Blood Vessels 13: 321-337 (1976)
Influence of Magnesium on Calcium- and Potassium-Related Responses in Vascular Smooth Muscle1 O liver C arrier, jr., R. K elly H ester, H elga A. J urevics and T homas E. T enner , jr. Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Tex.
Key Words. Vessel relaxation ■ Vessel contraction • Vascular muscle • Depolari zation • Lauthanum Abstract. The influence of Mg on Ca- and K-related responses of rabbit aortic strips was studied. Aortic strips bathed in either a K-free or 60.0 m M K solution were less sensitive to Ca in the presence of 1.2 m M Mg. Ca induced greater maximum tension in the aortic strips in the presence of Mg when bathed in 60.0 m M K. Addition of K to a K-free medium induced a slight relaxation at low concentrations « 1.0 m.M) which was Ca dependent. The relaxation was less in the presence of Mg. With Mg in the medium throughout the experiment (90 min), the threshold con centration of K, inducing a contractile response, was increased as was the maximum tension. Acute addition of Mg decreased the maximum response to K. When K was lowered from 5.4 to 0.0 m M a contractile response, absent in the presence of Mg, occurred. When the K was increased, the tension returned to baseline. At 2530 m M K, the tension again increased, reaching a maximum at 40-50 m M K. «Ca uptake was stimulated at all K concentrations producing a contraction. Mg de pressed this increase in uptake. «C a efflux was more rapid into a 50.0 m M than into 0.0 m M K solution. Mg delayed 15Ca efflux. It is concluded from these data that Mg has two components in its effect on the Ca associated with K responses. One is a competition with Ca at extracellular sites probably at the membrane, and the other is intracellular, probably a competition with Ca at sequestration sites.
> This work was supported in part by Air Force Contract No. AFOSR 71-2075.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Received: July 9, 1975; accepted: February 23, 1976.
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related responses of cardiac and vascular tissue, we had occasion to ob serve the responses of rabbit aortic strips to cumulative concentrations of calcium in both high (50 mM) and low (0.0 mM) potassium Ringer solu tions. We found that aortae responded to calcium in both instances, but that magnesium affected the responses differently. It is known that muscle membranes become depolarized when bathed in a high-potassium me dium [Su et al., 1964; A xelsson et al., 1967; H endrickx and C asteels, 1974]. It has been reported that there is an increase in «calcium (45Ca) uptake under these conditions [B riggs, 1962; van B reemen et al., 1972]. It has recently been shown that there is also a depolarization of the vascular smooth muscle membrane in a potassium-free medium [A xels son et al., 1967; Somlyo et al., 1972; H endrickx and C asteels, 1974]. However, as far as we can determine, nothing is known of how vascular muscle placed in a potassium-free solution handles calcium nor of the influence magnesium has on calcium movements in either a high- or lowpotassium environment. Recent reports in the literature have suggested that magnesium may play a role in modulating calcium availability to the contractile mech anism of smooth muscle [A ltura and A ltura, 1971, 1974; T urlapaty and C arrier, 1973], It is believed that magnesium can influence cal cium entrance into neural tissue [M üller and F inkelstein , 1973], and in doing so, affect neurotransmitter release. During cardiopul monary surgery, and as a result of cardiomyopathies, it has been sug gested that magnesium may be responsible for cardiac potassium move ments [Scheinman et al., 1971; Seelig , 1972a, b; K han et al., 1973], It is possible that the potassium losses which occur during open-heart surgery may occur due to a deficiency in magnesium in the bypass solutions used. The above findings make it apparent that further knowledge of the effects of magnesium at the cellular level are needed. In view of this, we have initiated a study to investigate the influence magnesium has on potassium- and calcium-related responses of vascular muscle, and how magnesium may modify calcium movements during such responses. The results of such a study are the subject of the present report. Though these results are still preliminary in nature, requiring more in depth investiga tion, they do suggest that magnesium may play a role in controlling ion movements in vascular smooth muscle.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
During the course of recent studies [J urevics and C arrier, 1973; T urlapaty and C arrier, 1973] of the effects of magnesium on calcium-
Magnesium, Calcium and Potassium Responses
323
Methods
Solutions. A normal Ringer solution of the following composition was used in these studies: NaCl, 154.0 m M ; KCI, 5.4 m M ; CaCI2, 2.4 m M ; NaHCOs, 6.0 m M ; and dextrose, 11.0 m M . This solution was used either with or without 1.2 m M MgS04. When lanthanum was used in the 45Ca uptake experiments, Tris buffer (5 m M ) was
