Pfl/igers Arch (1991) 419:230-234
Journal of Physiology
003167689100169E
9 Springer-Verlag1991
Lactate dehydrogenase activity and isoform distribution in normal and hypertrophic smooth muscle tissue from the rat Ulf Malmqvist 1, Anders Arner 1, and Bengt Uvelius ~ 1Department of Physiologyand Biophysics,and 2Department of Urology, Lund University, S61vegatan 19, S-22362 Lund, Sweden Received March 12, 1991/Receivedafter revision June 7, 1991/AcceptedJune 18, 1991
Abstract. The lactate dehydrogenase (LDH) activity and isoform distribution of L D H were investigated in tissue samples from the rat portal vein, aorta and urinary bladder. In addition, samples were obtained from hypertrophic urinary bladder. The total L D H activity per unit smooth muscle volume was higher in the urinary bladder compared to that in portal vein and aorta. Five L D H isoforms, reflecting different combinations of the two polypeptide chains denoted H and M, could be separated by agarose gel electrophoresis. The aorta contained more o f the H form compared to the portal vein and urinary bladder. This difference suggests that the aorta, which is a slow smooth muscle, is more adapted for aerobic metabolism than the faster muscles of portal vein and urinary bladder. In the hypertrophic urinary bladder a shift in L D H isoform pattern towards less of the H form was found, which correlates with a better maintenance of contraction in anoxia in this type of hypertrophic smooth muscle.
Key words: Smooth muscle - Lactate dehydrohgenase Isoforms - Hypertrophy, Rat urinary bladder portal vein - Rat arota
Rat
Introduction Smooth muscle exhibits a large variation in contractile properties and in the metabolic correlates o f contraction (see [7, 201). In general, muscles with high shortening velocity have a correspondingly high metabolic tension cost [tension-related adenosine 5'-triphosphate (ATP) hydrolysis/active force], whereas slow muscles have a low tension cost (see [71). Smooth muscle has, during optimal oxygenation, a considerable lactate formation (aerobic glycolysis, see [21]). In comparison with the oxidative glucose metabolism, aerobic glycolysis is energetically un-
Offprint requests to: U. Malmqvist
favourable, since less ATP is formed for a given amount o f glucose. The reason for the aerobic glycolysis is unclear at present, although it has been suggested that it is linked to membrane ion pump activity [21]. During contraction the relative contribution of glycolysis to the ATP formation differs among muscles; e.g. K+-induced contractions are associated with a decrease in lactate formation in the rat aorta and an increase in the rat portal vein
[1]. Lactate dehydrogenase (LDH) catalyzes the reduction o f pyruvate to lactate or the oxidation of lactate to pyruvate. L D H consists of four subunits composed of two different polypeptide chains (denoted H, M; see [5]). Five different forms (M4, M3H, M2H2, MH3, H4) can be formed which differ in electrophoretic mobility. Compared to the M subunit, the H subunit has a higher affinity for lactate, for the formation of pyruvate and a larger product inhibition by lactate when forming lactate from pyruvate. Thus, with increasing amount of H subunits in LDH, the enzyme is less capable of forming lactate from pyruvate. The H and M forms are encoded by two different genes [5] and the relative amount of the two forms, and L D H isoforms, can thus vary between cells. In tissues dependent on oxidative metabolism, such as the heart, the H forms of L D H dominate. The expression of L D H isoforms in striated muscle can be influenced by alterations in the environment or in the functional demands on the muscle. During the transition of "fast twich" to "slow twitch" of fast twitch skeletal muscle a shift in L D H pattern towards more of the H forms is observed [22]. In cardiac muscle, hypertension causes an increase of the M form of L D H [9]. In smooth muscle the L D H isoform distribution in homologous muscle is found to vary between species [12]. L D H isoform shifts have been observed in smooth muscle when exposed to low oxygen tension [11]. During atherosclerosis an increase in the M form is observed in the smooth muscle cells in the atherosclerotic plaque [6]. In the human myometrium the L D H activity increases and the L D H isoform pattern shifts towards the M form during pregnancy [8, 14].
