Membrane-bound WOLFGANG
carbonic
BRUNS
AND
anhydrase
GEROLF
Zentrum Physiologie, Medizinische Federal Republic of Germany
GROS
Hochschule
Bruns, Wolfgang, and Gerolf Gros. Membrane-bound carbonic anhydrase in the heart. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H577-H584, 1992.-Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed. sarcolemma; sarcoplasmic reticulum; ton buffering; cattle; rabbit
dansylsulfonamide;
pro-
MUSCLE, like skeletal muscle, has long been thought to contain no carbonic anhydrase (CA). Although during the last 15 years at least three isozymes have been identified in skeletal muscle, i.e., two cytosolic forms (18, 27) and one that is bound to the sarcolemma and sarcoplasmic reticulum (3, 12, 3l), several publications dealing with heart muscle seem to confirm the view that this tissue is devoid of CA (24, 35). Geers et al. (13) included cardiac tissue in a study on the distribution of the different isozymes in individual blood-free perfused muscles of the rabbit. In the heart, they found no cytosolic CA, which is in line with previous reports (5, 15, 18, 24), but a high CA activity, which could be extracted from the particulate fraction using Triton X-100. The failure of the enzyme to appear in the supernatant of the homogenate in the absence of Triton implies that it is membrane bound. We therefore prepared purified sarcolemmal and sarcoplasmic reticulum vesicles from the bovine heart and investigated the CA activity of these membrane fractions.
CARDIAC
METHODS Membrane preparations. For each preparation, one fresh bovine heart was obtained from the local slaughterhouse, placed in ice, transferred to the laboratory, and used within 1.5 h after the animal’s death. All subsequent steps of trimming and homogenization were performed in a room refrigerated to 4°C. Left ventricular muscle was cleaned of fat and connective tissue and minced with a meat grinder. The material amounted to 180 g/preparation. The membrane preparations were done essentially as described by Jones and co-workers (20-22). 0363-6135/92
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in the heart
Hannover,
D-3000
Hannover 61,
Sarcophsmic reticulum membranes. The principle of the preparation (20, 21) is to homogenize the tissue vigorously by mechanical means in hypotonic buffer and to prepare a microsomal fraction from the homogenate by differential centrifugation. After the microsomes have been washed in a buffer of high KC1 concentration to dissolve contractile proteins, the material is incubated for 10 min at 37°C in a medium containing 50 mM histidine, 100 mM KCl, 65 mM MgC1,, 60 mM NanATP, 25 mM tris(hydroxymethyl)aminomethane (Tris), 25 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 20 mM CaC12, and 5 mM Tris-oxalate (pH 7.1) to load sarcoplasmic reticulum (SR) vesicles selectively with calcium oxalate; this serves to increase their density. Deviating from the protocol of Jones and co-workers (20, 21), the incubated vesicles were then not sedimented and resuspended, but the suspension was divided in four portions of about 10 ml which were immediately layered on four discontinuous sucrose gradients each consisting of 10 ml 1.5 M sucrose, 0.3 M KCl, 50 mM sodium pyrophosphate, 0.1 M Tris (pH 7.2) at the bottom plus, at the top, 5 ml of an identical solution but with only 1.0 M sucrose. The gradients were centrifuged in a Kontron TFT 65.38 fixed-angle rotor at 170,000 g,,, for 2 h. After the run each tube contained three bands plus a pellet of membrane material. The uppermost band was called fraction I, the intermediate band was called fraction II, and the lowest band, located at the 1.0 M-l.5 M sucrose interface, was called fraction III. The bands were collected by aspiration and diluted in 4 vol of ice-cold distilled water. The pellets, termed SR fraction, were resuspended in 0.25 M sucrose and 10 mM histidine (pH 7.5; subsequently called sucrose medium). All fractions were sedimented at 170,000 g,,, for 30 min and finally resuspended in sucrose medium. Of the microsomal suspension (see above) 2.8 ml were not placed on the gradient after oxalate loading but were sedimented in a separate tube during the gradient run. This pellet was resuspended in sucrose medium and subjected to the same final sedimentation and resuspension step as the other fractions. A small amount of the cytosolic fraction from the initial homogenate was also preserved. All samples were frozen in liquid N, and stored at -80°C. A further modification of the original method was to use an Ultra Turrax T 45 equipped with a TP 45/2G generator (Jahnke & Kunkel, Staufen, FRG) at three-fourths maximal speed for homogenization instead of a Polytron. SarcolemmaZ membranes. The principle of the preparation (22) is to homogenize the tissue gently in a buffer with high NaCl concentration and to wash the particulate fraction several times, first with the high-NaCl buffer and then with a hypotonic NaHC03 buffer. The particulate fraction is then homogenized vigorously, and subsequently differential centrifugation is performed. The loose upper layer of the resulting microsomal pellet, which we call crude sarcolemma (crude SL), is resuspended in sucrose medium to give a volume of 30 ml and divided in three portions, each of which is layered over 10 ml 0.6 M sucrose, 0.3 M NaCl, 0.05 M sodium pyrophosphate, and 0.1 M Tris (pH 7.1) and spun in a fixed-angle rotor at 170,000 g,,, for 90 min. The whitish band at the 0.25 M-O.6 M sucrose interface, termed sarcolemmal (SL) fraction, was collected by aspiration and diluted in 4 vol of ice-cold H,Q. The pellet, termed P fraction, was resuspended in sucrose medium. Both fractions were sedimented at 170,000 g,,, for 30 min and finally
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resuspended in sucrose medium. Of the original crude SL suspension (see above) 3 ml were retained for comparative purposes and treated as described for the microsomes from the SR preparation. Again, a small amount of the cytosolic fraction from the primary homogenate was retained. Freezing and storage were as described for the SR preparation. Determination of carbonic anhydrase activity. CA activity was determined using the modification of Maren’s micromethod that has been described in detail previously (3,4). The method utilizes the change of pH in the assay volume caused by hydration of CO, (continuously supplied as gas) following the addition of HCO;-CO:buffer. The time needed for the indicator phenol red to change color from red to yellow depends on CA activity. Temperature is 0°C. Immediately before the measurements, samples of the membrane fractions were solubilized by slowly adding 10% (wt/vol) Triton X-100 in water to a final concentration of 1% (wt/vol), followed by a lo-min incubation time at room temperature. One unit of CA activity was defined as the enzyme concentration in the final assay volume of 0.4 ml that cuts down to one-half the time necessary with the uncatalyzed CO, hydration. Corrections were made for the prolongation of the reaction time due to the presence of sucrose medium (4). The specific CA activity was obtained by dividing the number of units by the protein concentration in the reaction chamber. The amount of CA activity present in a given sample was calculated by multiplying the number of units (a value that is proportional to enzyme concentration) by the volume of the sample. Determination of subcellular markers. Adenosinetriphosphatase (ATPase) activities were determined at 37°C in reaction media essentially identical with those of Jones and Besch (19). The reaction time was 10 min. Reactions were stopped, and the amounts of inorganic phosphate liberated from ATP were determined as described previously (31). The specific ATPase activity was calculated as the amount of inorganic phosphate released per hour per milligram protein. Ca2’-Mg2’-ATPase activity, as a SR marker, was routinely stimulated by the addition of 5.7 PM calcium ionophore A23187 to the assay medium. Determinations were done in the presence of 1 mM ouabain and 5 mM sodium azide. Activity was calculated as the difference in ATP splitting in the presence of 50 PM CaCl, and in the absence of Ca2+ (1 mM EGTA