3oumalof
Altered
Molecular
and Cellular Cardiology
( 1976) 8,65 l-66 1
Calcium Sequestration By Subcellular Fractions Smooth Muscle From Spontaneously Hypertensive R. CLINTON Department
of Anatomy
(Received R. C. WEBB
WEBBt
and Cardiovascular of Iowa, Iowa Cip,
Altered
C. BHALLA
Center, College of Medicine, Iowa 52242, USA
1975, and acceped
3 November
AND R. C. BWALLA.
RAMESH
AND
of Vascular Rats*
Calcium
18 December
Sequestration
University
1975)
By Subcellular
Fractions of
Vascular Smooth Muscle From Spontaneously Hypertensive Rats. Journal and Cellular
Cardiology
(1976)
8, 651-660.
The
calcium
sequestration
of Molecular characteristics of
microsomal and mitochondrial fractious prepared from aortae of spontaneously hypertensive (SHR), Kyoto Wistar normotensive (NWR), and normotensive Sprague-Dawley (NSDR) r&s w&e studied. Calcium uptake by the microsomal vesicles of SHR was signiflcantlv lower as comuared to NWR and NSDR. However. mitochondria isolated from SHR aortae did not di#er from NWR in their calcium uptake characteristics. Microsomal vesicles of NSDR aortae accumulated consistently more calcium than NWR. These findings illustrate activity in
the significance
of relevant
controls
for SHR.
Calcium-dependent
ATPase
the microsomal fraction was significantly increased in SHR as compared to NWR. This may indicate a compensatory phenomenon on the part of the smooth muscle cell to increase Car+ uptake by the microsomal vesicles. These results suggest that a decreased ability to sequester calcium by the microsomal fraction of vascular smooth muscle of SHR may result in altered calcium distribution in the muscle cell. KBY
vesicles;
WORDS: Aortic smooth muscle; Subcellular Mitochondria; Spontaneously hypertensive
calcium distribution; Microsomal rats; Calcium sequestration.
1. Introduction There is considerable evidence that calcium ions have a central role in the control of contractile processes in both striated [II, 13, 17,23,27] and smooth muscle [I, 6, 10, 21, 281. In striated muscle the source of calcium for muscular activation has been determined to be the sarcoplasmic reticulum [32]. Although there is an apparent lack of well-defined sarcoplasmic reticulum in smooth muscle [IO], several reports indicate that microsomal and mitochondrial fractions isolated from uterine [8], intestinal [I], and vascular smooth muscle [15, 18, 20, 30, 331 actively sequester Cazf in the presence of ATP and Mgs+. Many factors undoubtedly modiG the pathogenesis of hypertension, however, it appears that Ca2+ and the mechanisms related to its regulation of action may be * These studies were supported by NIH __ Association. t NIH Predoctoral trainee. NIH-GM00148.
Grant
HL-14388
and a grant-in-aid
from the Iowa
Heart
652
R. C. WEBB
AND
R. C. BHALLA
intimately involved in the maintenance of hypertension [16, 291. Recent studies by Aoki and others [Z-4] indicate that the microsomal fractions of vascular smooth muscle from spontaneously hypertensive rats (SHR) show reduced Gas+ accumulating ability compared to that from normotensive control rats. Mangelsen and Bohr [25] have observed a similar decrease in the rate of calcium uptake by microsomal vesicles isolated from thoracic aortae of renal hypertensive rabbits. In terms of vascular smooth muscle function, these investigators suggest that impaired uptake of calcium by the sequestering system may produce a shift in the distribution in intracellular activator calcium, thereby increasing vascular sensitivity to vasoconstrictor agents. Evidence from physiological and pharmacological investigations [5, 9, 221 indicate that the appropriate controls for studies involving SHR are controversial. However, Kyoto Wistar normotensive rats (NWR), which were derived from the same strain as SHR, are probably the best controls available. In the present study, we have re-examined the Gas+ sequestering ability of subcellular fractions prepared from vascular smooth muscle of SHR and compared the results with those obtained Sprague-Dawley rats using Kyoto Wistar normotensive (NWR) and normotensive (NSDR) .