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Tissue preparation and procedure. New Zealand white rabbits of either sex weighing approximately 1.5 kg were sacrificed by a blow on the head and then exsanguinated. A section of the thoracic aorta between the aortic arch and the diaphragm was removed and placed in oxygenated Ringer solution. Helical aortic strips were then prepared according to the method of F urchgott [I960]. Aortic strips were divided into two equal segments which were approximately 5 mm in width and 40-50 mm in length. In all of the experimental procedures to follow, one segment of each aorta served as a paired control (no magnesium). Ligatures were placed on both ends of the muscle segments. The muscle was mounted verti cally on a glass rod and placed in a tissue organ bath containing 20 ml of normal Ringer solution with or without 1.2 m M MgS04, the end of each segment was then attached to a Grass FT-03 force-displacement transducer and isometric tension was recorded on a Grass Model 7 Polygraph. All muscle preparations were incubated 90 min under an initial tension of 2 g before the experimental procedures were initiated. During this period, the tissues were washed with fresh solution every 15 min to prevent toxic accumulation of waste products [A ltura and A ltura , 1970]. The solutions, both those in the chambers and those used for washing, were continuously oxygenated with a 95% 0 2-5% COa mixture and maintained at a constant temperature of 37 °C. At the end of the initial incubation period, dose-response relationships were obtained for calcium or for potassium, radioactive calcium procedures were initiated, or electrolyte content of the tissue assayed. Calcium responses were obtained in either a potassium-free or 50.0 m M potassium Ringer solution in the absence or presence of 1.2 m M MgS04. Potassium responses were obtained in a potassium-free Ringer solution containing 0.0, 0.3, 1.2 or 2.4 m M CaCl2 in the absence or presence of 1.2 m M MgS04. In one series of experiments (see ‘Results’), the calcium was not added until after the initial 90 min incubation period, and in some experiments (see ‘Results’), magnesium was not added until after the initial 90 min incubation period. In the experiments in which the potassium concentration of the Ringer solution was altered from 5.4 to 0.0 and then to 50.0 m M , the concentration of potassium was reduced by simply diluting the bathing medium with a potassium-free Ringer solution until the desired concentration was obtained. This was done without ex posing the tissue to air at any time during the experiment. In these experiments, the sodium content of the solution remained constant (154.0 m M ) at all concentra tions of potassium. In several test experiments the sodium was reduced as the potassium was increased to maintain osmolality constant. Results obtained in these experiments were not significantly different from those in which the sodium remained constant at 154.0 m M .
324
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substituted for the NaHCOs, and the solution was oxygenated with 100% O, (medical grade). In some experiments (see ‘Results’), the calcium and/or the potassium contents of the Ringer solution were varied. All solutions were prepared with double-distilled, deionized water with the pH adjusted and maintained at 7.4. 45Ca uptake. Aortic strips were incubated in a normal Ringer solution for 90 min. After this initial incubation, the bathing solution was changed to a similar solution containing various amounts of potassium (see ‘Results’) with or without 1.2 mM MgS04 and trace amounts of uniformly labeled Ca efflux. Aortic strips were incubated under 2 g tension for 90 min in a normal Ringer solution and then for 60 min (to insure complete exchange) in a similar solution containing «Ca. After this period of uptake, the strips were rinsed in four tubes containing 5 ml of normal Ringer solution (nonradioactive) within a total period of 40-50 sec and then placed in test tubes containing 5 ml of either potassium-free or 50.0 mM potassium Ringer solution with or without 1.2 mM MgSO,. The tissue was transferred to successive tubes containing 5 ml of these
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Calcium uptake [lanthanum method of van B reemen et al., 1972]. Aortic strips were incubated in a Tris-buffered Ringer solution for 90 min. At the end of this time, the tissues were incubated in a similar solution containing 0.0, 5.4, 25.0 or 50.0 mM potassium with or without 1.2 mM MgSO, and trace amounts of uniformly labeled calcium for 15 min. At the end of this time, LaCl3 (10 mM) was added to the same solution, and after 3 min the solution was changed to a calcium-free solution containing Tris buffer, 5 mM and LaCl3, 10 mM and incubated 45 min. The tissues were then blotted and weighed and radioactivity determined as above. The incubation time for 15 min used in both radioactive procedures above was determined from preliminary experiments in which calcium uptakes (both by the conventional and lanthanum methods) were determined at 5, 10, 15, 20, and 30 min. It was found that there was no further increase in calcium uptake into the tissues by either method, in the absence or presence of 1.2 mM MgSO,, after 10 min. There were no significant differences between the uptakes at 10 min and at 15, 20, or 30 min. Therefore, in view of the experiment being designed to test the effects of magnesium on calcium uptakes associated with potassium and calcium responses, we chose to use the 15-min incubation time in all uptake experiments.