added). Ouabain-sensitive Na’-K’-ATPase activity, as a SL marker, was maximized by the unmasking of latent activity by preincubation of the membranes with 1 mg alamethicin/mg membrane protein. Azide was included in the reaction medium. Azide-sensitive ATPase activity, as a mitochondrial marker, was determined in the presence of ouabain. Lactate dehydrogenase (LDH) activity, as a marker for the cytosolic fraction, was determined at 27°C in a coupled photometric assay. The samples were preincubated in 0.1% (wt/vol) Triton X-100 in assay buffer to make accessible enzyme molecules possibly caged inside the vesicles. The final Triton concentration in the assay was 0.003% (wt/vol). It was ascertained that this procedure did not cause any loss of LDH activities present in the cytosolic fractions (data not shown). One unit of LDH activity was defined as the amount of enzyme that catalyzes the oxidation of 1 pmol NADH2/min. The specific activity was obtained by dividing the activity by the amount of protein present in the assay. Heme content, which was regarded as another cytosolic marker, was estimated from photometric absorption spectra in the presence of 0.1% (wt/vol) Triton X100 in sucrose medium, using an absorbancy coefficient of 100 rnM-l*crn-l at 415 nm. Protein. Protein concentrations were determined by the Lowry method using the assay kit from Sigma Chemical. Samples were solubilized with sodium deoxycholate and protein was precipitated with trichloroacetic acid before the assay (Sigma
ANHYDRASE
procedure P 5656). Triton X-114 phase separation. The method of Bordier (2) was simplified. The Triton X-114 (Sigma) was not pretreated. To 100 ~1 of freshly thawed SL membranes (1.1-3.5 mg protein/ ml) or 100 ~1 of bovine erythrocytes (lysed and diluted 1:500 in distilled water) in an Eppendorf test tube was added 167 ~1 of chilled 1.6% (wt/vol) Triton X-114 in 0.3 M NaCl, and 20 mM Tris (pH 7.4). The mixture was shaken, and the tube was placed in ice water for 5 min, incubated at 31°C in a water bath for 4 min, and centrifuged at 300 g for 3 min in a swing-out rotor. The supernatant (-240 ~1) was carefully transferred into another tube using a micropipette, mixed with a 0.25-fold volume of chilled 2.5% (wt/vol) Triton X-114 in 0.3 M NaCl-20 mM Tris (pH 7.4), placed in ice water for 5 min, and layered over the sediment from the first centrifugation. After 4 min at 31°C the centrifugation step was repeated. The final supernatant, termed water phase, and the final sediment, termed Triton phase, were separated and assayed for CA activity immediately thereafter. In the original method the mixture of sample and Triton is layered over a cushion of 6% (wt/vol) sucrose before incubation in the warm. After centrifugation, the Triton phase is found at the bottom of the tube, whereas the water phase remains above the sucrose cushion. This procedure is designed to exclude water soluble enzymes from the water space of the Triton phase. We omitted the sucrose cushion, since it caused difficulties in identifying the water phase after centrifugation, probably because our samples themselves were suspended in sucrose. Histochemical Localization of CA. Cryosections of left ventricular cardiac muscle from a rabbit were stained using the dansylsulfonamide (DNSA) method described by Dermietzel et al. (8). The specific CA inhibitor DNSA exhibits an intense blue fluorescence when bound to the enzyme, whereas the yellowgreen fluorescence of the free DNSA is faint. A stock solution of 8 x 10m4 M DNSA in 0.1 M HCl was prepared by heating to 60°C for 1 h. This solution was diluted at least 30-fold in phosphate buffer (53 mM Na2HP04-13 mM KH2P04, pH 7.4) at room temperature immediately before the experiments. The DNSA concentration in phosphate buffer was checked photometrically at 323 nm using an absorbancy coefficient of 4,650 M-l cm-l. Th e st oc k so 1u t’ion was stable for at least 1 wk when stored in the dark at room temperature. Sections (10 pm) of tissue were preincubated in 0.1 ml phosphate buffer at room temperature for 10 min. They were then rinsed once with l2.7 x lo+ M DNSA in phosphate buffer. Subsequently 0.1 ml of the DNSA solution was pipetted onto the sections, and a glass cover slip was mounted using nail polish. Controls for the specificity of the DNSA staining were performed by including lO-4 M L-645,151, another specific CA inhibitor (1), in both incubation media. The specimens were viewed and photographed in a Leitz Orthoplan microscope equipped with Fluotar objectives, a high-pressure mercury lamp (HBO 50, Osram) as ultraviolet source, and a Ploemopak 2.1 fluorescence illuminator. The latter consisted of a BP 270-380 filter in the excitation light path, a RKP 380 dichroic mirror, and a BP 410-580 filter in the observation light path. RESULTS
Characteristics of membrane fractions. Data from the SR preparations are shown in Table 1. The lightest band in the density gradient, fraction I, is enriched in Na+K+-ATPase and reduced in azide-sensitive ATPase compared with the microsomes. Its Ca’+-Mg2+-ATPase activity is lower than that of the SR fraction. The data indicate that fraction I is enriched in SL membranes, mainly at the expense of mitochondrial and SR compo-
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Table
1. Properties
CARBONIC
of membrane fractions obtained with SR preparation pmol
Fraction
Microsome I II III SR
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ANHYDRASE
ProteinY ield, mg
CA, U.ml.mg protein-’
111.1246.1 10.4t2.9 39.8t23.0 42.0k16.7 3.5k1.7
phosphate.
Na’-K’-ATPase
1.6t0.5 12.0t3.1 2.2t0.7 0.6tO.l 0.9kO.4
h-l.
mg protein-l Azide-sensitive ATPase
Ca2’-Mg2’-ATPase
8t3 53k20 9t4 2t2 3t1
23t6 24t7 20t6 21t8 102t38
LDH, U/mg protein
54t13 13t5 32t4 54k9 14k6
0.5t0.1 2.2t0.6 0.6kO.2 0.4kO.l 0.2kO.l
Values are means t SD from 5 preparations for total amounts of protein recovered in each fraction and specific enzyme activities of the individual fractions. Parameters were determined as described in METHODS. One unit (U) of carbonic anhydrase (CA) activity was defined as the enzyme concentration in the final assay volume that cuts down to one-half the time of reaction necessary with the uncatalyzed CO2 hydration. Microsome fraction refers to crude microsomal pellet obtained by differential centrifugation. Note that only 93% of this material was actually subjected to the density gradient centrifugation, which yielded the other fractions listed. LDH, lactate dehydrogenase. In sarcoplasmic reticulum (SR) fraction Ca2’-Mg2’-ATPase is significantly enriched (P c 0.001) compared with microsome fraction, whereas Na’-K’-ATPase (P < 0.01) and azide-sensitive ATPase (P < 0.005) are significantly reduced.
nents. Its CA activity is 7.5fold higher than that of the microsomes. The heaviest fraction, SR, as expected, contains the highest activity of Ca2+-Mg2+-ATPase, whereas the activities of the other two membrane markers are here markedly lower than in the microsomes. The SR fraction contains CA activity, but no enrichment in relation to the crude membranes can be observed. Most of the mitochondrial material present in microsomes partitions into fraction III, which contains the lowest specific CA activity. In the cytosolic supernatants from the SR preparations the CA activity was 49 t 13 U, and the LDH activity was 69 t 13 U/ml (means t SD). The ratio CA/LDH in the cytosolic supernatant, in the microsomes and in the SR fraction is shown in Table 2. It can be seen that the membrane fractions are enriched in CA with respect to LDH. If all the CA present in the supernatant is assumed to be of cytosolic origin, then any CA/LDH in the membrane fraction that is greater than the one found in the supernatant indicates the presence of CA in excess of what can be expected from cytosolic contamination of the vesicle preparations. Based on this approach, the percentage contribution of contaminating cytosolic CA to total CA of a given membrane fraction is given by (CA/LDH) cytosol/(CA/LDH)f,,,ti,, . 100%. These percentages were calculated for each single preparation. The data are summarized in Table 2. It can be seen that only about one-fifth of CA activity in the SR fraction may be attributed to contamination with cytosolic CA.