2. Materials
and
Methods
The spontaneously hypertensive rats (SHR) maintained at the University of Iowa are inbred descendents of the hypertensive Wistar strain developed by Okamoto and Aoki [26], and they correspond to the Fss and Fss generation at this time from the original pairing. This colony is maintained by brother-sister mating only. There is no random breeding. Kyoto Wistar normotensive rats (NWR), which were derived originally from the same strain as SHR were used in these studies as relevant controls and were raised under conditions identical to those of SHR. In some experiments, normotensive Sprague-Dawley rats (NSDR) were used to compare with both Kyoto Wistar strains. Preoperative systolic blood pressure was determined in the unanesthetized state by the tail plethysmographic method using an automated cuff inflator-pulse reading system manufactured by Technilab Instruments. The animals were stunned and sacrificed by cervical dislocation; and aortae, from arch to bifurcation, were removed. The aortae were trimmed of adherent fat and loose connective tissue, and washed twice in cold homogenizing buffer (0.25 M sucrose, 0.02 M Tris-HCl, pH 7.4). The tissue was finely minced in 10 volumes of buffer and homogenized with a Polytron (Brinkman) at a rheostat setting of 2.0 for three 10 s pulses. The microsomal vesicles and mitochondria were prepared according to the procedure of Fitzpatrick et al. [1.5] as described earlier [3U]. The homogenate was
CALCIUM
IN
VASCULAR
SMOOTH
MUSCLE
OF SHR
653
centrifuged at 1500 g for 10 min, the supernatant at 27 000 g for 10 min, and the new supernatant at 105 000 g for 60 min to yield the pelleted microsomal vesicles. Mitochondria were isolated by centrifuging the homogenate at 1500 g for 10 min and the supernatant at 9500 g for 10 min. Pelleted fractions were resuspended in cold homogenizing buffer for immediate use. Sample protein content was determined by the procedure of Lowry et al. [%I, with bovine serum albumin as a standard.
Microsomal
and mitochondrial
calcium uptake
Except where stated in results, microsomal calcium uptake was studied at 30°C in a reaction mixture of 1 ml containing 20 mM Tris (hydroxymethyl) aminomethane (Tris-HCl, pH 7.4); 100 mM KCl, 2 mM MgC12; 2 mM ATP; 0.05 to 10.0 PM CaCls; 0.2 $Zi 45Caaf (New England Nuclear) ; 15 mM NaNa; 7.5 mM oxalate; and 80 to 100 pg protein. Mitochondrial calcium uptake was determined by the same method as microsomal calcium uptake except that sodium azide, a potent inhibitor of mitochondrial sequestration, was omitted. The amount of 45Ca2+associated with microsomal vesicles and mitochondria was separated from free 45Ca2+by Millipore filtration (HAWP, 0.45 pg). The Millipore filters were dissolved in Bray’s solution [7] and radioactivity was determined by liquid scintillation spectrometry (Packard Tri-Carb Spectrometer). Non-specific binding of 4sCa2+ was determined by filtering samplesimmediately after the addition of protein to the assaytubes, and thesecounts were subtracted from each sample.
Calcium-stimulated
A TPa.re activity of microsomal fractions
Calcium-stimulated adenosinetriphosphatase (ATPase) activity was determined at 30°C from the rate of liberation of inorganic phosphate. The reaction was initiated by the addition of protein, and terminated by the addition of 0.5 ml of 20% trichloroacetic acid. The precipitate was removed by centrifuging at 2000 g for 10 min and the soluble phosphorus was measured by the procedure of Fiske and Subbarow [24]. ATP hydrolysis associatedwith calcium uptake was calculated as the difference between activity in the presence of calcium and the activity in its absence.
3. Results
The average blood pressure of SHR was 189 f 4 (SE.) mm Hg compared to 114 f 5 for NWR and 126 f 2 for NSDR. The blood pressuresof SHR were significantly higher than those of NWR and NSDR.
654
R. C. WEBB
AND
R.