Magnesium, Calcium and Potassium Responses
325
same solutions at designated time intervals for 60 min. At the end of the final wash out period, the tissue was blotted with mineral-free filter paper for 90 sec and the wet weight determined. The tissue was then digested in NCS tissue solubilizer and the radioactivity determined as above. 1 ml of each washout solution was added to 14 ml of liquid scintillation fluid containing 40% ethanol and the radioactivity determined. -tsCa washout data was obtained by determining the total «Ca content of each washout solution and that remaining in the tissue after the 60-min washout. Curves were plotted which express the decline in tissue radioactivity with time (desatura tion curves). Tissue electrolyte content. Tissue calcium, magnesium, potassium and sodium were determined by atomic absorption spectroscopy using a Pcrkin-Elmer Model 303. Tissues were incubated in a normal Ringer or a potassium-free Ringer (with or without 2.4 mM CaCl„) for 90 min under a resting tension of 2 g. They were then blotted on mineral-free filter paper and wet weights obtained. They were then digested in 100 u\ of concentrated nitric acid and the contents evaporated to dryness over a hot plate. This required about 2 h total time. The residue was allowed to cool for 15 min and resuspended in 1.0 ml of 0.1 n HC1 plus 9.0 ml 0.1% lanthanum. The electrolyte contents of this solution were then determined. Statistical significance of data was determined by the use of Student’s t-test. Differences were considered significant at p < 0.05.
Influence of Magnesium on Calcium-Induced Responses of Rabbit Aortic Strips The responses of rabbit aortic strips to cumulative concentrations of calcium in the absence and presence of 1.2 mM magnesium are illustrated in figure 1. The aortic strips were bathed in either a 0.0 or 60.0 mM potassium, calcium-free Ringer solution for 90 min before the addition of calcium. The presence of magnesium in the medium decreased the sensitivity of the aortic strips (7 preparations) to calcium 2.23 (p < 0.005) times at the EDr>0 level when they were tested in the 60.0 mM potassium solution, and 18.0 times (p < 0.001) when they (9 preparations) were tested in the potassium-free medium. The maximum tension developed by the aortae in the 60.0 mM potassium solution was increased signifi cantly (p < 0.001) from 3.38 + 0.30 to 5.29±0.25g in the presence of magnesium. Magnesium had no significant effect on the maximum tension (2.36 ± 0.19 g) developed by the aortae tested in the potassium-free medium.
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Results
C ariuer/H ester/J urevics/T enner
Calcium, mM
b
Fig. I. The effect of 1.2 mM MgS04 on calcium-induced contractile responses of rabbit aortic strips. The open circles represent the responses obtained in the absence of magnesium, the closed circles represent the responses obtained in the presence of 1.2 m M MgSO,. Vertical bars represent the standard error of the mean, a Aortic strips were incubated and tested in a potassium-free medium, b Aortic strips were incubated and tested in a 60.0 m M potassium medium.
