European Journal of Pharmacology, 37 (1976) 213--216
213
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication CALCIUM INFLUX AND POSTJUNCTIONAL SUPERSENSITIVITY IN GUINEA PIG AORTIC STRIPS MARVIN KAIMAN and SHOJI SHIBATA *
Department of Pharmacology, School of Medicine, University of Hawaii, Honolulu, Hawaii, U.S.A. Received 16 March 1976, accepted 18 March 1976
M. KAIMAN and S. SHIBATA, Calcium influx and postjunctional supersensitivity in guinea pig aortic strips, European J. Pharmacol. 37 (1976) 213--216. Both reserpine and 6-hydroxydopamine (6-OHDA) pretreatment potentiated the sensitivity of guinea pig aortic strips to norepinephrine (NE), barium, methoxamine and potassium indicating postjunctional supersensitivity. However, cocaine treatment only potentiated the NE response indicating prejunctional supersensitivity. 6-OHDA and reserpine-induced supersensitivity but not cocaine-induced supersensitivity was accompanied by an increase in 4SCa influx. Postjunctional supersensitivity Guinea pig aortic strips
6-Hydroxydopamine
Reserpine
Cocaine
4SCa influx
1. Introduction
2. Materials and methods
It is well known that reserpine, 6-hydroxydopamine (6-OHDA) and cocaine induce supersensitivity in vascular smooth muscle (Carrier and Shibata, 1967; Maling et al., 1971; Shibata et al., 1972). Carrier and Shibata (1967), Hudgins and Harris (1970) and Carrier and Jurevics (1973) have suggested that a change in the permeability of vascular smooth muscle sarcolemma to calcium may account for reserpine-induced supersensitivity. In spite of this suggestion, no reports have appeared in the literature which deal with the influx of calcium in supersensitive vascular smooth muscle. In this report, the influx of 4SCa in reserpine, 6-OHDA and cocaine-induced supersensitive guinea pig aortic strips are presented.
Helically cut strips (3.5 cm × 0.3 cm) were prepared from aortae obtained from guinea pigs with or without pretreatment with either one 4 mg/kg i.m. dose of reserpine (Serpasil, CIBA) 24 h prior to sacrifice or two 50 mg/kg i.p. doses of 6-hydroxydopamine hydrobromide (Regis) dissolved in 0.1% ascorbic acid 24 and 48 h prior to sacrifice. The strips were then placed into 25 ml organ baths which contained Krebs--Ringer bicarbonate buffer solution at pH 7.4. The organ baths were maintained at 37°C and aerated with a 95% 02 and 5% CO2 gas mixture. A loading tension of 1.5 g was obtained by elevating Grass FT.03 transducers. Isometric contractions were recorded by attaching a 6-channel Grass polygraph to the transducers. The strips were equilibrated for 1.5 h, the medium being replaced every 15 min prior to the addition of norepinephrine bitartrate (NE), methoxamine hydrochloride (ME, Vasoxyl), barium chloride (Ba 2÷) or potassium chloride (K÷). The contractile responses are expressed
* Please send reprint requests and correspondence t o : Dr. S. Shibata, University of Hawaii, Department of Pharmacology, School of Medicine, Biomedical Sciences Building, 1960 East-West Road, Honolulu, Hawaii 96822, U.S.A.