C. BHALLA
Electron micrographs of the 105 000 g pellet (microsomal fraction) of NSDR aortic homogenates showed that this fraction consisted of vesicular structures. Most vesicles consisted of smooth membranes but occasional rough membrane vesicles were encountered. No intact mitochondrial fragments could be identified in this preparation. Electron microscopic examination of the mitochondrial pellet (9500 g pellet) showed that it contained mostly intact mitochondria, however, fragmented mitochondria could also be seen. Biochemical characterization of subcellular fractions, as reported earlier [30] was carried out by the determination of cytochrome oxidase activity [31]. The activities per milligram of protein in the microsomal fraction was less than 11 y0 of that in the mitochondrial fraction (SHR, 10%; NWR, 9%; NSDR, 11%). In addition, the lack of mitochondrial contamination was also demonstrated by the azide insensitivity [12] of calcium sequestration in the presence of oxalate by the microsomal fractions (Table 1). Protein content of subcellular fractions isolated from aortae of SHR, NWR, and NSDR is given in Table 2. There were no significant differences between the three strains of rats in the yield of protein in different subcellular fractions isolated through the centrifugation procedure.
TABLE
1. The effect of sodium azide on calcium uptake by mitochondria and microsomes isolated from aortae of SHR, NWR, and NSDR. Calcium uptake assays, in the presence and absence of sodium azide, were performed as described in text. Incubation time was 15 min. Each value is the mean of 5 determinations f S.E.M. Non-specific calcium binding was 12.08 nmol Caz+/g protein for SHR mitochondria; 8.43 for NWR mitochondria, 8.10 for NSDR mitochondria; 4.62 for SHR microsomes; 3.49 for NWR microsomes and 5.12 for NSDR microsomes Calcium uptake (nmol Ca2+/mg protein/l5 min) Mitochondria Microsomes 15 mu NaNs 0 n-m NaGa 15 mu NaNs 0 msx NaN3
SHR NWR NSDR
.27 f .02 .29 & .05 .27 f .lO
2.99 & .I5 2.98 f .30 3.23 * .32
2.11 f .ll 3.86 f .13 4.36 f .27
2.25 & .I7 3.94 * .13 4.46 f .26
Calcium uptake by microsomal vesicles isolated from aortae of SHR was significantly lower (P
Altered
Molecular
and Cellular Cardiology
( 1976) 8,65 l-66 1
Calcium Sequestration By Subcellular Fractions Smooth Muscle From Spontaneously Hypertensive R. CLINTON Department
of Anatomy
(Received R. C. WEBB
WEBBt
and Cardiovascular of Iowa, Iowa Cip,
Altered
C. BHALLA
Center, College of Medicine, Iowa 52242, USA
1975, and acceped
3 November
AND R. C. BWALLA.
RAMESH
AND
of Vascular Rats*
Calcium
18 December
Sequestration
University
1975)
By Subcellular
Fractions of
Vascular Smooth Muscle From Spontaneously Hypertensive Rats. Journal and Cellular
Cardiology
(1976)
8, 651-660.
The
calcium
sequestration
of Molecular characteristics of
microsomal and mitochondrial fractious prepared from aortae of spontaneously hypertensive (SHR), Kyoto Wistar normotensive (NWR), and normotensive Sprague-Dawley (NSDR) r&s w&e studied. Calcium uptake by the microsomal vesicles of SHR was signiflcantlv lower as comuared to NWR and NSDR. However. mitochondria isolated from SHR aortae did not di#er from NWR in their calcium uptake characteristics. Microsomal vesicles of NSDR aortae accumulated consistently more calcium than NWR. These findings illustrate activity in
the significance
of relevant
controls
for SHR.
Calcium-dependent
ATPase
the microsomal fraction was significantly increased in SHR as compared to NWR. This may indicate a compensatory phenomenon on the part of the smooth muscle cell to increase Car+ uptake by the microsomal vesicles. These results suggest that a decreased ability to sequester calcium by the microsomal fraction of vascular smooth muscle of SHR may result in altered calcium distribution in the muscle cell. KBY
vesicles;
WORDS: Aortic smooth muscle; Subcellular Mitochondria; Spontaneously hypertensive
calcium distribution; Microsomal rats; Calcium sequestration.