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Magnesium, Calcium and Potassium Responses
327
Table /. Effect of magnesium on potassium-induced responses to rabbit aortic strips
threshold, mM
extent, g
Potassium contraction threshold, extent, g mM
0.0 1.2
0.30 ±0.03 0.50±0.1
0.5 ±0.0* 0.4 ±0.09
42.0 ±3.2 60.0±3.02
1.3 ±0.11 1.1 ±0.1
1.2
0.0 1.2
0.20 ±0.06 0.25 ±0.1
0.4 ±0.1 0.1 ±0.073
42.0 ±2.0 55.0±3.02
1.4 ±0.09 1.8±0.093
2.4
0.0 1.2
0.1 ±0.02 0.05 ±0.01 no relaxation
42.0 ±2.0 51.0±2.02
1.1 ±0.08 1.9± l 3
Calcium in Ringer solution mM
Magnesium in Ringer solution mM
0.3
Potassium relaxation
Effect of Magnesium and Calcium on Potassium-Induced Tension Responses of Rabbit Aortic Strips Potassium-induced relaxation. When paired aortic strips were in cubated 90 min in a potassium-free Ringer solution in either the absence or presence of 1.2 mM magnesium, and then potassium was added to the solution in a cumulative manner, a slight relaxation of the muscle was seen. The threshold concentration of potassium producing this relaxation was dependent upon the calcium content of the Ringer solu tion. That is, the higher the calcium content the lower was the threshold. The magnitude of the relaxation was also inversely related to the calcium present in the bathing medium. Magnesium decreased the magnitude of the relaxation observed (table I). Potassium-induced contraction. When the potassium concentration of the solution bathing the aortic strips was increased several times above the concentration inducing a relaxation of the strips (see above), a contractile response occurred. The magnitude of this response was signifi-
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Aortic strips were incubated 90 min in a potassium-free Ringer solution with the indicated calcium and with or without 1.2 mM MgS04. 1 Mean values with SEM of potassium required to initiate a contractile or relaxation response and the grams tension lost or gained in 4 paired experiments. 2 The threshold of the response was significantly different (p
Influence of Magnesium on Calcium- and Potassium-Related Responses in Vascular Smooth Muscle1 O liver C arrier, jr., R. K elly H ester, H elga A. J urevics and T homas E. T enner , jr. Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Tex.
Key Words. Vessel relaxation ■ Vessel contraction • Vascular muscle • Depolari zation • Lauthanum Abstract. The influence of Mg on Ca- and K-related responses of rabbit aortic strips was studied. Aortic strips bathed in either a K-free or 60.0 m M K solution were less sensitive to Ca in the presence of 1.2 m M Mg. Ca induced greater maximum tension in the aortic strips in the presence of Mg when bathed in 60.0 m M K. Addition of K to a K-free medium induced a slight relaxation at low concentrations « 1.0 m.M) which was Ca dependent. The relaxation was less in the presence of Mg. With Mg in the medium throughout the experiment (90 min), the threshold con centration of K, inducing a contractile response, was increased as was the maximum tension. Acute addition of Mg decreased the maximum response to K. When K was lowered from 5.4 to 0.0 m M a contractile response, absent in the presence of Mg, occurred. When the K was increased, the tension returned to baseline. At 2530 m M K, the tension again increased, reaching a maximum at 40-50 m M K. «Ca uptake was stimulated at all K concentrations producing a contraction. Mg de pressed this increase in uptake. «C a efflux was more rapid into a 50.0 m M than into 0.0 m M K solution. Mg delayed 15Ca efflux. It is concluded from these data that Mg has two components in its effect on the Ca associated with K responses. One is a competition with Ca at extracellular sites probably at the membrane, and the other is intracellular, probably a competition with Ca at sequestration sites.
> This work was supported in part by Air Force Contract No. AFOSR 71-2075.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Received: July 9, 1975; accepted: February 23, 1976.