214
M. K A I M A N , S. S H I B A T A
in terms of the mean ED~0 of geometrically determined means. Aortic strips were incubated in Krebs-Ringer for 1.5 h. The strips were then cut into pieces (6--8 mg wet weight) and placed into a Tris-modified Krebs--Ringer buffer solution at pH 7.4 with the following composition (mM): NaC1, 144.5; KC1, 6.0; CaC12, 1.2; MgSO4 7 H20, 1.3; tris-(hydroxymethyl)aminomethane, 5 ; and glucose, 5.6 in distilled, deionized water. The medium was maintained at 37°C and oxygenated with 100% 02. The strips were incubated for 15 min prior to the addition of 4SCa (0.5 pCi/ml) and NE or K ÷. In some experiments cocaine was present during the last 10 min of the 15 min incubation period. Tissue samples were removed 1 and 5 min after the addition of 4SCa and placed into calcium-free Tris Ringer containing 2 mM LaC13 for 30 min in order to remove extracellularly bound 4SCa (Van Breemen et al., 1972). The tissues were then blotted, quickly dipped into four successive ice-cold Tris Ringer media, reblotted and weighed. The tissues were then placed into scintillation vials and 0.5 ml of Soluene-100 (Packard) was added and the tissues were digested overnight at 45°C. 15 ml of toluene-based scintillation cocktail containing 5 g PPO and 50 mg
POPOP per liter of toluene was added to the vials and the amount of radioactivity was determined in a scintillation counter (Packard TriCarb 3380). From the 4SCa retained by the tissue after exposure to the lanthanum-Tris media, 4SCa influx was calculated and expressed in terms of nmole/g wet weight of tissue as follows: tissue 45 Ca content after La 3 + (dpm/g wet weight of tissue) specific activity of 4 SCa in Tris media (dpm/nmole Ca 2 +) = 45Ca influx
3. Results Table 1 shows the EDs0's foi the norepinephrine, methoxamine, barium and potassium contractile responses of guinea pig aortic strips after treatment with reserpine, 6-hydroxydopamine and cocaine. In the strips pretreated with reserpine or 6-OHDA the EDs0's for NE, ME, Ba 2+ and K ÷ were decreased but the maximal contractile responses were not altered thus indicating the development of supersensitivity. In the strips treated with cocaine only the EDs0 for NE was decreased while the EDs0 for K ÷ was increased. Cocaine treatment had no effect on the EDs0 for ME or Ba 2+. Maximal contractile response to NE, ME, Ba 2÷ and K ÷ were not altered by cocaine.
TABLE 1 Effect o f reserpine, 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) a n d c o c a i n e o n t h e EDs0 values o f n o r e p i n e p h r i n e ( N E ) , m e t h o x a m i n e (ME), b a r i u m (Ba 2+) a n d p o t a s s i u m (K ÷) in g u i n e a pig a o r t i c strips. G u i n e a pigs were e i t h e r t r e a t e d w i t h 4 m g / k g i.m. reserpine for 24 h or 50 m g / k g i.p. 6-OHDA, twice, 24 h apart. A o r t i c strips were t r e a t e d w i t h cocaine at a final b a t h c o n c e n t r a t i o n o f 1 × 10 -s M for 10 min. All values are e x p r e s s e d as EDs0 (95% C.I.) t . Treatment
NE
ME
Ba 2+
K÷
Control
6.7 ( 4 . 5 - - 9 . 7 ) x 10 - T M
3.7 ( 2 . 9 - - 4 . 8 ) X 10 -6 M
1.4 ( 1 . 1 - - 1 . 7 ) x 10 - 3 M
21.3 ( 1 9 . 9 - - 2 2 . 9 ) x 10 - 3 M
Reserpine
1.3 ( 0 . 7 - - 1 . 8 ) x 10 -7 M *
0.8 ( 0 . 5 - - 1 . 2 ) × 10 -6 M *
0.4 ( 0 . 2 - - 0 . 7 ) × 10 -3 M *
9.4 ( 7 . 2 - - 1 2 . 2 ) x 10 -3 M *
6-OHDA
1.8 ( 1 . 2 - - 2 . 2 ) × 10-7M *
1.2 ( 0 . 8 - - 1 . 7 ) x 10-6M *
0.7 ( 0 . 5 - - 1 . 0 ) × 10-3M *
14.6 ( 1 3 . 6 - - 1 5 . 7 ) × 10-3M *
Cocaine
3.5 ( 2 . 8 - - 4 . 3 ) × 10 - T M *
3.9 ( 3 . 1 - - 5 . 0 ) x 10 - 6 M
1.5 ( 1 . 1 - - 1 . 8 ) × 10 - 3 M
25.7 ( 2 2 . 7 - - 2 9 . 8 ) × 10 - 3 M * *
t" Each value r e p r e s e n t s t h e g e o m e t r i c m e a n value w i t h 95% c o n f i d e n c e intervals of 10 e x p e r i m e n t s . * Strips supersensitive, EDs0 significantly l o w e r t h a n c o n t r o l , p < 0.05 ( S t u d e n t ' s t-test). ** Strips subsensitive, EDs0 significantly higher t h a n c o n t r o l , p < 0.05 ( S t u d e n t ' s t-test).