231 T h e p u r p o s e o f the present study was to determine the L D H activity a n d i s o f o r m d i s t r i b u t i o n in different s m o o t h muscle tissues f r o m one species in order to investigate a possible correlation with contractile a n d m e t a b o l ic properties o f the muscles. I n addition, we c o m p a r e d the activity a n d isoforms o f L D H i n n o r m a l a n d hypertrophic rat u r i n a r y b l a d d e r to investigate if alterations i n these metabolic properties c a n occur d u r i n g growth o f s m o o t h muscle.
Materials and methods Adult female Sprague-Dawleyrats weighing 220-250 g were used. The animals were killed by cervical fracture and the following tissues were dissected: aorta, portal vein and urinary bladder. For comparison the rat heart, rat psoas and rabbit soleus muscles were analysed in some experiments. Hypertrophy of the urinary bladder was induced by urinary outflow obstruction as described previously [3]. The animals were anaesthetized by intraperitoneal injection of methohexital sodium (Brietal E. Lilly AB, Stockholm, Sweden) and via a lower abdominal incision a ligature of 4/0 surgical silk was tied around the proximal urethra. The degree of obstruction was determined by an indwellingrod inserted in the urethra during the operation. After 10 days the animals were killed and the urinary bladders removed for analysis. The tissue samples were weighed and homogenized in 1500 Ixl of a solution containing 72 mM Na2HPO4 and 28 mM NaH2PO4 at pH 7.2. The samples were then centrifuged using an Eppendorf (Netheler-Hinz GmbH, Hamburg, Germany) 5415 C centrifuge. The supernatant was further diluted 1 : 10 for urinary bladder, 1 : 3 for aorta and 1 : 1 for portal vein with the phosphate buffer to give about 3 mg muscle wet weight per ml. The samples were then used for electrophoresis and assay of LDH activity. Electrophoretic separation of LDH isozymes was performed on 11 • 20.5 cm agarose gels essentially as described by van der Helm [25]. The electrophoresis buffer contained 410 mM Na2HPO4 and 90 mM KH2PO4 at pH 7.4. About 5 ~tl of each tissue sample was loaded on the gel and separated for about 30 min with 260 V giving about 100 mA. The gels were stained with nitroblue tetrazolium as described in [25] and scanned using a Hoeffer GS-300 densitometer (Hoeffer Scientific Instruments, San Francisco, Calif., USA). The relative percentage of the H form was calculated assuming 0, 25, 50, 75 and 100% in the respective LDH isoforms (M4, M3H, M2H2, MH3, H4). LDH activity was determined using a photometrical multiple point rate test (Ectachem, Eastman Kodak, Rochester, New York, N.Y. USA). Activities are given in units (1 unit corresponds to 1 mol of pyruvate converted to lactate per s at 37~C). Tissue activities are given in units/mg wet weight or in units/smooth muscle cell volume.
Statbstics. Valuesare reported as mean___SEM with the number of observations (n) given within parentheses. Statistical comparisons were made using the Student's t-test for unpaired data or analysis of variance using the Scheff6 method of determining simultaneous confidence limits.
