Blood Vessels 12: 279-289 (1975)
Calcium Flux and Binding in the Aortic Smooth Muscle from the Spontaneously Hypertensive Rat1 S. Shibata , M. K uchii and T. T aniguchi Department of Pharmacology, School of Medicine, Biomedical Sciences Building, University of Hawaii, Honolulu, Hawaii
Key Words. Spontaneously hypertensive rat aorta • Norepinephrine • 45Ca influx and efflux • Potassium • Microsomal 45Ca uptake • Cations (Mn++, Co**", La*” ). Abstract. There was no significant difference in the tissue calcium content be tween spontaneously hypertensive rat (SHR) and normotensive rat aortae. 45Ca uptake was significantly greater, whereas 4SCa influx was significantly less in SHR than in control aortae. There was no difference in 45Ca efflux in SHR and control tissues. 4SCa influx in both SHR and control tissues was increased by potassium, not increased by norepinephrine and inhibited by Co” , Mn++ and La+” (all 0.5 mM). Both SHR and control aortae were equally inhibited by M n" and Co”' (0.1 mM). There was no difference in the 45Ca uptake by microsome fraction from both SHR and control tissue. These data indicate some differences in the translocation of Ca” at the cellular level in SHR and control aortae.
Introduction Previous experiments have indicated that the aorta of the spontaneously hypertensive rat (SHR) shows a lower reactivity to certain stimuli than that of the normotensive rat [Spector et al., 1969; Shibata et al., 1973]. There was a characteristic difference in the configuration of the contractile response especially to norepinephrine and potassium. The response of the control aortic strip to agonists was characterized by a fast and a slow 1 This work was partly supported by grants from the Hawaii Heart Association and Takeda Chemical Industries Ltd.
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Received: December 20,1974; accepted: April 23,1975.
280
S hibata/K uchii /T aniguchi
component; however, in the SHR aorta, the slow component was dimin ished or frequently, completely absent as is seen with the rabbit aorta when extracellularly bound Ca** is replaced by La***. Moreover, it has been demonstrated that vascular smooth muscle of SHR contracts in re sponse to certain cations, whereas that of the normotensive rat does not [Shibata et al., 1973; B o h r , 1974], On this basis it was postulated that the difference in vascular reactivity of hypertensive and normotensive rats may reflect a difference in calcium metabolism in vascular smooth muscles [Shibata et al., 1973]. To test this hypothesis, the present investigation was undertaken to study the flux and binding of Ca** in aortae of SHR and normotensive rats.
SHR and Kyoto and Hawaii normotensive Wistar rats (NWR) of either sex, 3 months in age, weighing 250-300 g, were used in this study. SHR and NWR were obtained from stocks originally contributed by NIH and Kyoto University animal centers and inbred at the University of Hawaii animal colony. In some cases, Hawaii NWR also were used. Prior to their use, the blood pressures of the animals were measured by use of a sphygmomanometer (NARCO Biosystem Inc.) without anesthe sia. The systolic blood pressure of SHR and NWR were 208 ± 12 mm Hg (n = 30) and 127 ± 6 mm Hg (n = 30), respectively. The animals were sacrificed by a blow on the head and the carotid artery cut. For uptake and flux studies the aortae were freed of excess fat and connective tissues and prepared into helical strips about 3 mm in width and 40 mm in length. The aortic strips were placed in Tris-buffered physiological solution (pH 7.4) containing in mM: NaCl, 160; MgCl2, 1; CaCl2, 1.5; KC1, 4.6; dextrose, 10; Tris, 5. The solution was maintained at 37 °C and aerated with pure oxygen throughout the experiment. Tissue calcium content. To determine the total tissue calcium, the thoracic aortae were removed, weighed and dried in an incubator for 48 h at 100-105 °C. Dry tissue weights were measured and tissues then digested in nitric and perchloric acid until almost a colorless solution. Calcium concentration was then determined on an atomic absorption spectrometer (Shimazu model AA-610S). isCa uptake. 45Ca uptake by aortic strips was determined according to the methods described by van Breemen et al. [1972]. In this method, after an equilibration period of 1 h in physiological solution, the aortic strips were incubated with ,5Ca (0.5 «Ci/ml) in 2.5 ml of Tris-buffered solution or calcium-free solution for periods varying up to 1 h. This Ca-free medium was prepared simply by removing Ca** from Trisbuffered medium while retaining the radioactive 4SCa. Following the incubation in radioactive medium, the strips were blotted, weighed and ashed; the ash was then dissolved in 1 ml of 1 mM LaCl, in 0.1 N HC1 to which 10 ml of Bray’s solution was added. The mixture was then counted in a Packard liquid scintillation counter. Tissue radioactivity was expressed as milliliter of bath fluid cleared per gram wet weight. This expression is numerically the same as the tissue/medium ratio.