In contrast to the SR preparation, the procedure of the SL preparation tends to result in a high proportion of light membrane material, namely, SL vesicles, in the microsomal fraction (20). The latter was therefore designated crude SL. The data from our SL preparations are shown in Table 3. The lightest band, the SL fraction, comprises 20% of the protein recovered after the density gradient centrifugation of the crude SL. This value is only 11% for fraction I of the SR preparations (see Table 1). In addition, the purity of the SL fraction is higher than that of fraction I judged from a comparison of their Na+-K+ATPase activities. This is paralleled by higher specific CA activity and lower Ca2+-Mg2+- and azide-sensitive ATPase activities of the SL fraction. Of the CA activity present in the crude SL and layered onto the gradient 72% is recovered in the SL fraction. There is no distinct distribution pattern of Ca”+ -Mg2+-ATPase. This can be expected, since the repeated mild homogenizations and washings of the particulate fraction tend to reduce the microsomal SR content (20) and since no calcium oxalate loading step is included in this preparation procedure. The CA/LDH in the crude SL and the SL fraction and the same value for the cytosolic supernatant (CA 27 t 10 U, LDH 69 t 13 U/ml, means t SD) are shown in Table 2. The percentage contaminations of the fractions with cytosolic CA, as calculated from the CA/LDH for each single preparation, are summarized in Table 2. A negligible proportion of the CA activity in the SL fraction
Table 2. Contamination of subcellular fractions with cytosolic CA
Fraction
CA/LDH, U/W/ml)
Contamination With Cytosolic CA From CA/ LDH, %
Heme,
Supernatant Microsome SR
0.720.3 3.221.3 4.8t1.6
100 28t21 18&9
ND ND ND
Supernatant Crude SL SL
0.4tO.l 6.5tl.2 11.9k1.9
100 6t2 3tl
llOk20 ND 2tl
Values are means t SD from 5 sarcoplasmic reticulum (SR) calculations of contamination from ratios of carbonic anhydrase heme. For calculations see text. Supernatant refers to supernatant see Table 3.
PM
CA/Heme, U/PM
Contamination With Cytosolic CA From CA/ Heme, %
0.3tO.l
100
14.224.4
221
preparations (top) and 4 sarcolemmal (SL) preparations (bottom). On left are to lactate dehydrogenase (CA/LDH); on right are calculations from ratios CA/ from primary homogenate; for microsome fraction see Table 1; for crude SL
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Table
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ANHYDRASE
3. Properties of membrane fractions obtained with SL preparation Fraction
Protein Yield, mg
CA, U. ml-l. mg protein-l
pmol phosphate. Na’-K+-ATPase
h-l. mg protein-’
LDH, U/mg protein
Azide-sensitive ATPase
Ca2’-M$‘-ATPase
4.122.0 14t7 l&10 113219 0.6t0.3 Crude SL 25.1t17.1 4.5t1.3 15.1t3.6 99*52 15t4 lOZk5 1.320.1 SL P 18.lt12.2 0.8t0.3 4t2 21z!zll 106t13 0.2tO.l Values are means t SD from 4 preparations. Crude SL refers to loose deposit of sarcolemmal membrane material on the microsomal pellet resulting from differential centrifugation. Note that only 90% of this material was subjected to density gradient centrifugation, which yielded the other fractions listed. LDH, lactate dehydrogenase. In SL fraction carbonic anhydrase (CA) activity (P < 0.001) and Na’-K+-ATPase (P < 0.05) are significantly enriched compared with crude SL, whereas azide-sensitive ATPase is significantly reduced (P < 0.001). For further explanations see Table 1.
appears to originate from the cytosol. An analogous calculation, based on the heme contents of the supernatant and the SL fraction, gives the same result. Triton X-l 14 phase-separation studies. The studies were performed with SL membranes and, as a control, with lysed bovine erythrocytes. In the case of membranes the total amount of CA activity introduced into each experiment ranged from 1.3 to 5.2 U/ml. After separation, the volumes of the Triton phase and the water phase were ~0.06 and 0.26 ml, respectively. It can be seen in Table 4 that most of the SL CA partitions into the Triton phase. In the case of red blood cells the starting amount of CA was 2.1 U/ml. In contrast to the behavior of SL CA, most of the cytosolic CA from erythrocytes remains in the water phase. This is mainly due to the greater volume of the water phase (0.29 ml) compared with the Triton phase (0.04 ml), whereas the CA activities in the water phase and in the Triton phase (6 and 5.1 U, respectively) do not differ very much in this case. This is compatible with the observation of Bordier (2) that water occupies -90% (vol/vol) of the Triton phase. Histochemistry. When applying the DNSA technique to the slaughterhouse bovine hearts we found that after the incubation of the unfixed sections the tissue was poorly preserved. We therefore examined rabbit heart specimens that had been deep-frozen immediately after excision. A micrograph of ventricular muscle incubated with 2.7 x 10m5 M DNSA is presented in Fig. 1. Intense blue fluorescence was observed at the borders of muscle cells. Cells appear delimited by fine lines, which we interpret as SL membranes that are stained due to their content of CA. Intracellular staining can also be observed (see below). In Fig. 2A it is apparent that the space between many cells is tinged in a bulky manner. This Table
4. Comparison of phase-separation behavior CA in Triton X-114 and water
of SL and erythrocyte
observation may result from an oblique projection onto the horizontal plane of stained surfaces of cells that extend into the depth of the section. Another explanation may be the staining of some extracellular structures. Again there is intracellular fluorescence. The staining is not uniformly distributed across the cytosol but appears as though associated with intracellular structures. This staining pattern could indicate the presence of CA associated with intracellular membranes, such as the transverse tubular system or the SR. A micrograph of a control section that was prepared from the same specimen as the one in Fig. 2A is shown in Fig. 2B. Preincubation with the CA inhibitor L645,151 results in an almost complete loss of the SL and intracellular DNSA staining of the myocytes. On the other hand, there is now a diffuse homogenous blue background fluorescence that is caused by the L-645,151 alone. This was verified by performing control sections incubated with L-645,151 in the absence of DNSA (results not shown). Other inhibitors that are nonfluorescent, such as ethoxzolamide and methazolamide, which have been used successfully to suppress the DNSA staining in different tissues (3, 8), did not yield satisfactory results, when we applied them to cardiac tissue. We suppose that their lack of effectiveness is related to their lower lipophilicity compared with L-645,151 (1). L645,151 in this study proved to be the only inhibitor that caused a definite suppression of the DNSA staining of cellular structures, thus demonstrating the specificity of the DNSA staining. DISCUSSION
Quality of membrane preparations. The SR fraction (Table 1) exhibits a high Ca’+-Mg2+-ATPase activity that is in line with data from the literature for oxalate-loaded SR from dog heart [92 pmol h-l mg-’ (21)] and bovine heart [ 75 pmol h-l mg-’ (30)]. The azide-sensitive ATPase activity indicates the presence of some mitochondrial material in this fraction. Jones and Besch (19) reported a specific activity of 140 pmol h-l mg protein-’ in purified mitochondria. If our P fraction (Table 3) was assumed to consist of mitochondrial material only and its azide-sensitive ATPase activity used to calculate mitochondrial contamination in the SR fraction, a maximal value of -15% would result. The azide-sensitive ATPase activity of fraction 111 (Table 1) reveals the presence of a higher proportion of mitochondrial membranes in this band. In addition, fraction III is expected to contain l
l
Total CA Activity Source of CA
Added, %
n
Triton
phase
Water phase
lltl 91t9 87 9 11 85 Values for sarcolemmal (SL) carbonic anhydrase (CA) are means t SD; values for erythrocyte CA are from 2 experiments. Phase-separation method of Bordier (2) was modified as described in METHODS. Amounts of CA activity determined in Triton and water phases, respectively, are expressed as percentage of total amount of CA activity applied. SL Bovine erythrocytes
4 2
l
l
l
l
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ANHYDRASE
H581
Fig. 1. Micrograph showing carbonic anhydrase (CA) dansylsulfonamide (DNSA) fluorescence of mainly cross-sectioned left ventricular tissue. Section (10 Frn) incubated with 2.7 x low5 M DNSA. White sites correspond to blue fluorescence and indicate presence of CA. Myocytes are stained at their borders. Intracellular staining occurs also. Bar = 50 pm.