322
C arrier/H ester/J urevics/T enner
related responses of cardiac and vascular tissue, we had occasion to ob serve the responses of rabbit aortic strips to cumulative concentrations of calcium in both high (50 mM) and low (0.0 mM) potassium Ringer solu tions. We found that aortae responded to calcium in both instances, but that magnesium affected the responses differently. It is known that muscle membranes become depolarized when bathed in a high-potassium me dium [Su et al., 1964; A xelsson et al., 1967; H endrickx and C asteels, 1974]. It has been reported that there is an increase in «calcium (45Ca) uptake under these conditions [B riggs, 1962; van B reemen et al., 1972]. It has recently been shown that there is also a depolarization of the vascular smooth muscle membrane in a potassium-free medium [A xels son et al., 1967; Somlyo et al., 1972; H endrickx and C asteels, 1974]. However, as far as we can determine, nothing is known of how vascular muscle placed in a potassium-free solution handles calcium nor of the influence magnesium has on calcium movements in either a high- or lowpotassium environment. Recent reports in the literature have suggested that magnesium may play a role in modulating calcium availability to the contractile mech anism of smooth muscle [A ltura and A ltura, 1971, 1974; T urlapaty and C arrier, 1973], It is believed that magnesium can influence cal cium entrance into neural tissue [M üller and F inkelstein , 1973], and in doing so, affect neurotransmitter release. During cardiopul monary surgery, and as a result of cardiomyopathies, it has been sug gested that magnesium may be responsible for cardiac potassium move ments [Scheinman et al., 1971; Seelig , 1972a, b; K han et al., 1973], It is possible that the potassium losses which occur during open-heart surgery may occur due to a deficiency in magnesium in the bypass solutions used. The above findings make it apparent that further knowledge of the effects of magnesium at the cellular level are needed. In view of this, we have initiated a study to investigate the influence magnesium has on potassium- and calcium-related responses of vascular muscle, and how magnesium may modify calcium movements during such responses. The results of such a study are the subject of the present report. Though these results are still preliminary in nature, requiring more in depth investiga tion, they do suggest that magnesium may play a role in controlling ion movements in vascular smooth muscle.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
During the course of recent studies [J urevics and C arrier, 1973; T urlapaty and C arrier, 1973] of the effects of magnesium on calcium-
Magnesium, Calcium and Potassium Responses
323
Methods
Solutions. A normal Ringer solution of the following composition was used in these studies: NaCl, 154.0 m M ; KCI, 5.4 m M ; CaCI2, 2.4 m M ; NaHCOs, 6.0 m M ; and dextrose, 11.0 m M . This solution was used either with or without 1.2 m M MgS04. When lanthanum was used in the 45Ca uptake experiments, Tris buffer (5 m M ) was
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Tissue preparation and procedure. New Zealand white rabbits of either sex weighing approximately 1.5 kg were sacrificed by a blow on the head and then exsanguinated. A section of the thoracic aorta between the aortic arch and the diaphragm was removed and placed in oxygenated Ringer solution. Helical aortic strips were then prepared according to the method of F urchgott [I960]. Aortic strips were divided into two equal segments which were approximately 5 mm in width and 40-50 mm in length. In all of the experimental procedures to follow, one segment of each aorta served as a paired control (no magnesium). Ligatures were placed on both ends of the muscle segments. The muscle was mounted verti cally on a glass rod and placed in a tissue organ bath containing 20 ml of normal Ringer solution with or without 1.2 m M MgS04, the end of each segment was then attached to a Grass FT-03 force-displacement transducer and isometric tension was recorded on a Grass Model 7 Polygraph. All muscle preparations were incubated 90 min under an initial tension of 2 g before the experimental procedures were initiated. During this period, the tissues were washed with fresh solution every 15 min to prevent toxic accumulation of waste products [A ltura and A ltura , 1970]. The solutions, both those in the chambers and those used for washing, were continuously oxygenated with a 95% 0 2-5% COa mixture and maintained at a constant temperature of 37 °C. At the end of the initial incubation period, dose-response relationships were obtained for calcium or for potassium, radioactive calcium procedures were initiated, or electrolyte content of the tissue assayed. Calcium responses were obtained in either a potassium-free or 50.0 m M potassium Ringer solution in the absence or presence of 1.2 m M MgS04. Potassium responses were obtained in a potassium-free Ringer solution containing 0.0, 0.3, 1.2 or 2.4 m M CaCl2 in the absence or presence of 1.2 m M MgS04. In one series of experiments (see ‘Results’), the calcium was not added until after the initial 90 min incubation period, and in some experiments (see ‘Results’), magnesium was not added until after the initial 90 min incubation period. In the experiments in which the potassium concentration of the Ringer solution was altered from 5.4 to 0.0 and then to 50.0 m M , the concentration of potassium was reduced by simply diluting the bathing medium with a potassium-free Ringer solution until the desired concentration was obtained. This was done without ex posing the tissue to air at any time during the experiment. In these experiments, the sodium content of the solution remained constant (154.0 m M ) at all concentra tions of potassium. In several test experiments the sodium was reduced as the potassium was increased to maintain osmolality constant. Results obtained in these experiments were not significantly different from those in which the sodium remained constant at 154.0 m M .