1. Introduction There is considerable evidence that calcium ions have a central role in the control of contractile processes in both striated [II, 13, 17,23,27] and smooth muscle [I, 6, 10, 21, 281. In striated muscle the source of calcium for muscular activation has been determined to be the sarcoplasmic reticulum [32]. Although there is an apparent lack of well-defined sarcoplasmic reticulum in smooth muscle [IO], several reports indicate that microsomal and mitochondrial fractions isolated from uterine [8], intestinal [I], and vascular smooth muscle [15, 18, 20, 30, 331 actively sequester Cazf in the presence of ATP and Mgs+. Many factors undoubtedly modiG the pathogenesis of hypertension, however, it appears that Ca2+ and the mechanisms related to its regulation of action may be * These studies were supported by NIH __ Association. t NIH Predoctoral trainee. NIH-GM00148.
Grant
HL-14388
and a grant-in-aid
from the Iowa
Heart
652
R. C. WEBB
AND
R. C. BHALLA
intimately involved in the maintenance of hypertension [16, 291. Recent studies by Aoki and others [Z-4] indicate that the microsomal fractions of vascular smooth muscle from spontaneously hypertensive rats (SHR) show reduced Gas+ accumulating ability compared to that from normotensive control rats. Mangelsen and Bohr [25] have observed a similar decrease in the rate of calcium uptake by microsomal vesicles isolated from thoracic aortae of renal hypertensive rabbits. In terms of vascular smooth muscle function, these investigators suggest that impaired uptake of calcium by the sequestering system may produce a shift in the distribution in intracellular activator calcium, thereby increasing vascular sensitivity to vasoconstrictor agents. Evidence from physiological and pharmacological investigations [5, 9, 221 indicate that the appropriate controls for studies involving SHR are controversial. However, Kyoto Wistar normotensive rats (NWR), which were derived from the same strain as SHR, are probably the best controls available. In the present study, we have re-examined the Gas+ sequestering ability of subcellular fractions prepared from vascular smooth muscle of SHR and compared the results with those obtained Sprague-Dawley rats using Kyoto Wistar normotensive (NWR) and normotensive (NSDR) .
2. Materials
and
Methods
The spontaneously hypertensive rats (SHR) maintained at the University of Iowa are inbred descendents of the hypertensive Wistar strain developed by Okamoto and Aoki [26], and they correspond to the Fss and Fss generation at this time from the original pairing. This colony is maintained by brother-sister mating only. There is no random breeding. Kyoto Wistar normotensive rats (NWR), which were derived originally from the same strain as SHR were used in these studies as relevant controls and were raised under conditions identical to those of SHR. In some experiments, normotensive Sprague-Dawley rats (NSDR) were used to compare with both Kyoto Wistar strains. Preoperative systolic blood pressure was determined in the unanesthetized state by the tail plethysmographic method using an automated cuff inflator-pulse reading system manufactured by Technilab Instruments. The animals were stunned and sacrificed by cervical dislocation; and aortae, from arch to bifurcation, were removed. The aortae were trimmed of adherent fat and loose connective tissue, and washed twice in cold homogenizing buffer (0.25 M sucrose, 0.02 M Tris-HCl, pH 7.4). The tissue was finely minced in 10 volumes of buffer and homogenized with a Polytron (Brinkman) at a rheostat setting of 2.0 for three 10 s pulses. The microsomal vesicles and mitochondria were prepared according to the procedure of Fitzpatrick et al. [1.5] as described earlier [3U]. The homogenate was
CALCIUM
IN
VASCULAR
SMOOTH
MUSCLE
OF SHR
653
centrifuged at 1500 g for 10 min, the supernatant at 27 000 g for 10 min, and the new supernatant at 105 000 g for 60 min to yield the pelleted microsomal vesicles. Mitochondria were isolated by centrifuging the homogenate at 1500 g for 10 min and the supernatant at 9500 g for 10 min. Pelleted fractions were resuspended in cold homogenizing buffer for immediate use. Sample protein content was determined by the procedure of Lowry et al. [%I, with bovine serum albumin as a standard.