junctional SR, whereas those vesicles that accumulate calcium oxalate and exhibit high Ca’+-Mg’+-ATPase activity (SR fraction, Table 1) are considered to represent free SR (21). Because of the probably substantial heterogeneity of fraction III, we can only discuss the occurrence of CA activity in free SR but not in junctional SR. The SL fraction (Table 3) has a specific ouabainsensitive Na’+-K2’-ATPase activity, which is in the range of values reported for canine heart SL [140 prnol. h-l. mg-l (19)] and bovine heart SL [55 prnol. h-l +mg-l
(2311.
The large scatter in our values may partly be due to variable time intervals (max 1.5 h) between the death of the animals, when anoxia starts, and homogenization, when reoxygenation occurs. Dhalla et al. (9) found a decrease of Na+-K+-ATPase activity to 70% of the control level after 90 min of ischemia in dog hearts, and the activity was further reduced to 30% upon reperfusion for 60 min. The Ca’+-Mg2+-ATPase activity in our SL fraction is comparable to the levels found by other authors in SL fractions from bovine heart [27 pmol ah-l. mg-l(30)]. Data on the azide-sensitive ATPase activities in SL fractions from dog heart (19) are in good agreement with our values. A calculation similar to that given above for the SR fraction yields a maximal mitochondrial contamination of -10% in the SL fraction. Another source of contamination is to be expected from recent reports which revealed that cardiac SL fractions obtained by different methods, including the one used by us, are considerably contaminated with endothelial plasma membranes (26,29). Thus it is likely that our SL fraction also contains significant amounts of this contaminant. Carbonic anhydrase associated with cardiac sarcolemma. Tables 1 and 3 indicate that there is a positive
correlation of CA activity with the surface membrane marker Na+-K+-ATPase. The SL fraction, with the highest Na+-K+-ATPase activity (Table 3), exhibits the highest CA activity, a value that is 9.5-fold the one found in the microsomes (Table 1). Three sources must be considered from which CA in this fraction could arise: 1) erythrocyte cytosol, 2) endothelial membranes, and 3) the SL. 1) Two markers have been determined to assess the cytosolic contamination of the SL fraction. One of these, LDH, derives almost entirely from the cytosol of the myocytes (data not shown), whereas the other, heme, to a large proportion originates in the cytosol of erythrocytes. The levels of both markers indicate essentially no contamination of the SL fraction with soluble CA, and this is further substantiated by the Triton X-114 phaseseparation studies showing that the CA of the SL fraction is not cytosolic but membrane bound (see below). 2) Endothelial CA has been reported to occur in lung, skeletal muscle (for a review see Ref. 14), and brain (16, 17). Our biochemical data do not exclude the possibility of an endothelial CA because endothelial membranes are likely to contaminate our SL fraction. On the other hand, the present histochemical results do not support the notion of an endothelial CA. With the present data, we cannot definitely resolve the question as to the existence of endothelial CA in the heart. 3) In skeletal muscle the existence of SL CA has recently been established by functional, histochemical, and biochemical evidence (8, 12,31). Zborowska-Sluis et al. (34) had first observed that, in skeletal muscle perfused with either blood or plasma, the space of distribution of Hl*CO: was reduced after administration of acetazolamide by 32 and 39%, respectively, and this was
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for propionate, there was a transient rise in pH, and fall in pHi due to the combination of propionate with H+ at the cell surface, followed by nonionic diffusion of propionic acid into the cell interior. In the presence of 10e4 M acetazolamide the transient surface alkalinization was more pronounced, provided the superfusion solution was mainly buffered by HCOTCO,. A computer model was used to reproduce the experimental pH tracings. This took into account the bulk superfusate compartment, the cell surface compartment, and the intracellular compartment. The action of the CA inhibitor could only be explained by the presence of CA at the external cell surface, whereas the calculations were insensitive to any other possible localization of the enzyme in the tissue (6). Our observation that the SL fraction is enriched in CA activity fits well with these results. Further evidence for the existence of SL CA is provided by the histochemical observations on rabbit heart reported here. We thus conclude that, as in skeletal muscle, there is functional as well as biochemical and histochemical evidence that in the heart there is CA associated with the SL. The specific activity in cardiac SL is of the same order of magnitude as the values reported by Wetzel and Gros (31) for SL fractions from red and white rabbit skeletal muscle (18 and 38 U . ml. mg-‘, respectively). Our results seem to contrast with an earlier report by Moynihan (24). From measurements in homogenates of unperfused rat muscles, which had been prepared in the presence of 0.5% (vol/vol) Triton X-100, he concluded that skeletal muscle contains intrinsic CA, whereas cardiac muscle does not. He quantitated the contribution of the erythrocyte enzyme to the total CA activity in the homogenate by measurements of two different markers of the blood space. From the data of Geers et al. (13) it becomes obvious that particulate CA will contribute only little (
carbonic
BRUNS
AND
anhydrase
GEROLF
Zentrum Physiologie, Medizinische Federal Republic of Germany
GROS
Hochschule
Bruns, Wolfgang, and Gerolf Gros. Membrane-bound carbonic anhydrase in the heart. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H577-H584, 1992.-Microsomal membranes from bovine heart homogenates were subfractionated by density gradient centrifugation. Fractions with high levels of a sarcolemmal (SL) marker are enriched in specific carbonic anhydrase (CA) activity up to ninefold compared with the microsomes. Fractions with high levels of a sarcoplasmic reticulum marker and a mitochondrial marker, respectively, exhibit specific CA activities that are similar to the one found in the microsomes. Determination of cytosolic markers reveals that the CA activity in the SL fraction is not due to contamination by cytosolic CA, and it is shown by Triton X-114 phase separation that the CA activity is due to an integral membrane protein. In cryosections from rabbit heart the SL region of cardiomyocytes is stained by the fluorescent CA inhibitor dansylsulfonamide. Intracellular staining occurs also, with a pattern suggesting the presence of CA associated with intracellular membranes. Although it cannot be excluded that there is a contribution by endothelial membranes, it appears likely that most CA of the heart is bound to the SL. The possible involvement of the enzyme in extracellular proton buffering is discussed. sarcolemma; sarcoplasmic reticulum; ton buffering; cattle; rabbit
dansylsulfonamide;
pro-
MUSCLE, like skeletal muscle, has long been thought to contain no carbonic anhydrase (CA). Although during the last 15 years at least three isozymes have been identified in skeletal muscle, i.e., two cytosolic forms (18, 27) and one that is bound to the sarcolemma and sarcoplasmic reticulum (3, 12, 3l), several publications dealing with heart muscle seem to confirm the view that this tissue is devoid of CA (24, 35). Geers et al. (13) included cardiac tissue in a study on the distribution of the different isozymes in individual blood-free perfused muscles of the rabbit. In the heart, they found no cytosolic CA, which is in line with previous reports (5, 15, 18, 24), but a high CA activity, which could be extracted from the particulate fraction using Triton X-100. The failure of the enzyme to appear in the supernatant of the homogenate in the absence of Triton implies that it is membrane bound. We therefore prepared purified sarcolemmal and sarcoplasmic reticulum vesicles from the bovine heart and investigated the CA activity of these membrane fractions.
