Gerba and E. D. McNaughton 1972 Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx. Circ. Res., 30: 44-54. Yarom, R., D. Ben-Ishay and 0. Zinder 1972 Myocardial cationic shifts induced by isoproterenol. Electron microscopic and electron probe studies. J. Molec. Cell. Card., 4: 559-570. Yarom, R., and K. Braun 1971 Ca*+ changes in myocardium following scorpion venom in jections. J. Molec. Cell. Card., 2: 177-179. Yarom, R., and U. Meiri 1971 N lines in striated muscle: a site of intracellular CaZ+. Nature New Biol., 234: 254-256. 1972 Ultrastructure cation precipitation i n frog skeletal muscle. I. Localization of pyroantimonate precipitate a t rest and in Tetanus. J. Ultrastruct. Res., 39: 430-442. Zadunaisky, J. A., J. F. Gennaro, Jr., N. Bashirelahi and M. Hilton 1968 Intracellular redistribution of sodium and calcium during stimulation of sodium transport in epithelial cells. J. Gen. Physiol., 51: 290s-302s.

PLATE 1 EXPLANATION O F FIGURES

1 Aortic smooth muscle fixed in potassium pyroantimonate-0s01 (pro-

cedure A). The nuclei ( N ) and nucleolus ( N u ) contain abundant reaction product. Non-specific reaction product in the cytoplasm (CP) is abundant. Reaction product also occurs in the mitochondria ( M ) and at the plasma membrane (arrows). The sarcoplasmic reticulum (SR) has a slight amount of reaction product. x 22,500. 2

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Aortic smooth muscle fixed in potassium pyroantimonate-Os04 (procedure A ) . Variability with respect to the mitochondrial ( M ) and the plasma membrane (arrow) deposition of reaction product as described under RESULTS is apparent in this electron micrograph. The mitochondria ( M ) have very little reaction product and the plasma membrane ( arrow) has virtually no reaction product. Non-specific cytoplasmic reaction product (CP) is abundant as also shown in figure 1. Surface vesicles (SV) are seen tangentially sectioned. x 33,750.

LOCALIZATION OF CALCIUM IN SMOOTH MUSCLE G. Debbas, L. Hoffman, E. J. Landon and L. Hurwitz

PLATE 1

463

PLATE 2 EXPLANATION OF FIGURE

3 Aortic smooth muscle incubated for 50 minutes at 25°C in calciumdepleted depolarizing solution prior to fixation in potassium pyroantimonate-Os04 (procedure C ) . The nucleus ( N ) and nucleolus ( N u ) contain much more reaction product than the rest of the cellular organelles. The mitochondria ( M ) have little or no reaction product, while the sarcoplasmic reticulum ( S R ) has none. Surface vesicles (SV), plasma membrane (arrow). x 27,825.

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LOCALIZATION OF CALCIUM I N SMOOTH MUSCLE G. Debbas, L. Hoffman, E. J. Landon and L. Hurwitz

PLATE 2

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PLATE 3 EXPLANATION O F FIGURES

4

Aortic smooth muscle incubated for 50 minutes at 25°C in a depolarizing solution containing 10.8 mM CaClz prior to fixation in potassium pyroantimonate-OsOa (procedure B ( 1 ) ) . Portions of four smooth muscle cells are shown. Reaction product is abundant on the inner surface of the plasma membrane (arrows) as well as within mitochondria ( M ) . Reaction product is also present on profiles of the sarcoplasmic reticulum ( S R ) and to a lesser extent i n the nucleus ( N ) . x 27,000.

5 Aortic smooth muscle incubated for 50 minutes at 25°C in a depolark i n g solution containing 10.8 mM CaClz prior to fixation in potassium pyroantimonate-OsOe (procedure B ( 1 ) ). Peripherally located sarcoplasmic reticulum (SR) is dilated and contains dense accumulation of reaction product as do the plasma membranes (arrows) of the three cells in the field. x 38,000.

466

LOCALIZATION OF CALCIUM IN SMOOTH MUSCLE G. Debbas, L. Hoffman, E. J. Landon and L. Hurwitz

PLATE 3

467

PLATE 4 EXPLANATION OF FIGURES

468

6

Aortic smooth muscle incubated for 45 minutes at 25°C in a depolarizing solution containing 10.8 mM CaCL. After incubation the tissue was washed for 15 minutes in a calcium-depleted depolarizing solution prior to fixation in potassium pyroantimonate-0s01 (procedure B ( 2 ) a ) . Reaction product is present on the inner aspect of the plasm a membrane (arrows) as well as within mitochondria ( M) . Surface vesicles (SV). x 32,400.

7

Aortic smooth muscle incubated for 45 minutes at 25°C in a depolarizing solution containing 10.8 mM CaC12. After incubation the tissue was washed for 10 minutes in a calcium-depleted depolarizing solution containing 10-3 M EGTA. It was further washed for another 5 minutes in a calcium-depleted depolarizing solution (without any added EGTA) prior to being fixed in potassium pyroantimonate-Os04 (procedure B ( 2 ) b ) . In this high power electron micrograph reaction product is prominent in several dilated profiles of the sarcoplasmic reticulum (SR). Reaction product appears to be predominantly adhering to the inner and outer surface of the membrane (MEM) which is barely visible. x 64,600.

LOCALIZATION OF CALCIUM IN SMOOTH MUSCLE G. Debbas, L. Hoffman, E. J. Landon and L. Hurwitz

PLATE 4

469

PLATE 5 EXPLANATlON

470

O F FIGURES

8

Aortic smooth muscle incubated, washed and fixed as that in figure 7 (procedure B ( 2 ) b ) . Reaction product is apparent at the inner surface of the plasma membrane (arrows) and in the mitochondria ( M ) . Nucleus ( N ) . x 27,000.

9

Aortic smooth muscle incubated for 45 minutes at 25°C in a depolarizing solution containing 10.8 mM CaC12. After incubation the tissue was washed for 5 minutes in a calcium-depleted depolarizing solution containing M EGTA, followed by another 5 minute wash in a calcium-depleted depolarizing solution (without any added EGTA). The tissue was subsequently immersed for 5 minutes at 4°C in a potassium pyroantimonate solution containing no OsO4 and then fixed in potassium pyroantimonate-OsO4 (procedure B ( 2 ) c ) . Reaction product is readily visible in the nucleus ( N ) , although slight in quantity. Reaction product is quite prominent at the inner surface of the plasma membrane (arrows) and in mitochondria ( M ) . x 36,450.

LOCALIZATION OF CALCIUM I N SMOOTH MUSCLE G. Debbas, L. Hoffman, E. J. Landon and L. Hurwitz

PLATE 5

473