CALCIUM AND S U P E R S E N S I T I V I T Y
215
TABLE 2 E f f e c t o f r e s e r p i n e , 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) and cocaine o n ~SCa influx in guinea pig aortic strips in the a b s e n c e or p r e s e n c e o f n o r e p i n e p h r i n e (NE} o r p o t a s s i u m (K÷). Strips were placed i n t o Tris Ringer c o n t a i n i n g 4SCa (0.5 p C i / m l ) for 1 or 5 min. The strips were t h e n placed i n t o a calcium-free Tris Ringer c o n t a i n i n g 2 mM LaCl3 for 30 min. 4SCa influx was calculated f r o m the l a n t h a n u m - r e s i s t a n t 4SCa radioactivity (see Materials and methods}. Treatment
Time (min)
4SCa influx ( n m o ] e / g + S.E.M.) ~f No agonist
NE (7.2 × 10 -7 M)
K ÷ (21.5 raM) 7 1 . 6 ± 4.9 181.5 ± 1 0 . 7 "
Control
1 5
3 8 . 9 ± 3.7 86.8 ± 6.5
54.7 ± 116.1±
Reserpine
1 5
40.1 ± 4.6 90.3 ± 5.4
86.0 ± 4.3 * , * * 191.0 ± 10.5 * , * *
140.6 ± 4.9 * , * * 401.3 ± 20.7 * , * *
6-OHDA
1 5
74.8 ± 158.5 ±
5.4",** 8.6 * , * *
100.1 ± 5 . 8 " , * * 320.5 ± 15.3 * , * *
Cocaine
1 5
58.6 ± 120.2 ±
2.9 * 6.4 *
52.9 _+ 4.1 * , * * 140.7 ± 6.7 * , * *
49.0± 3.2"* 118.1 _+ 6.5 ** 36.6 ± 2.8 84.2 ± 5.4
2.7" 7.5"
t Each value r e p r e s e n t s the m e a n ± S.E.M. o f 7 e x p e r i m e n t s . * Value significantly d i f f e r e n t f r o m n o agonist, p < 0.05 ( S t u d e n t ' s t-test). ** Value significantly d i f f e r e n t f r o m no p r e t r e a t m e n t , p < 0.05 ( S t u d e n t ' s t-test).
These results indicate that cocaine treatment causes supersensitivity to NE and subsensitivity to K* in guinea pig aortic strips. Table 2 shows the effect of reserpine, 6OHDA and cocaine treatment on 4SCa influx in guinea pig aortic strips in the absence or presence of NE (7.2 × 10 -~ M) or K ÷ (21.5 mM). 4SCa influx was increased by 6-OHDA treatment b u t was not altered by reserpine or cocaine treatment. 4SCa influx was increased b y K ÷, and to a lesser extent b y NE in strips with or without reserpine, 6-OHDA or cocaine treatment. The increase in 4SCa influx b y NE or K ÷ was greater in reserpine and 6OHDA-treated strips than in the control strips. Furthermore, the increase in 4SCa influx by NE or K ÷ was greater in reserpine-treated strips than in 6-OHDA-treated strips. On the other hand, the increase in 4SCa influx by K ÷ was greater in untreated strips than in cocainetreated strips. The increase in 4SCa influx b y NE in cocaine-treated strips did n o t differ from that of the control strips.