CARDIAC
METHODS Membrane preparations. For each preparation, one fresh bovine heart was obtained from the local slaughterhouse, placed in ice, transferred to the laboratory, and used within 1.5 h after the animal’s death. All subsequent steps of trimming and homogenization were performed in a room refrigerated to 4°C. Left ventricular muscle was cleaned of fat and connective tissue and minced with a meat grinder. The material amounted to 180 g/preparation. The membrane preparations were done essentially as described by Jones and co-workers (20-22). 0363-6135/92
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in the heart
Hannover,
D-3000
Hannover 61,
Sarcophsmic reticulum membranes. The principle of the preparation (20, 21) is to homogenize the tissue vigorously by mechanical means in hypotonic buffer and to prepare a microsomal fraction from the homogenate by differential centrifugation. After the microsomes have been washed in a buffer of high KC1 concentration to dissolve contractile proteins, the material is incubated for 10 min at 37°C in a medium containing 50 mM histidine, 100 mM KCl, 65 mM MgC1,, 60 mM NanATP, 25 mM tris(hydroxymethyl)aminomethane (Tris), 25 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 20 mM CaC12, and 5 mM Tris-oxalate (pH 7.1) to load sarcoplasmic reticulum (SR) vesicles selectively with calcium oxalate; this serves to increase their density. Deviating from the protocol of Jones and co-workers (20, 21), the incubated vesicles were then not sedimented and resuspended, but the suspension was divided in four portions of about 10 ml which were immediately layered on four discontinuous sucrose gradients each consisting of 10 ml 1.5 M sucrose, 0.3 M KCl, 50 mM sodium pyrophosphate, 0.1 M Tris (pH 7.2) at the bottom plus, at the top, 5 ml of an identical solution but with only 1.0 M sucrose. The gradients were centrifuged in a Kontron TFT 65.38 fixed-angle rotor at 170,000 g,,, for 2 h. After the run each tube contained three bands plus a pellet of membrane material. The uppermost band was called fraction I, the intermediate band was called fraction II, and the lowest band, located at the 1.0 M-l.5 M sucrose interface, was called fraction III. The bands were collected by aspiration and diluted in 4 vol of ice-cold distilled water. The pellets, termed SR fraction, were resuspended in 0.25 M sucrose and 10 mM histidine (pH 7.5; subsequently called sucrose medium). All fractions were sedimented at 170,000 g,,, for 30 min and finally resuspended in sucrose medium. Of the microsomal suspension (see above) 2.8 ml were not placed on the gradient after oxalate loading but were sedimented in a separate tube during the gradient run. This pellet was resuspended in sucrose medium and subjected to the same final sedimentation and resuspension step as the other fractions. A small amount of the cytosolic fraction from the initial homogenate was also preserved. All samples were frozen in liquid N, and stored at -80°C. A further modification of the original method was to use an Ultra Turrax T 45 equipped with a TP 45/2G generator (Jahnke & Kunkel, Staufen, FRG) at three-fourths maximal speed for homogenization instead of a Polytron. SarcolemmaZ membranes. The principle of the preparation (22) is to homogenize the tissue gently in a buffer with high NaCl concentration and to wash the particulate fraction several times, first with the high-NaCl buffer and then with a hypotonic NaHC03 buffer. The particulate fraction is then homogenized vigorously, and subsequently differential centrifugation is performed. The loose upper layer of the resulting microsomal pellet, which we call crude sarcolemma (crude SL), is resuspended in sucrose medium to give a volume of 30 ml and divided in three portions, each of which is layered over 10 ml 0.6 M sucrose, 0.3 M NaCl, 0.05 M sodium pyrophosphate, and 0.1 M Tris (pH 7.1) and spun in a fixed-angle rotor at 170,000 g,,, for 90 min. The whitish band at the 0.25 M-O.6 M sucrose interface, termed sarcolemmal (SL) fraction, was collected by aspiration and diluted in 4 vol of ice-cold H,Q. The pellet, termed P fraction, was resuspended in sucrose medium. Both fractions were sedimented at 170,000 g,,, for 30 min and finally
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resuspended in sucrose medium. Of the original crude SL suspension (see above) 3 ml were retained for comparative purposes and treated as described for the microsomes from the SR preparation. Again, a small amount of the cytosolic fraction from the primary homogenate was retained. Freezing and storage were as described for the SR preparation. Determination of carbonic anhydrase activity. CA activity was determined using the modification of Maren’s micromethod that has been described in detail previously (3,4). The method utilizes the change of pH in the assay volume caused by hydration of CO, (continuously supplied as gas) following the addition of HCO;-CO:buffer. The time needed for the indicator phenol red to change color from red to yellow depends on CA activity. Temperature is 0°C. Immediately before the measurements, samples of the membrane fractions were solubilized by slowly adding 10% (wt/vol) Triton X-100 in water to a final concentration of 1% (wt/vol), followed by a lo-min incubation time at room temperature. One unit of CA activity was defined as the enzyme concentration in the final assay volume of 0.4 ml that cuts down to one-half the time necessary with the uncatalyzed CO, hydration. Corrections were made for the prolongation of the reaction time due to the presence of sucrose medium (4). The specific CA activity was obtained by dividing the number of units by the protein concentration in the reaction chamber. The amount of CA activity present in a given sample was calculated by multiplying the number of units (a value that is proportional to enzyme concentration) by the volume of the sample. Determination of subcellular markers. Adenosinetriphosphatase (ATPase) activities were determined at 37°C in reaction media essentially identical with those of Jones and Besch (19). The reaction time was 10 min. Reactions were stopped, and the amounts of inorganic phosphate liberated from ATP were determined as described previously (31). The specific ATPase activity was calculated as the amount of inorganic phosphate released per hour per milligram protein. Ca2’-Mg2’-ATPase activity, as a SR marker, was routinely stimulated by the addition of 5.7 PM calcium ionophore A23187 to the assay medium. Determinations were done in the presence of 1 mM ouabain and 5 mM sodium azide. Activity was calculated as the difference in ATP splitting in the presence of 50 PM CaCl, and in the absence of Ca2+ (1 mM EGTA added). Ouabain-sensitive Na’-K’-ATPase activity, as a SL marker, was maximized by the unmasking of latent activity by preincubation of the membranes with 1 mg alamethicin/mg membrane protein. Azide was included in the reaction medium. Azide-sensitive ATPase activity, as a mitochondrial marker, was determined in the presence of ouabain. Lactate dehydrogenase (LDH) activity, as a marker for the cytosolic fraction, was determined at 27°C in a coupled photometric assay. The samples were preincubated in 0.1% (wt/vol) Triton X-100 in assay buffer to make accessible enzyme molecules possibly caged inside the vesicles. The final Triton concentration in the assay was 0.003% (wt/vol). It was ascertained that this procedure did not cause any loss of LDH activities present in the cytosolic fractions (data not shown). One unit of LDH activity was defined as the amount of enzyme that catalyzes the oxidation of 1 pmol NADH2/min. The specific activity was obtained by dividing the activity by the amount of protein present in the assay. Heme content, which was regarded as another cytosolic marker, was estimated from photometric absorption spectra in the presence of 0.1% (wt/vol) Triton X100 in sucrose medium, using an absorbancy coefficient of 100 rnM-l*crn-l at 415 nm. Protein. Protein concentrations were determined by the Lowry method using the assay kit from Sigma Chemical. Samples were solubilized with sodium deoxycholate and protein was precipitated with trichloroacetic acid before the assay (Sigma
ANHYDRASE
procedure P 5656). Triton X-114 phase separation. The method of Bordier (2) was simplified. The Triton X-114 (Sigma) was not pretreated. To 100 ~1 of freshly thawed SL membranes (1.1-3.5 mg protein/ ml) or 100 ~1 of bovine erythrocytes (lysed and diluted 1:500 in distilled water) in an Eppendorf test tube was added 167 ~1 of chilled 1.6% (wt/vol) Triton X-114 in 0.3 M NaCl, and 20 mM Tris (pH 7.4). The mixture was shaken, and the tube was placed in ice water for 5 min, incubated at 31°C in a water bath for 4 min, and centrifuged at 300 g for 3 min in a swing-out rotor. The supernatant (-240 ~1) was carefully transferred into another tube using a micropipette, mixed with a 0.25-fold volume of chilled 2.5% (wt/vol) Triton X-114 in 0.3 M NaCl-20 mM Tris (pH 7.4), placed in ice water for 5 min, and layered over the sediment from the first centrifugation. After 4 min at 31°C the centrifugation step was repeated. The final supernatant, termed water phase, and the final sediment, termed Triton phase, were separated and assayed for CA activity immediately thereafter. In the original method the mixture of sample and Triton is layered over a cushion of 6% (wt/vol) sucrose before incubation in the warm. After centrifugation, the Triton phase is found at the bottom of the tube, whereas the water phase remains above the sucrose cushion. This procedure is designed to exclude water soluble enzymes from the water space of the Triton phase. We omitted the sucrose cushion, since it caused difficulties in identifying the water phase after centrifugation, probably because our samples themselves were suspended in sucrose. Histochemical Localization of CA. Cryosections of left ventricular cardiac muscle from a rabbit were stained using the dansylsulfonamide (DNSA) method described by Dermietzel et al. (8). The specific CA inhibitor DNSA exhibits an intense blue fluorescence when bound to the enzyme, whereas the yellowgreen fluorescence of the free DNSA is faint. A stock solution of 8 x 10m4 M DNSA in 0.1 M HCl was prepared by heating to 60°C for 1 h. This solution was diluted at least 30-fold in phosphate buffer (53 mM Na2HP04-13 mM KH2P04, pH 7.4) at room temperature immediately before the experiments. The DNSA concentration in phosphate buffer was checked photometrically at 323 nm using an absorbancy coefficient of 4,650 M-l cm-l. Th e st oc k so 1u t’ion was stable for at least 1 wk when stored in the dark at room temperature. Sections (10 pm) of tissue were preincubated in 0.1 ml phosphate buffer at room temperature for 10 min. They were then rinsed once with l2.7 x lo+ M DNSA in phosphate buffer. Subsequently 0.1 ml of the DNSA solution was pipetted onto the sections, and a glass cover slip was mounted using nail polish. Controls for the specificity of the DNSA staining were performed by including lO-4 M L-645,151, another specific CA inhibitor (1), in both incubation media. The specimens were viewed and photographed in a Leitz Orthoplan microscope equipped with Fluotar objectives, a high-pressure mercury lamp (HBO 50, Osram) as ultraviolet source, and a Ploemopak 2.1 fluorescence illuminator. The latter consisted of a BP 270-380 filter in the excitation light path, a RKP 380 dichroic mirror, and a BP 410-580 filter in the observation light path. RESULTS
Characteristics of membrane fractions. Data from the SR preparations are shown in Table 1. The lightest band in the density gradient, fraction I, is enriched in Na+K+-ATPase and reduced in azide-sensitive ATPase compared with the microsomes. Its Ca’+-Mg2+-ATPase activity is lower than that of the SR fraction. The data indicate that fraction I is enriched in SL membranes, mainly at the expense of mitochondrial and SR compo-
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Table
1. Properties
CARBONIC
of membrane fractions obtained with SR preparation pmol
Fraction
Microsome I II III SR
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ProteinY ield, mg
CA, U.ml.mg protein-’
111.1246.1 10.4t2.9 39.8t23.0 42.0k16.7 3.5k1.7
phosphate.
Na’-K’-ATPase
1.6t0.5 12.0t3.1 2.2t0.7 0.6tO.l 0.9kO.4
h-l.
mg protein-l Azide-sensitive ATPase
Ca2’-Mg2’-ATPase
8t3 53k20 9t4 2t2 3t1
23t6 24t7 20t6 21t8 102t38
LDH, U/mg protein
54t13 13t5 32t4 54k9 14k6
0.5t0.1 2.2t0.6 0.6kO.2 0.4kO.l 0.2kO.l
Values are means t SD from 5 preparations for total amounts of protein recovered in each fraction and specific enzyme activities of the individual fractions. Parameters were determined as described in METHODS. One unit (U) of carbonic anhydrase (CA) activity was defined as the enzyme concentration in the final assay volume that cuts down to one-half the time of reaction necessary with the uncatalyzed CO2 hydration. Microsome fraction refers to crude microsomal pellet obtained by differential centrifugation. Note that only 93% of this material was actually subjected to the density gradient centrifugation, which yielded the other fractions listed. LDH, lactate dehydrogenase. In sarcoplasmic reticulum (SR) fraction Ca2’-Mg2’-ATPase is significantly enriched (P c 0.001) compared with microsome fraction, whereas Na’-K’-ATPase (P < 0.01) and azide-sensitive ATPase (P < 0.005) are significantly reduced.