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Methods
Calcium Flux and SHR Aortae
281
iiCa influx (“Ca retained). The aortic strips were exposed to 45Ca for 30 min and placed in Ca-free medium containing 2 mM LaCl3 for 1 h. The strips were then blotted, weighed, ashed and counted. From the 4SCa taken up, influx was calculated and expressed as mmol/g tissue (45Ca, 100cpm/ml tissue equals Ca** 0.113 mmol/g tissue). 4SCa efflux. Aortic strips were allowed to take up 45Ca from the Ca-free physio logical medium for 30 min and dipped three times in nonradioactive solution in rapid sequence to rinse the radioactivity adhering to the tissue surface. The strips were then transferred to the counting vials containing 1 ml of Ca-free medium. They were moved at 5-min intervals up to 100 min through a succession of vials after which 10 ml of Bray’s solution was added and the vials prepared for counting. At the end of the final washing period, the tissue was weighed, ashed and the radioactivity counted. Data for washout of 45Ca were obtained by determining the 45Ca in the wash samples and the 45Ca remaining in the tissue after 100 min washout. Binding of 4SCa to microsome fractions. 45Ca binding to microsome fractions was determined by the method of F itzpatrick et al. [1972], Approximately 1.5 g of vascular tissue was homogenated in ten volumes of cold isotonic sucrose medium in a potter homogenizer fitted with a Teflon pestle. To prepare the microsome frac tions the homogenate was spun at 1,500 g for 10 min. The supernatant of this procedure was subjected to centrifugation at 27,000 g for 10 min and the microsomes obtained from the subsequent supernatant spun at 105,000 g for 60 min. The final microsomal pellet was resuspended in 2 ml of isotonic sucrose and used immediately. 45Ca uptake was studied at 37 °C in a 3-ml incubation mixture containing Tris, 10 mM; KC1, 100 mM; Mg-ATP, 3 mM; ammonium oxalate, 5 mM; sodium azide, 5 mM; 45Ca, 0.4 //Ci; CaCI2 0.1 mM and 0.2 ml of the microsomal fraction. The uptake of calcium by vesicles was determined by a radioisotopic method. The samples of particulate matter were filtered in the incubation medium, washed on a Millipore filter and control for radioactivity on a liquid scintillation counter. Protein was determined by the method of L owry et al. [1951]. Ca uptake by microsome is expressed as «mol/g protein per 15 min.
Results
>sCa Uptake Aortic strips were incubated in 45Ca Tris buffer solution for varying periods of time and tissue 45Ca content was measured to determine the uptake of Ca**. As shown in figure 1, the 45Ca uptake of SHR aortic strips was significantly greater than that of the control. In both strips the 45Ca
Downloaded by: MacQuarie University 137.111.162.20 - 1/17/2020 3:13:13 PM
Tissue Calcium Content In table I the total calcium content of SHR and Kyoto and Hawaii NWR aortae is presented. There was no significant difference in the tissue calcium content between SHR and control (Kyoto NWR and Hawaii NWR) aortae. This is also true of the atria and ventricle of these animals.
282
Shibata/K uchii/T aniguchi
Fig. 2. 45Ca uptake by aortae from SHR ( • ) and normotensive rats (O) in Ca” -free medium. Five aortae from different animals were used for each experi ment. 45Ca uptake of SHR aortae at 30 and 60 min after incubation was significantly different from control (p < 0.05 and < 0.02).
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Fig. 1. 45Ca uptake by aortae from SHR (# ) and normotensive rats (O) in normal Ringer’s medium. Five aortae from different animals were used for each experiment. All values of 45Ca uptake of SHR aortae at different incubation times are significantly different from the control (Student t-test, p < 0.05-< 0.02).