4. Discussion Van Breemen et al. (1972) have shown that cellular calcium influx can be estimated after the displacement of extracellulary bound calcium by lanthanum. One seemingly direct approach to assess the relationship between cell membrane permeability to calcium and vascular smooth muscle supersensitivity would then be to measure the amount of 4SCa retained by control and supersensitive tissues after a lanthanum wash. However, no such studies have appeared in the literature. In the present report, evidence was obtained which indicates that sarcolemma permeability to extracellular calcium plays a role in postjunctional supersensitivity but may not play a role in prejunctional supersensitivity. A marked increase in 4SCa influx accompanies the supersensitivity to NE and K ÷ of aortic strips from guinea pigs pretreated with 6OHDA or reserpine. Since these strips were also supersensitive to ME and Ba 2÷, therefore,
216
the supersensitivity induced by 6-OHDA and reserpine is postjunctional. Thus 4SCa influx is increased in postjunctionally supersensitive guinea pig aortic strips. Furthermore, supersensitivity and 4SCa influx were greater after reserpine than after 6-OHDA which indicates that a direct proportional relationship exists between 45Ca influx and postjunctional supersensitivity. An increase in 4SCa influx in supersensitive tissues may arise either in response to an increase in cell membrane permeability to calcium or to an increase in the effective concentration of an agonist at the effector cell. However, it is well known that postjunctional supersensitivity involves a change in the physiology of the effector cell. Thus, the increase in 4SCa influx after reserpine or 6OHDA indicates that an increase in the sarcolemma permeability to calcium occurs in the postjunctionally supersensitive aortic strips. No change in 45Ca influx accompanied the supersensitivity to NE of guinea pig aortic strips treated with cocaine. Since NE initiates aortic contraction in the rabbit by release of intracellularly sequestered calcium (Van Breemen et al., 1972}, therefore, supersensitivity to NE may reflect an enhanced release of calcium from intracellular stores. However, 4SCa influx in the presence of K ÷ was decreased which indicates that cocaine may decrease sarcolemma permeability to calcium. Since these strips were not supersensitive to ME, Ba 2÷ and K ÷, therefore cocaine-induced supersensitivity is prejunctional. As a consequence, we cannot definitely conclude that there is no change in 4SCa influx in prejunctionally supersensitive guinea pig aortic strips. It is interesting to note that in the absence of any agonist only 6-OHDA treatment increased 4SCa influx indicating that 6-OHDA
M. KAIMAN, S. SHIBATA
affects 4SCa influx differently than either reserpine or cocaine. In conclusion, our results show that supersensitivity to K ÷ and NE of aortic strips from reserpine- or 6-OHDA-pretreated guinea pigs involves an increase in calcium influx whereas supersensitivity to NE of aortic strips treated with cocaine may not involve any change in calcium influx.
Acknowledgement This investigation was partially supported by the Hawaii Heart Association.
References Carrier, O. and H.A. Jurevics, 1973, The role of calcium in "nonspecific" supersensitivity of vascular muscle, J. Pharmacol. Exptl. Therap. 184, 81. Carrier, O. and S. Shibata, 1967, A possible role for tissue calcium in reserpine supersensitivity, J. Pharmacol. Exptl. Therap. 155, 42. Hudgins, P.M. and T.M. Harris, 1970, Further studies on the effects of calcium removal upon vascular smooth muscle contraction induced by norepinephrine, histamine and potassium, J. Pharmacol. Exptl. Therap. 159, 91. Maling, H.M., J.H. Fleisch and W.F. Saul, 1971, Species differences in aortic responses to vasoactive amines: The effects of compound 48/80, cocaine, reserpine and 6-hydroxydopamine, J. Pharmacol. Exptl. Therap. 176,672. Shibata, S., M. Kuchii and K. Kurahashi, 1972, The supersensitivity of isolated rabbit atria and aortic strips produced by 6-hydroxydopamine, European J. Pharmacol. 18,271. Van Breemen, C., B.R. Farinas, P. Gerba and E. McNaughton, 1972, Excitation--contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx, Circulation Res. 30, 44.
213
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication CALCIUM INFLUX AND POSTJUNCTIONAL SUPERSENSITIVITY IN GUINEA PIG AORTIC STRIPS MARVIN KAIMAN and SHOJI SHIBATA *
Department of Pharmacology, School of Medicine, University of Hawaii, Honolulu, Hawaii, U.S.A. Received 16 March 1976, accepted 18 March 1976
M. KAIMAN and S. SHIBATA, Calcium influx and postjunctional supersensitivity in guinea pig aortic strips, European J. Pharmacol. 37 (1976) 213--216. Both reserpine and 6-hydroxydopamine (6-OHDA) pretreatment potentiated the sensitivity of guinea pig aortic strips to norepinephrine (NE), barium, methoxamine and potassium indicating postjunctional supersensitivity. However, cocaine treatment only potentiated the NE response indicating prejunctional supersensitivity. 6-OHDA and reserpine-induced supersensitivity but not cocaine-induced supersensitivity was accompanied by an increase in 4SCa influx. Postjunctional supersensitivity Guinea pig aortic strips
6-Hydroxydopamine
Reserpine
Cocaine
4SCa influx
1. Introduction
2. Materials and methods
It is well known that reserpine, 6-hydroxydopamine (6-OHDA) and cocaine induce supersensitivity in vascular smooth muscle (Carrier and Shibata, 1967; Maling et al., 1971; Shibata et al., 1972). Carrier and Shibata (1967), Hudgins and Harris (1970) and Carrier and Jurevics (1973) have suggested that a change in the permeability of vascular smooth muscle sarcolemma to calcium may account for reserpine-induced supersensitivity. In spite of this suggestion, no reports have appeared in the literature which deal with the influx of calcium in supersensitive vascular smooth muscle. In this report, the influx of 4SCa in reserpine, 6-OHDA and cocaine-induced supersensitive guinea pig aortic strips are presented.