Results T h e weight o f the u r i n a r y b l a d d e r in the 10-day ligated g r o u p was 248+_ 14 mg c o m p a r e d to 57_+3 m g i n the control a n i m a l s ( P < 0 . 0 0 1 , Students t-test, n = 6 in each group). This weight increase is similar to t h a t f o u n d in previous studies o f this a n i m a l model, (e.g. [3]) a n d reflects a m a r k e d h y p e r t r o p h y o f the s m o o t h muscle cells. L D H isozymes from different s m o o t h muscle tissues separated o n agarose gels are s h o w n in Fig. 1. Five different isoforms of L D H were f o u n d i n accordance with the literature: H4, H 3 M , H 2 M 2 , H M 3 a n d M4 (correspond-
Fig. 1A-D. Separation of LDH isoforms from different smooth muscle tissues from the rat on agarose gels. The photographs show original gels and gel scans from A portal vein, B aorta, C urinary bladder, D hypertrophic urinary bladder. The separated bands correspond, from left ( - ) to right (+) to the M4, M3H, M2H2, MH3, and H4 LDH isoforms respectively ing to L D H - 1 , L D H - 2 , L D H - 3 , L D H - 4 , L D H - 5 (see [10]). I n control experiments we separated extracts from rat psoas, rat h e a r t a n d rabbit soleus muscles. I n the psoas muscle o n l y the M4 form was observed whereas in the soleus the p a t t e r n was shifted towards the H forms. T h e heart c o n t a i n e d H 4 a n d small a m o u n t s of H 3 M a n d H2M2. I n the s m o o t h muscle samples different patterns were observed; in the a o r t a the isozyme p a t t e r n was shifted toward the H 4 forms a n d in the portal vein a n d urin a r y b l a d d e r the p a t t e r n was d o m i n a t e d by the M forms. C o m p a r e d with the controls, the hypertrophic u r i n a r y b l a d d e r c o n t a i n e d more o f the M4 form. D e n s i t o m e t r i c gelscans of the L D H isozymes from the s m o o t h muscle tissues, are also shown i n Fig. 1. T h e five forms could be clearly distinguished o n the scans. T h e areas of the peaks c o r r e s p o n d i n g to the different forms were q u a n t i t a t e d as the p r o d u c t o f peak height a n d the width at h a l f the height. T h e calculated relative
232 Table 1. Lactate dehydrogenase(LDH) isoform distribution in different smooth muscle tissues from the rat Preparation
LDH isoform M4
M3H
M2H2
MH3
H4
H / ( H + M)
8.0+ 1.4
33.1 + 1.6
33.1 + 1.4
25.2+2.2
0.6_+0.6
44.3 + 1.5
Portal vein
33.1 + 1.5
43.7-+ 1.9
19.7_+ 1.5
3.5_+ 1.0
0
23.4_+0.7
Control bladder
27.3 -+ 1.6
37.6 -+ 1.6
20.9 + 0.7
6.3 + 1.2
8.0 -+0.4
32.5 + 0.9
Hypertrophic bladder
48.9 +_2.6
33.4 _+1.0
10.9 -+ 1.0
3.5 + 0.9
3.4 +_1.1
19.7 -+2.4
Aorta
The relative proportions of the different LDH isozymes were determined as shown in Fig. 1 and are expressed as a percentage of total LDH. Values are shown as mean -+ SEM (n = 6 in each group). The relative content of H form was calculated as described in Methods. Statistical comparisons of the H / ( H + M) data, using analysis of variance, showed that significant ( P < 0.05) differences existed between all groups except between the portal vein and hypertrophic urinary bladder
amounts o f the different L D H isoforms for the different tissue samples are given in Table 1. The relative amount of the H form was calculated and shown also in Table 1. The isoform pattern in the aorta was shifted towards the H forms and the calculated relative content of the H form [ H / ( M + H ) in Table 1] was significantly higher than in the other muscles. In the hypertrophic urinary bladder a significant increase in the M4 content and a decrease in total H form was observed. The total L D H activities in the tissue samples are given in Table 2. The values are given per unit tissue wet weight and smooth muscle cell volume. The L D H activities per unit smooth muscle volume were calculated using previous structural data from these muscles reported from our laboratory (references given in the Table 2 legend).