Calcium Flux and SHR Aortae
283
Table I. Calcium content of aorta and heart from SHR and NWR Animal
SHR Kyoto NWR Hawaii NWR
Calcium content, mEq/kg dry weight (± SEM; n = 7) aorta
atria
ventricle
3.98 ± 0.4 3.38 ± 0.3 3.7 ±0.4
2.2 ± 0.6 2.2 ± 0.2 2.8 ± 0.4
0.67 ± 0.04 0.73 ± 0.03 0.72 ± 0.04
uptake was maximal within 10 min after exposure to the radioisotope medium. When the aortic strips were incubated in Ca-free Ringer’s solu tion (nonradioactive Ca-free medium), for 15 min prior to exposure to the 45Ca medium, the J5Ca uptake by both tissues increased nearly 20-fold over that in normal Ringer’s solution, and the peak 45Ca uptake did not occur until after 30 min (fig. 2). Under these conditions, although the difference in tissue 45Ca content between SHR and control was not sig nificant during the early incubation periods after 30 min incubation, this became statistically significant (n = 5, p < 0.05 ~ p < 0.02) (fig. 2).
Efflux of iSCa in Ca-free Medium Figure 3 shows the loss of 4SCa from aortic strips which had been first exposed to 45Ca for 30 min and subsequently placed in the Ca-free medium.
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4SCa Influx Table II shows the influx of 45Ca in aortic strips of NWR and SHR. Although the SHR aorta showed a greater 45Ca uptake than NWR, the 45Ca influx for SHR tissue was slightly but significantly less than that for NWR. Administration of norepinephrine (10~5m) failed to increase 45Ca influx in either SHR or NWR aorta. On the other hand, high potassium (60 him) increased the 45Ca influx by approximately 16% in NWR and by 86% in SHR aorta. In table III are shown the inhibitory effects of cations such as Co~, Mn ** and La*** on the 45Ca influx. When the SHR aorta was incubated with 45Ca in the presence of MnCl2, CoCl., and LaCl3 (all at 0.5 mM), the 45Ca influx was inhibited by approximately 21, 32 and 79% , respectively. These cations also affected the 45Ca influx in NWR aorta to nearly the same extent observed in SHR. La*++ was the most effective inhibitor of 45Ca influx in both aortic strips.
284
S hibata/K uchii/T aniguchi
Table 11. Effects of norepinephrine and excess potassium on the 15Ca influx for aortae from SHR and NWR Experi ments
Control Norepinephrine KC1 (60 mM)
7 7 7
nmol/g tissue (mean ± SEM) NWR
SHR
p values 1
79.0 ± 3.5 72.0 ± 6.2 86.5 ± 3.5
66.7 ± 5.3 54.5 ± 8.5 114.0 ±4.5
p
Calcium Flux and Binding in the Aortic Smooth Muscle from the Spontaneously Hypertensive Rat1 S. Shibata , M. K uchii and T. T aniguchi Department of Pharmacology, School of Medicine, Biomedical Sciences Building, University of Hawaii, Honolulu, Hawaii
Key Words. Spontaneously hypertensive rat aorta • Norepinephrine • 45Ca influx and efflux • Potassium • Microsomal 45Ca uptake • Cations (Mn++, Co**", La*” ). Abstract. There was no significant difference in the tissue calcium content be tween spontaneously hypertensive rat (SHR) and normotensive rat aortae. 45Ca uptake was significantly greater, whereas 4SCa influx was significantly less in SHR than in control aortae. There was no difference in 45Ca efflux in SHR and control tissues. 4SCa influx in both SHR and control tissues was increased by potassium, not increased by norepinephrine and inhibited by Co” , Mn++ and La+” (all 0.5 mM). Both SHR and control aortae were equally inhibited by M n" and Co”' (0.1 mM). There was no difference in the 45Ca uptake by microsome fraction from both SHR and control tissue. These data indicate some differences in the translocation of Ca” at the cellular level in SHR and control aortae.
Introduction Previous experiments have indicated that the aorta of the spontaneously hypertensive rat (SHR) shows a lower reactivity to certain stimuli than that of the normotensive rat [Spector et al., 1969; Shibata et al., 1973]. There was a characteristic difference in the configuration of the contractile response especially to norepinephrine and potassium. The response of the control aortic strip to agonists was characterized by a fast and a slow 1 This work was partly supported by grants from the Hawaii Heart Association and Takeda Chemical Industries Ltd.
Downloaded by: MacQuarie University 137.111.162.20 - 1/17/2020 3:13:13 PM
Received: December 20,1974; accepted: April 23,1975.
280
S hibata/K uchii /T aniguchi
component; however, in the SHR aorta, the slow component was dimin ished or frequently, completely absent as is seen with the rabbit aorta when extracellularly bound Ca** is replaced by La***. Moreover, it has been demonstrated that vascular smooth muscle of SHR contracts in re sponse to certain cations, whereas that of the normotensive rat does not [Shibata et al., 1973; B o h r , 1974], On this basis it was postulated that the difference in vascular reactivity of hypertensive and normotensive rats may reflect a difference in calcium metabolism in vascular smooth muscles [Shibata et al., 1973]. To test this hypothesis, the present investigation was undertaken to study the flux and binding of Ca** in aortae of SHR and normotensive rats.