Helically cut strips (3.5 cm × 0.3 cm) were prepared from aortae obtained from guinea pigs with or without pretreatment with either one 4 mg/kg i.m. dose of reserpine (Serpasil, CIBA) 24 h prior to sacrifice or two 50 mg/kg i.p. doses of 6-hydroxydopamine hydrobromide (Regis) dissolved in 0.1% ascorbic acid 24 and 48 h prior to sacrifice. The strips were then placed into 25 ml organ baths which contained Krebs--Ringer bicarbonate buffer solution at pH 7.4. The organ baths were maintained at 37°C and aerated with a 95% 02 and 5% CO2 gas mixture. A loading tension of 1.5 g was obtained by elevating Grass FT.03 transducers. Isometric contractions were recorded by attaching a 6-channel Grass polygraph to the transducers. The strips were equilibrated for 1.5 h, the medium being replaced every 15 min prior to the addition of norepinephrine bitartrate (NE), methoxamine hydrochloride (ME, Vasoxyl), barium chloride (Ba 2÷) or potassium chloride (K÷). The contractile responses are expressed
* Please send reprint requests and correspondence t o : Dr. S. Shibata, University of Hawaii, Department of Pharmacology, School of Medicine, Biomedical Sciences Building, 1960 East-West Road, Honolulu, Hawaii 96822, U.S.A.
214
M. K A I M A N , S. S H I B A T A
in terms of the mean ED~0 of geometrically determined means. Aortic strips were incubated in Krebs-Ringer for 1.5 h. The strips were then cut into pieces (6--8 mg wet weight) and placed into a Tris-modified Krebs--Ringer buffer solution at pH 7.4 with the following composition (mM): NaC1, 144.5; KC1, 6.0; CaC12, 1.2; MgSO4 7 H20, 1.3; tris-(hydroxymethyl)aminomethane, 5 ; and glucose, 5.6 in distilled, deionized water. The medium was maintained at 37°C and oxygenated with 100% 02. The strips were incubated for 15 min prior to the addition of 4SCa (0.5 pCi/ml) and NE or K ÷. In some experiments cocaine was present during the last 10 min of the 15 min incubation period. Tissue samples were removed 1 and 5 min after the addition of 4SCa and placed into calcium-free Tris Ringer containing 2 mM LaC13 for 30 min in order to remove extracellularly bound 4SCa (Van Breemen et al., 1972). The tissues were then blotted, quickly dipped into four successive ice-cold Tris Ringer media, reblotted and weighed. The tissues were then placed into scintillation vials and 0.5 ml of Soluene-100 (Packard) was added and the tissues were digested overnight at 45°C. 15 ml of toluene-based scintillation cocktail containing 5 g PPO and 50 mg
POPOP per liter of toluene was added to the vials and the amount of radioactivity was determined in a scintillation counter (Packard TriCarb 3380). From the 4SCa retained by the tissue after exposure to the lanthanum-Tris media, 4SCa influx was calculated and expressed in terms of nmole/g wet weight of tissue as follows: tissue 45 Ca content after La 3 + (dpm/g wet weight of tissue) specific activity of 4 SCa in Tris media (dpm/nmole Ca 2 +) = 45Ca influx
3. Results Table 1 shows the EDs0's foi the norepinephrine, methoxamine, barium and potassium contractile responses of guinea pig aortic strips after treatment with reserpine, 6-hydroxydopamine and cocaine. In the strips pretreated with reserpine or 6-OHDA the EDs0's for NE, ME, Ba 2+ and K ÷ were decreased but the maximal contractile responses were not altered thus indicating the development of supersensitivity. In the strips treated with cocaine only the EDs0 for NE was decreased while the EDs0 for K ÷ was increased. Cocaine treatment had no effect on the EDs0 for ME or Ba 2+. Maximal contractile response to NE, ME, Ba 2÷ and K ÷ were not altered by cocaine.