Microsomal
and mitochondrial
calcium uptake
Except where stated in results, microsomal calcium uptake was studied at 30°C in a reaction mixture of 1 ml containing 20 mM Tris (hydroxymethyl) aminomethane (Tris-HCl, pH 7.4); 100 mM KCl, 2 mM MgC12; 2 mM ATP; 0.05 to 10.0 PM CaCls; 0.2 $Zi 45Caaf (New England Nuclear) ; 15 mM NaNa; 7.5 mM oxalate; and 80 to 100 pg protein. Mitochondrial calcium uptake was determined by the same method as microsomal calcium uptake except that sodium azide, a potent inhibitor of mitochondrial sequestration, was omitted. The amount of 45Ca2+associated with microsomal vesicles and mitochondria was separated from free 45Ca2+by Millipore filtration (HAWP, 0.45 pg). The Millipore filters were dissolved in Bray’s solution [7] and radioactivity was determined by liquid scintillation spectrometry (Packard Tri-Carb Spectrometer). Non-specific binding of 4sCa2+ was determined by filtering samplesimmediately after the addition of protein to the assaytubes, and thesecounts were subtracted from each sample.
Calcium-stimulated
A TPa.re activity of microsomal fractions
Calcium-stimulated adenosinetriphosphatase (ATPase) activity was determined at 30°C from the rate of liberation of inorganic phosphate. The reaction was initiated by the addition of protein, and terminated by the addition of 0.5 ml of 20% trichloroacetic acid. The precipitate was removed by centrifuging at 2000 g for 10 min and the soluble phosphorus was measured by the procedure of Fiske and Subbarow [24]. ATP hydrolysis associatedwith calcium uptake was calculated as the difference between activity in the presence of calcium and the activity in its absence.
3. Results
The average blood pressure of SHR was 189 f 4 (SE.) mm Hg compared to 114 f 5 for NWR and 126 f 2 for NSDR. The blood pressuresof SHR were significantly higher than those of NWR and NSDR.
654
R. C. WEBB
AND
R.
C. BHALLA
Electron micrographs of the 105 000 g pellet (microsomal fraction) of NSDR aortic homogenates showed that this fraction consisted of vesicular structures. Most vesicles consisted of smooth membranes but occasional rough membrane vesicles were encountered. No intact mitochondrial fragments could be identified in this preparation. Electron microscopic examination of the mitochondrial pellet (9500 g pellet) showed that it contained mostly intact mitochondria, however, fragmented mitochondria could also be seen. Biochemical characterization of subcellular fractions, as reported earlier [30] was carried out by the determination of cytochrome oxidase activity [31]. The activities per milligram of protein in the microsomal fraction was less than 11 y0 of that in the mitochondrial fraction (SHR, 10%; NWR, 9%; NSDR, 11%). In addition, the lack of mitochondrial contamination was also demonstrated by the azide insensitivity [12] of calcium sequestration in the presence of oxalate by the microsomal fractions (Table 1). Protein content of subcellular fractions isolated from aortae of SHR, NWR, and NSDR is given in Table 2. There were no significant differences between the three strains of rats in the yield of protein in different subcellular fractions isolated through the centrifugation procedure.
TABLE
1. The effect of sodium azide on calcium uptake by mitochondria and microsomes isolated from aortae of SHR, NWR, and NSDR. Calcium uptake assays, in the presence and absence of sodium azide, were performed as described in text. Incubation time was 15 min. Each value is the mean of 5 determinations f S.E.M. Non-specific calcium binding was 12.08 nmol Caz+/g protein for SHR mitochondria; 8.43 for NWR mitochondria, 8.10 for NSDR mitochondria; 4.62 for SHR microsomes; 3.49 for NWR microsomes and 5.12 for NSDR microsomes Calcium uptake (nmol Ca2+/mg protein/l5 min) Mitochondria Microsomes 15 mu NaNs 0 n-m NaGa 15 mu NaNs 0 msx NaN3
SHR NWR NSDR
.27 f .02 .29 & .05 .27 f .lO
2.99 & .I5 2.98 f .30 3.23 * .32
2.11 f .ll 3.86 f .13 4.36 f .27
2.25 & .I7 3.94 * .13 4.46 f .26
Calcium uptake by microsomal vesicles isolated from aortae of SHR was significantly lower (P