324
C arrier/H ester/J urevics/T enner
substituted for the NaHCOs, and the solution was oxygenated with 100% O, (medical grade). In some experiments (see ‘Results’), the calcium and/or the potassium contents of the Ringer solution were varied. All solutions were prepared with double-distilled, deionized water with the pH adjusted and maintained at 7.4. 45Ca uptake. Aortic strips were incubated in a normal Ringer solution for 90 min. After this initial incubation, the bathing solution was changed to a similar solution containing various amounts of potassium (see ‘Results’) with or without 1.2 mM MgS04 and trace amounts of uniformly labeled Ca efflux. Aortic strips were incubated under 2 g tension for 90 min in a normal Ringer solution and then for 60 min (to insure complete exchange) in a similar solution containing «Ca. After this period of uptake, the strips were rinsed in four tubes containing 5 ml of normal Ringer solution (nonradioactive) within a total period of 40-50 sec and then placed in test tubes containing 5 ml of either potassium-free or 50.0 mM potassium Ringer solution with or without 1.2 mM MgSO,. The tissue was transferred to successive tubes containing 5 ml of these
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Calcium uptake [lanthanum method of van B reemen et al., 1972]. Aortic strips were incubated in a Tris-buffered Ringer solution for 90 min. At the end of this time, the tissues were incubated in a similar solution containing 0.0, 5.4, 25.0 or 50.0 mM potassium with or without 1.2 mM MgSO, and trace amounts of uniformly labeled calcium for 15 min. At the end of this time, LaCl3 (10 mM) was added to the same solution, and after 3 min the solution was changed to a calcium-free solution containing Tris buffer, 5 mM and LaCl3, 10 mM and incubated 45 min. The tissues were then blotted and weighed and radioactivity determined as above. The incubation time for 15 min used in both radioactive procedures above was determined from preliminary experiments in which calcium uptakes (both by the conventional and lanthanum methods) were determined at 5, 10, 15, 20, and 30 min. It was found that there was no further increase in calcium uptake into the tissues by either method, in the absence or presence of 1.2 mM MgSO,, after 10 min. There were no significant differences between the uptakes at 10 min and at 15, 20, or 30 min. Therefore, in view of the experiment being designed to test the effects of magnesium on calcium uptakes associated with potassium and calcium responses, we chose to use the 15-min incubation time in all uptake experiments.
Magnesium, Calcium and Potassium Responses
325
same solutions at designated time intervals for 60 min. At the end of the final wash out period, the tissue was blotted with mineral-free filter paper for 90 sec and the wet weight determined. The tissue was then digested in NCS tissue solubilizer and the radioactivity determined as above. 1 ml of each washout solution was added to 14 ml of liquid scintillation fluid containing 40% ethanol and the radioactivity determined. -tsCa washout data was obtained by determining the total «Ca content of each washout solution and that remaining in the tissue after the 60-min washout. Curves were plotted which express the decline in tissue radioactivity with time (desatura tion curves). Tissue electrolyte content. Tissue calcium, magnesium, potassium and sodium were determined by atomic absorption spectroscopy using a Pcrkin-Elmer Model 303. Tissues were incubated in a normal Ringer or a potassium-free Ringer (with or without 2.4 mM CaCl„) for 90 min under a resting tension of 2 g. They were then blotted on mineral-free filter paper and wet weights obtained. They were then digested in 100 u\ of concentrated nitric acid and the contents evaporated to dryness over a hot plate. This required about 2 h total time. The residue was allowed to cool for 15 min and resuspended in 1.0 ml of 0.1 n HC1 plus 9.0 ml 0.1% lanthanum. The electrolyte contents of this solution were then determined. Statistical significance of data was determined by the use of Student’s t-test. Differences were considered significant at p < 0.05.