TABLE 1 Effect o f reserpine, 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) a n d c o c a i n e o n t h e EDs0 values o f n o r e p i n e p h r i n e ( N E ) , m e t h o x a m i n e (ME), b a r i u m (Ba 2+) a n d p o t a s s i u m (K ÷) in g u i n e a pig a o r t i c strips. G u i n e a pigs were e i t h e r t r e a t e d w i t h 4 m g / k g i.m. reserpine for 24 h or 50 m g / k g i.p. 6-OHDA, twice, 24 h apart. A o r t i c strips were t r e a t e d w i t h cocaine at a final b a t h c o n c e n t r a t i o n o f 1 × 10 -s M for 10 min. All values are e x p r e s s e d as EDs0 (95% C.I.) t . Treatment
NE
ME
Ba 2+
K÷
Control
6.7 ( 4 . 5 - - 9 . 7 ) x 10 - T M
3.7 ( 2 . 9 - - 4 . 8 ) X 10 -6 M
1.4 ( 1 . 1 - - 1 . 7 ) x 10 - 3 M
21.3 ( 1 9 . 9 - - 2 2 . 9 ) x 10 - 3 M
Reserpine
1.3 ( 0 . 7 - - 1 . 8 ) x 10 -7 M *
0.8 ( 0 . 5 - - 1 . 2 ) × 10 -6 M *
0.4 ( 0 . 2 - - 0 . 7 ) × 10 -3 M *
9.4 ( 7 . 2 - - 1 2 . 2 ) x 10 -3 M *
6-OHDA
1.8 ( 1 . 2 - - 2 . 2 ) × 10-7M *
1.2 ( 0 . 8 - - 1 . 7 ) x 10-6M *
0.7 ( 0 . 5 - - 1 . 0 ) × 10-3M *
14.6 ( 1 3 . 6 - - 1 5 . 7 ) × 10-3M *
Cocaine
3.5 ( 2 . 8 - - 4 . 3 ) × 10 - T M *
3.9 ( 3 . 1 - - 5 . 0 ) x 10 - 6 M
1.5 ( 1 . 1 - - 1 . 8 ) × 10 - 3 M
25.7 ( 2 2 . 7 - - 2 9 . 8 ) × 10 - 3 M * *
t" Each value r e p r e s e n t s t h e g e o m e t r i c m e a n value w i t h 95% c o n f i d e n c e intervals of 10 e x p e r i m e n t s . * Strips supersensitive, EDs0 significantly l o w e r t h a n c o n t r o l , p < 0.05 ( S t u d e n t ' s t-test). ** Strips subsensitive, EDs0 significantly higher t h a n c o n t r o l , p < 0.05 ( S t u d e n t ' s t-test).
CALCIUM AND S U P E R S E N S I T I V I T Y
215
TABLE 2 E f f e c t o f r e s e r p i n e , 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) and cocaine o n ~SCa influx in guinea pig aortic strips in the a b s e n c e or p r e s e n c e o f n o r e p i n e p h r i n e (NE} o r p o t a s s i u m (K÷). Strips were placed i n t o Tris Ringer c o n t a i n i n g 4SCa (0.5 p C i / m l ) for 1 or 5 min. The strips were t h e n placed i n t o a calcium-free Tris Ringer c o n t a i n i n g 2 mM LaCl3 for 30 min. 4SCa influx was calculated f r o m the l a n t h a n u m - r e s i s t a n t 4SCa radioactivity (see Materials and methods}. Treatment
Time (min)
4SCa influx ( n m o ] e / g + S.E.M.) ~f No agonist
NE (7.2 × 10 -7 M)
K ÷ (21.5 raM) 7 1 . 6 ± 4.9 181.5 ± 1 0 . 7 "
Control
1 5
3 8 . 9 ± 3.7 86.8 ± 6.5
54.7 ± 116.1±
Reserpine
1 5
40.1 ± 4.6 90.3 ± 5.4
86.0 ± 4.3 * , * * 191.0 ± 10.5 * , * *
140.6 ± 4.9 * , * * 401.3 ± 20.7 * , * *
6-OHDA
1 5
74.8 ± 158.5 ±
5.4",** 8.6 * , * *
100.1 ± 5 . 8 " , * * 320.5 ± 15.3 * , * *
Cocaine
1 5
58.6 ± 120.2 ±
2.9 * 6.4 *
52.9 _+ 4.1 * , * * 140.7 ± 6.7 * , * *
49.0± 3.2"* 118.1 _+ 6.5 ** 36.6 ± 2.8 84.2 ± 5.4
2.7" 7.5"
t Each value r e p r e s e n t s the m e a n ± S.E.M. o f 7 e x p e r i m e n t s . * Value significantly d i f f e r e n t f r o m n o agonist, p < 0.05 ( S t u d e n t ' s t-test). ** Value significantly d i f f e r e n t f r o m no p r e t r e a t m e n t , p < 0.05 ( S t u d e n t ' s t-test).