SHR and Kyoto and Hawaii normotensive Wistar rats (NWR) of either sex, 3 months in age, weighing 250-300 g, were used in this study. SHR and NWR were obtained from stocks originally contributed by NIH and Kyoto University animal centers and inbred at the University of Hawaii animal colony. In some cases, Hawaii NWR also were used. Prior to their use, the blood pressures of the animals were measured by use of a sphygmomanometer (NARCO Biosystem Inc.) without anesthe sia. The systolic blood pressure of SHR and NWR were 208 ± 12 mm Hg (n = 30) and 127 ± 6 mm Hg (n = 30), respectively. The animals were sacrificed by a blow on the head and the carotid artery cut. For uptake and flux studies the aortae were freed of excess fat and connective tissues and prepared into helical strips about 3 mm in width and 40 mm in length. The aortic strips were placed in Tris-buffered physiological solution (pH 7.4) containing in mM: NaCl, 160; MgCl2, 1; CaCl2, 1.5; KC1, 4.6; dextrose, 10; Tris, 5. The solution was maintained at 37 °C and aerated with pure oxygen throughout the experiment. Tissue calcium content. To determine the total tissue calcium, the thoracic aortae were removed, weighed and dried in an incubator for 48 h at 100-105 °C. Dry tissue weights were measured and tissues then digested in nitric and perchloric acid until almost a colorless solution. Calcium concentration was then determined on an atomic absorption spectrometer (Shimazu model AA-610S). isCa uptake. 45Ca uptake by aortic strips was determined according to the methods described by van Breemen et al. [1972]. In this method, after an equilibration period of 1 h in physiological solution, the aortic strips were incubated with ,5Ca (0.5 «Ci/ml) in 2.5 ml of Tris-buffered solution or calcium-free solution for periods varying up to 1 h. This Ca-free medium was prepared simply by removing Ca** from Trisbuffered medium while retaining the radioactive 4SCa. Following the incubation in radioactive medium, the strips were blotted, weighed and ashed; the ash was then dissolved in 1 ml of 1 mM LaCl, in 0.1 N HC1 to which 10 ml of Bray’s solution was added. The mixture was then counted in a Packard liquid scintillation counter. Tissue radioactivity was expressed as milliliter of bath fluid cleared per gram wet weight. This expression is numerically the same as the tissue/medium ratio.
Downloaded by: MacQuarie University 137.111.162.20 - 1/17/2020 3:13:13 PM
Methods
Calcium Flux and SHR Aortae
281
iiCa influx (“Ca retained). The aortic strips were exposed to 45Ca for 30 min and placed in Ca-free medium containing 2 mM LaCl3 for 1 h. The strips were then blotted, weighed, ashed and counted. From the 4SCa taken up, influx was calculated and expressed as mmol/g tissue (45Ca, 100cpm/ml tissue equals Ca** 0.113 mmol/g tissue). 4SCa efflux. Aortic strips were allowed to take up 45Ca from the Ca-free physio logical medium for 30 min and dipped three times in nonradioactive solution in rapid sequence to rinse the radioactivity adhering to the tissue surface. The strips were then transferred to the counting vials containing 1 ml of Ca-free medium. They were moved at 5-min intervals up to 100 min through a succession of vials after which 10 ml of Bray’s solution was added and the vials prepared for counting. At the end of the final washing period, the tissue was weighed, ashed and the radioactivity counted. Data for washout of 45Ca were obtained by determining the 45Ca in the wash samples and the 45Ca remaining in the tissue after 100 min washout. Binding of 4SCa to microsome fractions. 45Ca binding to microsome fractions was determined by the method of F itzpatrick et al. [1972], Approximately 1.5 g of vascular tissue was homogenated in ten volumes of cold isotonic sucrose medium in a potter homogenizer fitted with a Teflon pestle. To prepare the microsome frac tions the homogenate was spun at 1,500 g for 10 min. The supernatant of this procedure was subjected to centrifugation at 27,000 g for 10 min and the microsomes obtained from the subsequent supernatant spun at 105,000 g for 60 min. The final microsomal pellet was resuspended in 2 ml of isotonic sucrose and used immediately. 45Ca uptake was studied at 37 °C in a 3-ml incubation mixture containing Tris, 10 mM; KC1, 100 mM; Mg-ATP, 3 mM; ammonium oxalate, 5 mM; sodium azide, 5 mM; 45Ca, 0.4 //Ci; CaCI2 0.1 mM and 0.2 ml of the microsomal fraction. The uptake of calcium by vesicles was determined by a radioisotopic method. The samples of particulate matter were filtered in the incubation medium, washed on a Millipore filter and control for radioactivity on a liquid scintillation counter. Protein was determined by the method of L owry et al. [1951]. Ca uptake by microsome is expressed as «mol/g protein per 15 min.