Discussion
In our comparative study of L D H isoforms were made measurements on a fast and a slow smooth muscle, the Table 2. Lactate dehydrogenase (LDH) activity in different smooth muscle tissues from the rat Preparation
LDH activity nU/mg wet weight
nU/~l smooth muscle
0.51 _+0.07
2.33 +- 0.30
Portal vein
1.21 _+0.14
2.20 _+0.25
Control bladder
2.22 +_0.14
5.42 _+0.35
Hypertrophic bladder
2.57+_ 0.28
5.05 _+0.54
Aorta
LDH activity is expressed in units (U) relative to tissue wet weight or unit smooth muscle volume. Values are shown as mean +_SEM (n = 6 in each group). The volume of smooth muscle in the samples was calculated using previously reported structural data regarding the relative smooth muscle volume in the respective tissue preparations (portal vein: 55% [16]; aorta: 22% [1, 2]; urinary bladder: 49% [18]. Statistical comparisons of the LDH activity data (both when related to wet weight and to smooth muscle volume) using analysis of variance, showed that significant (P
Journal of Physiology
003167689100169E
9 Springer-Verlag1991
Lactate dehydrogenase activity and isoform distribution in normal and hypertrophic smooth muscle tissue from the rat Ulf Malmqvist 1, Anders Arner 1, and Bengt Uvelius ~ 1Department of Physiologyand Biophysics,and 2Department of Urology, Lund University, S61vegatan 19, S-22362 Lund, Sweden Received March 12, 1991/Receivedafter revision June 7, 1991/AcceptedJune 18, 1991
Abstract. The lactate dehydrogenase (LDH) activity and isoform distribution of L D H were investigated in tissue samples from the rat portal vein, aorta and urinary bladder. In addition, samples were obtained from hypertrophic urinary bladder. The total L D H activity per unit smooth muscle volume was higher in the urinary bladder compared to that in portal vein and aorta. Five L D H isoforms, reflecting different combinations of the two polypeptide chains denoted H and M, could be separated by agarose gel electrophoresis. The aorta contained more o f the H form compared to the portal vein and urinary bladder. This difference suggests that the aorta, which is a slow smooth muscle, is more adapted for aerobic metabolism than the faster muscles of portal vein and urinary bladder. In the hypertrophic urinary bladder a shift in L D H isoform pattern towards less of the H form was found, which correlates with a better maintenance of contraction in anoxia in this type of hypertrophic smooth muscle.
Key words: Smooth muscle - Lactate dehydrohgenase Isoforms - Hypertrophy, Rat urinary bladder portal vein - Rat arota
Rat
Introduction Smooth muscle exhibits a large variation in contractile properties and in the metabolic correlates o f contraction (see [7, 201). In general, muscles with high shortening velocity have a correspondingly high metabolic tension cost [tension-related adenosine 5'-triphosphate (ATP) hydrolysis/active force], whereas slow muscles have a low tension cost (see [71). Smooth muscle has, during optimal oxygenation, a considerable lactate formation (aerobic glycolysis, see [21]). In comparison with the oxidative glucose metabolism, aerobic glycolysis is energetically un-
Offprint requests to: U. Malmqvist
favourable, since less ATP is formed for a given amount o f glucose. The reason for the aerobic glycolysis is unclear at present, although it has been suggested that it is linked to membrane ion pump activity [21]. During contraction the relative contribution of glycolysis to the ATP formation differs among muscles; e.g. K+-induced contractions are associated with a decrease in lactate formation in the rat aorta and an increase in the rat portal vein
[1]. Lactate dehydrogenase (LDH) catalyzes the reduction o f pyruvate to lactate or the oxidation of lactate to pyruvate. L D H consists of four subunits composed of two different polypeptide chains (denoted H, M; see [5]). Five different forms (M4, M3H, M2H2, MH3, H4) can be formed which differ in electrophoretic mobility. Compared to the M subunit, the H subunit has a higher affinity for lactate, for the formation of pyruvate and a larger product inhibition by lactate when forming lactate from pyruvate. Thus, with increasing amount of H subunits in LDH, the enzyme is less capable of forming lactate from pyruvate. The H and M forms are encoded by two different genes [5] and the relative amount of the two forms, and L D H isoforms, can thus vary between cells. In tissues dependent on oxidative metabolism, such as the heart, the H forms of L D H dominate. The expression of L D H isoforms in striated muscle can be influenced by alterations in the environment or in the functional demands on the muscle. During the transition of "fast twich" to "slow twitch" of fast twitch skeletal muscle a shift in L D H pattern towards more of the H forms is observed [22]. In cardiac muscle, hypertension causes an increase of the M form of L D H [9]. In smooth muscle the L D H isoform distribution in homologous muscle is found to vary between species [12]. L D H isoform shifts have been observed in smooth muscle when exposed to low oxygen tension [11]. During atherosclerosis an increase in the M form is observed in the smooth muscle cells in the atherosclerotic plaque [6]. In the human myometrium the L D H activity increases and the L D H isoform pattern shifts towards the M form during pregnancy [8, 14].