nents. Its CA activity is 7.5fold higher than that of the microsomes. The heaviest fraction, SR, as expected, contains the highest activity of Ca2+-Mg2+-ATPase, whereas the activities of the other two membrane markers are here markedly lower than in the microsomes. The SR fraction contains CA activity, but no enrichment in relation to the crude membranes can be observed. Most of the mitochondrial material present in microsomes partitions into fraction III, which contains the lowest specific CA activity. In the cytosolic supernatants from the SR preparations the CA activity was 49 t 13 U, and the LDH activity was 69 t 13 U/ml (means t SD). The ratio CA/LDH in the cytosolic supernatant, in the microsomes and in the SR fraction is shown in Table 2. It can be seen that the membrane fractions are enriched in CA with respect to LDH. If all the CA present in the supernatant is assumed to be of cytosolic origin, then any CA/LDH in the membrane fraction that is greater than the one found in the supernatant indicates the presence of CA in excess of what can be expected from cytosolic contamination of the vesicle preparations. Based on this approach, the percentage contribution of contaminating cytosolic CA to total CA of a given membrane fraction is given by (CA/LDH) cytosol/(CA/LDH)f,,,ti,, . 100%. These percentages were calculated for each single preparation. The data are summarized in Table 2. It can be seen that only about one-fifth of CA activity in the SR fraction may be attributed to contamination with cytosolic CA.
In contrast to the SR preparation, the procedure of the SL preparation tends to result in a high proportion of light membrane material, namely, SL vesicles, in the microsomal fraction (20). The latter was therefore designated crude SL. The data from our SL preparations are shown in Table 3. The lightest band, the SL fraction, comprises 20% of the protein recovered after the density gradient centrifugation of the crude SL. This value is only 11% for fraction I of the SR preparations (see Table 1). In addition, the purity of the SL fraction is higher than that of fraction I judged from a comparison of their Na+-K+ATPase activities. This is paralleled by higher specific CA activity and lower Ca2+-Mg2+- and azide-sensitive ATPase activities of the SL fraction. Of the CA activity present in the crude SL and layered onto the gradient 72% is recovered in the SL fraction. There is no distinct distribution pattern of Ca”+ -Mg2+-ATPase. This can be expected, since the repeated mild homogenizations and washings of the particulate fraction tend to reduce the microsomal SR content (20) and since no calcium oxalate loading step is included in this preparation procedure. The CA/LDH in the crude SL and the SL fraction and the same value for the cytosolic supernatant (CA 27 t 10 U, LDH 69 t 13 U/ml, means t SD) are shown in Table 2. The percentage contaminations of the fractions with cytosolic CA, as calculated from the CA/LDH for each single preparation, are summarized in Table 2. A negligible proportion of the CA activity in the SL fraction
Table 2. Contamination of subcellular fractions with cytosolic CA
Fraction
CA/LDH, U/W/ml)
Contamination With Cytosolic CA From CA/ LDH, %
Heme,
Supernatant Microsome SR
0.720.3 3.221.3 4.8t1.6
100 28t21 18&9
ND ND ND
Supernatant Crude SL SL
0.4tO.l 6.5tl.2 11.9k1.9
100 6t2 3tl
llOk20 ND 2tl
Values are means t SD from 5 sarcoplasmic reticulum (SR) calculations of contamination from ratios of carbonic anhydrase heme. For calculations see text. Supernatant refers to supernatant see Table 3.
PM
CA/Heme, U/PM
Contamination With Cytosolic CA From CA/ Heme, %
0.3tO.l
100
14.224.4
221
preparations (top) and 4 sarcolemmal (SL) preparations (bottom). On left are to lactate dehydrogenase (CA/LDH); on right are calculations from ratios CA/ from primary homogenate; for microsome fraction see Table 1; for crude SL
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3. Properties of membrane fractions obtained with SL preparation Fraction
Protein Yield, mg
CA, U. ml-l. mg protein-l
pmol phosphate. Na’-K+-ATPase
h-l. mg protein-’
LDH, U/mg protein
Azide-sensitive ATPase
Ca2’-M$‘-ATPase
4.122.0 14t7 l&10 113219 0.6t0.3 Crude SL 25.1t17.1 4.5t1.3 15.1t3.6 99*52 15t4 lOZk5 1.320.1 SL P 18.lt12.2 0.8t0.3 4t2 21z!zll 106t13 0.2tO.l Values are means t SD from 4 preparations. Crude SL refers to loose deposit of sarcolemmal membrane material on the microsomal pellet resulting from differential centrifugation. Note that only 90% of this material was subjected to density gradient centrifugation, which yielded the other fractions listed. LDH, lactate dehydrogenase. In SL fraction carbonic anhydrase (CA) activity (P < 0.001) and Na’-K+-ATPase (P < 0.05) are significantly enriched compared with crude SL, whereas azide-sensitive ATPase is significantly reduced (P < 0.001). For further explanations see Table 1.
appears to originate from the cytosol. An analogous calculation, based on the heme contents of the supernatant and the SL fraction, gives the same result. Triton X-l 14 phase-separation studies. The studies were performed with SL membranes and, as a control, with lysed bovine erythrocytes. In the case of membranes the total amount of CA activity introduced into each experiment ranged from 1.3 to 5.2 U/ml. After separation, the volumes of the Triton phase and the water phase were ~0.06 and 0.26 ml, respectively. It can be seen in Table 4 that most of the SL CA partitions into the Triton phase. In the case of red blood cells the starting amount of CA was 2.1 U/ml. In contrast to the behavior of SL CA, most of the cytosolic CA from erythrocytes remains in the water phase. This is mainly due to the greater volume of the water phase (0.29 ml) compared with the Triton phase (0.04 ml), whereas the CA activities in the water phase and in the Triton phase (6 and 5.1 U, respectively) do not differ very much in this case. This is compatible with the observation of Bordier (2) that water occupies -90% (vol/vol) of the Triton phase. Histochemistry. When applying the DNSA technique to the slaughterhouse bovine hearts we found that after the incubation of the unfixed sections the tissue was poorly preserved. We therefore examined rabbit heart specimens that had been deep-frozen immediately after excision. A micrograph of ventricular muscle incubated with 2.7 x 10m5 M DNSA is presented in Fig. 1. Intense blue fluorescence was observed at the borders of muscle cells. Cells appear delimited by fine lines, which we interpret as SL membranes that are stained due to their content of CA. Intracellular staining can also be observed (see below). In Fig. 2A it is apparent that the space between many cells is tinged in a bulky manner. This Table
4. Comparison of phase-separation behavior CA in Triton X-114 and water
of SL and erythrocyte
observation may result from an oblique projection onto the horizontal plane of stained surfaces of cells that extend into the depth of the section. Another explanation may be the staining of some extracellular structures. Again there is intracellular fluorescence. The staining is not uniformly distributed across the cytosol but appears as though associated with intracellular structures. This staining pattern could indicate the presence of CA associated with intracellular membranes, such as the transverse tubular system or the SR. A micrograph of a control section that was prepared from the same specimen as the one in Fig. 2A is shown in Fig. 2B. Preincubation with the CA inhibitor L645,151 results in an almost complete loss of the SL and intracellular DNSA staining of the myocytes. On the other hand, there is now a diffuse homogenous blue background fluorescence that is caused by the L-645,151 alone. This was verified by performing control sections incubated with L-645,151 in the absence of DNSA (results not shown). Other inhibitors that are nonfluorescent, such as ethoxzolamide and methazolamide, which have been used successfully to suppress the DNSA staining in different tissues (3, 8), did not yield satisfactory results, when we applied them to cardiac tissue. We suppose that their lack of effectiveness is related to their lower lipophilicity compared with L-645,151 (1). L645,151 in this study proved to be the only inhibitor that caused a definite suppression of the DNSA staining of cellular structures, thus demonstrating the specificity of the DNSA staining. DISCUSSION