Results
>sCa Uptake Aortic strips were incubated in 45Ca Tris buffer solution for varying periods of time and tissue 45Ca content was measured to determine the uptake of Ca**. As shown in figure 1, the 45Ca uptake of SHR aortic strips was significantly greater than that of the control. In both strips the 45Ca
Downloaded by: MacQuarie University 137.111.162.20 - 1/17/2020 3:13:13 PM
Tissue Calcium Content In table I the total calcium content of SHR and Kyoto and Hawaii NWR aortae is presented. There was no significant difference in the tissue calcium content between SHR and control (Kyoto NWR and Hawaii NWR) aortae. This is also true of the atria and ventricle of these animals.
282
Shibata/K uchii/T aniguchi
Fig. 2. 45Ca uptake by aortae from SHR ( • ) and normotensive rats (O) in Ca” -free medium. Five aortae from different animals were used for each experi ment. 45Ca uptake of SHR aortae at 30 and 60 min after incubation was significantly different from control (p < 0.05 and < 0.02).
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Fig. 1. 45Ca uptake by aortae from SHR (# ) and normotensive rats (O) in normal Ringer’s medium. Five aortae from different animals were used for each experiment. All values of 45Ca uptake of SHR aortae at different incubation times are significantly different from the control (Student t-test, p < 0.05-< 0.02).
Calcium Flux and SHR Aortae
283
Table I. Calcium content of aorta and heart from SHR and NWR Animal
SHR Kyoto NWR Hawaii NWR
Calcium content, mEq/kg dry weight (± SEM; n = 7) aorta
atria
ventricle
3.98 ± 0.4 3.38 ± 0.3 3.7 ±0.4
2.2 ± 0.6 2.2 ± 0.2 2.8 ± 0.4
0.67 ± 0.04 0.73 ± 0.03 0.72 ± 0.04
uptake was maximal within 10 min after exposure to the radioisotope medium. When the aortic strips were incubated in Ca-free Ringer’s solu tion (nonradioactive Ca-free medium), for 15 min prior to exposure to the 45Ca medium, the J5Ca uptake by both tissues increased nearly 20-fold over that in normal Ringer’s solution, and the peak 45Ca uptake did not occur until after 30 min (fig. 2). Under these conditions, although the difference in tissue 45Ca content between SHR and control was not sig nificant during the early incubation periods after 30 min incubation, this became statistically significant (n = 5, p < 0.05 ~ p < 0.02) (fig. 2).
Efflux of iSCa in Ca-free Medium Figure 3 shows the loss of 4SCa from aortic strips which had been first exposed to 45Ca for 30 min and subsequently placed in the Ca-free medium.
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4SCa Influx Table II shows the influx of 45Ca in aortic strips of NWR and SHR. Although the SHR aorta showed a greater 45Ca uptake than NWR, the 45Ca influx for SHR tissue was slightly but significantly less than that for NWR. Administration of norepinephrine (10~5m) failed to increase 45Ca influx in either SHR or NWR aorta. On the other hand, high potassium (60 him) increased the 45Ca influx by approximately 16% in NWR and by 86% in SHR aorta. In table III are shown the inhibitory effects of cations such as Co~, Mn ** and La*** on the 45Ca influx. When the SHR aorta was incubated with 45Ca in the presence of MnCl2, CoCl., and LaCl3 (all at 0.5 mM), the 45Ca influx was inhibited by approximately 21, 32 and 79% , respectively. These cations also affected the 45Ca influx in NWR aorta to nearly the same extent observed in SHR. La*++ was the most effective inhibitor of 45Ca influx in both aortic strips.
284
S hibata/K uchii/T aniguchi
Table 11. Effects of norepinephrine and excess potassium on the 15Ca influx for aortae from SHR and NWR Experi ments
Control Norepinephrine KC1 (60 mM)
7 7 7
nmol/g tissue (mean ± SEM) NWR
SHR
p values 1
79.0 ± 3.5 72.0 ± 6.2 86.5 ± 3.5
66.7 ± 5.3 54.5 ± 8.5 114.0 ±4.5
p