231 T h e p u r p o s e o f the present study was to determine the L D H activity a n d i s o f o r m d i s t r i b u t i o n in different s m o o t h muscle tissues f r o m one species in order to investigate a possible correlation with contractile a n d m e t a b o l ic properties o f the muscles. I n addition, we c o m p a r e d the activity a n d isoforms o f L D H i n n o r m a l a n d hypertrophic rat u r i n a r y b l a d d e r to investigate if alterations i n these metabolic properties c a n occur d u r i n g growth o f s m o o t h muscle.
Materials and methods Adult female Sprague-Dawleyrats weighing 220-250 g were used. The animals were killed by cervical fracture and the following tissues were dissected: aorta, portal vein and urinary bladder. For comparison the rat heart, rat psoas and rabbit soleus muscles were analysed in some experiments. Hypertrophy of the urinary bladder was induced by urinary outflow obstruction as described previously [3]. The animals were anaesthetized by intraperitoneal injection of methohexital sodium (Brietal E. Lilly AB, Stockholm, Sweden) and via a lower abdominal incision a ligature of 4/0 surgical silk was tied around the proximal urethra. The degree of obstruction was determined by an indwellingrod inserted in the urethra during the operation. After 10 days the animals were killed and the urinary bladders removed for analysis. The tissue samples were weighed and homogenized in 1500 Ixl of a solution containing 72 mM Na2HPO4 and 28 mM NaH2PO4 at pH 7.2. The samples were then centrifuged using an Eppendorf (Netheler-Hinz GmbH, Hamburg, Germany) 5415 C centrifuge. The supernatant was further diluted 1 : 10 for urinary bladder, 1 : 3 for aorta and 1 : 1 for portal vein with the phosphate buffer to give about 3 mg muscle wet weight per ml. The samples were then used for electrophoresis and assay of LDH activity. Electrophoretic separation of LDH isozymes was performed on 11 • 20.5 cm agarose gels essentially as described by van der Helm [25]. The electrophoresis buffer contained 410 mM Na2HPO4 and 90 mM KH2PO4 at pH 7.4. About 5 ~tl of each tissue sample was loaded on the gel and separated for about 30 min with 260 V giving about 100 mA. The gels were stained with nitroblue tetrazolium as described in [25] and scanned using a Hoeffer GS-300 densitometer (Hoeffer Scientific Instruments, San Francisco, Calif., USA). The relative percentage of the H form was calculated assuming 0, 25, 50, 75 and 100% in the respective LDH isoforms (M4, M3H, M2H2, MH3, H4). LDH activity was determined using a photometrical multiple point rate test (Ectachem, Eastman Kodak, Rochester, New York, N.Y. USA). Activities are given in units (1 unit corresponds to 1 mol of pyruvate converted to lactate per s at 37~C). Tissue activities are given in units/mg wet weight or in units/smooth muscle cell volume.
Statbstics. Valuesare reported as mean___SEM with the number of observations (n) given within parentheses. Statistical comparisons were made using the Student's t-test for unpaired data or analysis of variance using the Scheff6 method of determining simultaneous confidence limits.