Influence of Magnesium on Calcium-Induced Responses of Rabbit Aortic Strips The responses of rabbit aortic strips to cumulative concentrations of calcium in the absence and presence of 1.2 mM magnesium are illustrated in figure 1. The aortic strips were bathed in either a 0.0 or 60.0 mM potassium, calcium-free Ringer solution for 90 min before the addition of calcium. The presence of magnesium in the medium decreased the sensitivity of the aortic strips (7 preparations) to calcium 2.23 (p < 0.005) times at the EDr>0 level when they were tested in the 60.0 mM potassium solution, and 18.0 times (p < 0.001) when they (9 preparations) were tested in the potassium-free medium. The maximum tension developed by the aortae in the 60.0 mM potassium solution was increased signifi cantly (p < 0.001) from 3.38 + 0.30 to 5.29±0.25g in the presence of magnesium. Magnesium had no significant effect on the maximum tension (2.36 ± 0.19 g) developed by the aortae tested in the potassium-free medium.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Results
C ariuer/H ester/J urevics/T enner
Calcium, mM
b
Fig. I. The effect of 1.2 mM MgS04 on calcium-induced contractile responses of rabbit aortic strips. The open circles represent the responses obtained in the absence of magnesium, the closed circles represent the responses obtained in the presence of 1.2 m M MgSO,. Vertical bars represent the standard error of the mean, a Aortic strips were incubated and tested in a potassium-free medium, b Aortic strips were incubated and tested in a 60.0 m M potassium medium.
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
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Magnesium, Calcium and Potassium Responses
327
Table /. Effect of magnesium on potassium-induced responses to rabbit aortic strips
threshold, mM
extent, g
Potassium contraction threshold, extent, g mM
0.0 1.2
0.30 ±0.03 0.50±0.1
0.5 ±0.0* 0.4 ±0.09
42.0 ±3.2 60.0±3.02
1.3 ±0.11 1.1 ±0.1
1.2
0.0 1.2
0.20 ±0.06 0.25 ±0.1
0.4 ±0.1 0.1 ±0.073
42.0 ±2.0 55.0±3.02
1.4 ±0.09 1.8±0.093
2.4
0.0 1.2
0.1 ±0.02 0.05 ±0.01 no relaxation
42.0 ±2.0 51.0±2.02
1.1 ±0.08 1.9± l 3
Calcium in Ringer solution mM
Magnesium in Ringer solution mM
0.3
Potassium relaxation
Effect of Magnesium and Calcium on Potassium-Induced Tension Responses of Rabbit Aortic Strips Potassium-induced relaxation. When paired aortic strips were in cubated 90 min in a potassium-free Ringer solution in either the absence or presence of 1.2 mM magnesium, and then potassium was added to the solution in a cumulative manner, a slight relaxation of the muscle was seen. The threshold concentration of potassium producing this relaxation was dependent upon the calcium content of the Ringer solu tion. That is, the higher the calcium content the lower was the threshold. The magnitude of the relaxation was also inversely related to the calcium present in the bathing medium. Magnesium decreased the magnitude of the relaxation observed (table I). Potassium-induced contraction. When the potassium concentration of the solution bathing the aortic strips was increased several times above the concentration inducing a relaxation of the strips (see above), a contractile response occurred. The magnitude of this response was signifi-
Downloaded by: Temple University 155.247.166.234 - 1/7/2020 5:00:20 AM
Aortic strips were incubated 90 min in a potassium-free Ringer solution with the indicated calcium and with or without 1.2 mM MgS04. 1 Mean values with SEM of potassium required to initiate a contractile or relaxation response and the grams tension lost or gained in 4 paired experiments. 2 The threshold of the response was significantly different (p