Quality of membrane preparations. The SR fraction (Table 1) exhibits a high Ca’+-Mg2+-ATPase activity that is in line with data from the literature for oxalate-loaded SR from dog heart [92 pmol h-l mg-’ (21)] and bovine heart [ 75 pmol h-l mg-’ (30)]. The azide-sensitive ATPase activity indicates the presence of some mitochondrial material in this fraction. Jones and Besch (19) reported a specific activity of 140 pmol h-l mg protein-’ in purified mitochondria. If our P fraction (Table 3) was assumed to consist of mitochondrial material only and its azide-sensitive ATPase activity used to calculate mitochondrial contamination in the SR fraction, a maximal value of -15% would result. The azide-sensitive ATPase activity of fraction 111 (Table 1) reveals the presence of a higher proportion of mitochondrial membranes in this band. In addition, fraction III is expected to contain l
l
Total CA Activity Source of CA
Added, %
n
Triton
phase
Water phase
lltl 91t9 87 9 11 85 Values for sarcolemmal (SL) carbonic anhydrase (CA) are means t SD; values for erythrocyte CA are from 2 experiments. Phase-separation method of Bordier (2) was modified as described in METHODS. Amounts of CA activity determined in Triton and water phases, respectively, are expressed as percentage of total amount of CA activity applied. SL Bovine erythrocytes
4 2
l
l
l
l
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Fig. 1. Micrograph showing carbonic anhydrase (CA) dansylsulfonamide (DNSA) fluorescence of mainly cross-sectioned left ventricular tissue. Section (10 Frn) incubated with 2.7 x low5 M DNSA. White sites correspond to blue fluorescence and indicate presence of CA. Myocytes are stained at their borders. Intracellular staining occurs also. Bar = 50 pm.
junctional SR, whereas those vesicles that accumulate calcium oxalate and exhibit high Ca’+-Mg’+-ATPase activity (SR fraction, Table 1) are considered to represent free SR (21). Because of the probably substantial heterogeneity of fraction III, we can only discuss the occurrence of CA activity in free SR but not in junctional SR. The SL fraction (Table 3) has a specific ouabainsensitive Na’+-K2’-ATPase activity, which is in the range of values reported for canine heart SL [140 prnol. h-l. mg-l (19)] and bovine heart SL [55 prnol. h-l +mg-l
(2311.
The large scatter in our values may partly be due to variable time intervals (max 1.5 h) between the death of the animals, when anoxia starts, and homogenization, when reoxygenation occurs. Dhalla et al. (9) found a decrease of Na+-K+-ATPase activity to 70% of the control level after 90 min of ischemia in dog hearts, and the activity was further reduced to 30% upon reperfusion for 60 min. The Ca’+-Mg2+-ATPase activity in our SL fraction is comparable to the levels found by other authors in SL fractions from bovine heart [27 pmol ah-l. mg-l(30)]. Data on the azide-sensitive ATPase activities in SL fractions from dog heart (19) are in good agreement with our values. A calculation similar to that given above for the SR fraction yields a maximal mitochondrial contamination of -10% in the SL fraction. Another source of contamination is to be expected from recent reports which revealed that cardiac SL fractions obtained by different methods, including the one used by us, are considerably contaminated with endothelial plasma membranes (26,29). Thus it is likely that our SL fraction also contains significant amounts of this contaminant. Carbonic anhydrase associated with cardiac sarcolemma. Tables 1 and 3 indicate that there is a positive
correlation of CA activity with the surface membrane marker Na+-K+-ATPase. The SL fraction, with the highest Na+-K+-ATPase activity (Table 3), exhibits the highest CA activity, a value that is 9.5-fold the one found in the microsomes (Table 1). Three sources must be considered from which CA in this fraction could arise: 1) erythrocyte cytosol, 2) endothelial membranes, and 3) the SL. 1) Two markers have been determined to assess the cytosolic contamination of the SL fraction. One of these, LDH, derives almost entirely from the cytosol of the myocytes (data not shown), whereas the other, heme, to a large proportion originates in the cytosol of erythrocytes. The levels of both markers indicate essentially no contamination of the SL fraction with soluble CA, and this is further substantiated by the Triton X-114 phaseseparation studies showing that the CA of the SL fraction is not cytosolic but membrane bound (see below). 2) Endothelial CA has been reported to occur in lung, skeletal muscle (for a review see Ref. 14), and brain (16, 17). Our biochemical data do not exclude the possibility of an endothelial CA because endothelial membranes are likely to contaminate our SL fraction. On the other hand, the present histochemical results do not support the notion of an endothelial CA. With the present data, we cannot definitely resolve the question as to the existence of endothelial CA in the heart. 3) In skeletal muscle the existence of SL CA has recently been established by functional, histochemical, and biochemical evidence (8, 12,31). Zborowska-Sluis et al. (34) had first observed that, in skeletal muscle perfused with either blood or plasma, the space of distribution of Hl*CO: was reduced after administration of acetazolamide by 32 and 39%, respectively, and this was
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for propionate, there was a transient rise in pH, and fall in pHi due to the combination of propionate with H+ at the cell surface, followed by nonionic diffusion of propionic acid into the cell interior. In the presence of 10e4 M acetazolamide the transient surface alkalinization was more pronounced, provided the superfusion solution was mainly buffered by HCOTCO,. A computer model was used to reproduce the experimental pH tracings. This took into account the bulk superfusate compartment, the cell surface compartment, and the intracellular compartment. The action of the CA inhibitor could only be explained by the presence of CA at the external cell surface, whereas the calculations were insensitive to any other possible localization of the enzyme in the tissue (6). Our observation that the SL fraction is enriched in CA activity fits well with these results. Further evidence for the existence of SL CA is provided by the histochemical observations on rabbit heart reported here. We thus conclude that, as in skeletal muscle, there is functional as well as biochemical and histochemical evidence that in the heart there is CA associated with the SL. The specific activity in cardiac SL is of the same order of magnitude as the values reported by Wetzel and Gros (31) for SL fractions from red and white rabbit skeletal muscle (18 and 38 U . ml. mg-‘, respectively). Our results seem to contrast with an earlier report by Moynihan (24). From measurements in homogenates of unperfused rat muscles, which had been prepared in the presence of 0.5% (vol/vol) Triton X-100, he concluded that skeletal muscle contains intrinsic CA, whereas cardiac muscle does not. He quantitated the contribution of the erythrocyte enzyme to the total CA activity in the homogenate by measurements of two different markers of the blood space. From the data of Geers et al. (13) it becomes obvious that particulate CA will contribute only little (