Results T h e weight o f the u r i n a r y b l a d d e r in the 10-day ligated g r o u p was 248+_ 14 mg c o m p a r e d to 57_+3 m g i n the control a n i m a l s ( P < 0 . 0 0 1 , Students t-test, n = 6 in each group). This weight increase is similar to t h a t f o u n d in previous studies o f this a n i m a l model, (e.g. [3]) a n d reflects a m a r k e d h y p e r t r o p h y o f the s m o o t h muscle cells. L D H isozymes from different s m o o t h muscle tissues separated o n agarose gels are s h o w n in Fig. 1. Five different isoforms of L D H were f o u n d i n accordance with the literature: H4, H 3 M , H 2 M 2 , H M 3 a n d M4 (correspond-
Fig. 1A-D. Separation of LDH isoforms from different smooth muscle tissues from the rat on agarose gels. The photographs show original gels and gel scans from A portal vein, B aorta, C urinary bladder, D hypertrophic urinary bladder. The separated bands correspond, from left ( - ) to right (+) to the M4, M3H, M2H2, MH3, and H4 LDH isoforms respectively ing to L D H - 1 , L D H - 2 , L D H - 3 , L D H - 4 , L D H - 5 (see [10]). I n control experiments we separated extracts from rat psoas, rat h e a r t a n d rabbit soleus muscles. I n the psoas muscle o n l y the M4 form was observed whereas in the soleus the p a t t e r n was shifted towards the H forms. T h e heart c o n t a i n e d H 4 a n d small a m o u n t s of H 3 M a n d H2M2. I n the s m o o t h muscle samples different patterns were observed; in the a o r t a the isozyme p a t t e r n was shifted toward the H 4 forms a n d in the portal vein a n d urin a r y b l a d d e r the p a t t e r n was d o m i n a t e d by the M forms. C o m p a r e d with the controls, the hypertrophic u r i n a r y b l a d d e r c o n t a i n e d more o f the M4 form. D e n s i t o m e t r i c gelscans of the L D H isozymes from the s m o o t h muscle tissues, are also shown i n Fig. 1. T h e five forms could be clearly distinguished o n the scans. T h e areas of the peaks c o r r e s p o n d i n g to the different forms were q u a n t i t a t e d as the p r o d u c t o f peak height a n d the width at h a l f the height. T h e calculated relative
232 Table 1. Lactate dehydrogenase(LDH) isoform distribution in different smooth muscle tissues from the rat Preparation
LDH isoform M4
M3H
M2H2
MH3
H4
H / ( H + M)
8.0+ 1.4
33.1 + 1.6
33.1 + 1.4
25.2+2.2
0.6_+0.6
44.3 + 1.5
Portal vein
33.1 + 1.5
43.7-+ 1.9
19.7_+ 1.5
3.5_+ 1.0
0
23.4_+0.7
Control bladder
27.3 -+ 1.6
37.6 -+ 1.6
20.9 + 0.7
6.3 + 1.2
8.0 -+0.4
32.5 + 0.9
Hypertrophic bladder
48.9 +_2.6
33.4 _+1.0
10.9 -+ 1.0
3.5 + 0.9
3.4 +_1.1
19.7 -+2.4
Aorta
The relative proportions of the different LDH isozymes were determined as shown in Fig. 1 and are expressed as a percentage of total LDH. Values are shown as mean -+ SEM (n = 6 in each group). The relative content of H form was calculated as described in Methods. Statistical comparisons of the H / ( H + M) data, using analysis of variance, showed that significant ( P < 0.05) differences existed between all groups except between the portal vein and hypertrophic urinary bladder
amounts o f the different L D H isoforms for the different tissue samples are given in Table 1. The relative amount of the H form was calculated and shown also in Table 1. The isoform pattern in the aorta was shifted towards the H forms and the calculated relative content of the H form [ H / ( M + H ) in Table 1] was significantly higher than in the other muscles. In the hypertrophic urinary bladder a significant increase in the M4 content and a decrease in total H form was observed. The total L D H activities in the tissue samples are given in Table 2. The values are given per unit tissue wet weight and smooth muscle cell volume. The L D H activities per unit smooth muscle volume were calculated using previous structural data from these muscles reported from our laboratory (references given in the Table 2 legend).
Discussion
In our comparative study of L D H isoforms were made measurements on a fast and a slow smooth muscle, the Table 2. Lactate dehydrogenase (LDH) activity in different smooth muscle tissues from the rat Preparation
LDH activity nU/mg wet weight
nU/~l smooth muscle
0.51 _+0.07
2.33 +- 0.30
Portal vein
1.21 _+0.14
2.20 _+0.25
Control bladder
2.22 +_0.14
5.42 _+0.35
Hypertrophic bladder
2.57+_ 0.28
5.05 _+0.54
Aorta
LDH activity is expressed in units (U) relative to tissue wet weight or unit smooth muscle volume. Values are shown as mean +_SEM (n = 6 in each group). The volume of smooth muscle in the samples was calculated using previously reported structural data regarding the relative smooth muscle volume in the respective tissue preparations (portal vein: 55% [16]; aorta: 22% [1, 2]; urinary bladder: 49% [18]. Statistical comparisons of the LDH activity data (both when related to wet weight and to smooth muscle volume) using analysis of variance, showed that significant (P