These results indicate that cocaine treatment causes supersensitivity to NE and subsensitivity to K* in guinea pig aortic strips. Table 2 shows the effect of reserpine, 6OHDA and cocaine treatment on 4SCa influx in guinea pig aortic strips in the absence or presence of NE (7.2 × 10 -~ M) or K ÷ (21.5 mM). 4SCa influx was increased by 6-OHDA treatment b u t was not altered by reserpine or cocaine treatment. 4SCa influx was increased b y K ÷, and to a lesser extent b y NE in strips with or without reserpine, 6-OHDA or cocaine treatment. The increase in 4SCa influx b y NE or K ÷ was greater in reserpine and 6OHDA-treated strips than in the control strips. Furthermore, the increase in 4SCa influx by NE or K ÷ was greater in reserpine-treated strips than in 6-OHDA-treated strips. On the other hand, the increase in 4SCa influx by K ÷ was greater in untreated strips than in cocainetreated strips. The increase in 4SCa influx b y NE in cocaine-treated strips did n o t differ from that of the control strips.
4. Discussion Van Breemen et al. (1972) have shown that cellular calcium influx can be estimated after the displacement of extracellulary bound calcium by lanthanum. One seemingly direct approach to assess the relationship between cell membrane permeability to calcium and vascular smooth muscle supersensitivity would then be to measure the amount of 4SCa retained by control and supersensitive tissues after a lanthanum wash. However, no such studies have appeared in the literature. In the present report, evidence was obtained which indicates that sarcolemma permeability to extracellular calcium plays a role in postjunctional supersensitivity but may not play a role in prejunctional supersensitivity. A marked increase in 4SCa influx accompanies the supersensitivity to NE and K ÷ of aortic strips from guinea pigs pretreated with 6OHDA or reserpine. Since these strips were also supersensitive to ME and Ba 2÷, therefore,
216
the supersensitivity induced by 6-OHDA and reserpine is postjunctional. Thus 4SCa influx is increased in postjunctionally supersensitive guinea pig aortic strips. Furthermore, supersensitivity and 4SCa influx were greater after reserpine than after 6-OHDA which indicates that a direct proportional relationship exists between 45Ca influx and postjunctional supersensitivity. An increase in 4SCa influx in supersensitive tissues may arise either in response to an increase in cell membrane permeability to calcium or to an increase in the effective concentration of an agonist at the effector cell. However, it is well known that postjunctional supersensitivity involves a change in the physiology of the effector cell. Thus, the increase in 4SCa influx after reserpine or 6OHDA indicates that an increase in the sarcolemma permeability to calcium occurs in the postjunctionally supersensitive aortic strips. No change in 45Ca influx accompanied the supersensitivity to NE of guinea pig aortic strips treated with cocaine. Since NE initiates aortic contraction in the rabbit by release of intracellularly sequestered calcium (Van Breemen et al., 1972}, therefore, supersensitivity to NE may reflect an enhanced release of calcium from intracellular stores. However, 4SCa influx in the presence of K ÷ was decreased which indicates that cocaine may decrease sarcolemma permeability to calcium. Since these strips were not supersensitive to ME, Ba 2÷ and K ÷, therefore cocaine-induced supersensitivity is prejunctional. As a consequence, we cannot definitely conclude that there is no change in 4SCa influx in prejunctionally supersensitive guinea pig aortic strips. It is interesting to note that in the absence of any agonist only 6-OHDA treatment increased 4SCa influx indicating that 6-OHDA
M. KAIMAN, S. SHIBATA
affects 4SCa influx differently than either reserpine or cocaine. In conclusion, our results show that supersensitivity to K ÷ and NE of aortic strips from reserpine- or 6-OHDA-pretreated guinea pigs involves an increase in calcium influx whereas supersensitivity to NE of aortic strips treated with cocaine may not involve any change in calcium influx.
Acknowledgement This investigation was partially supported by the Hawaii Heart Association.
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