4 C o n t r o l of Intracellular C a l c i u m i n Smooth Muscle
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Ε. E. DANIEL, A. K. GROVER, and C. Y. KWAN McMaster University Health Science Centre, Department of Neurosciences, Hamilton, Ontario, Canada L8N 3Z5 Control of intracellular calcium in smooth muscle cells is essential for control of the contractile state of the cells. Elevation of intra-cellular Ca is achieved by opening membrane channels for transport of Ca down its electrochemical gradient. The nature of these channels and their interaction with drugs including Ca antagonists are briefly considered. In some smooth muscles, elevation of intra-cellular Ca can occur by release of Ca sequestered in the cell; the role of this process in initiation of contraction is unclear. Lowering of intracellular Ca to maintain or allow relax ation may utilize a variety of mechanisms. The major focus of this article is to summarize and eval uate the data showing that the plasma membrane plays a major role in lowering intracellular Ca . This evidence has been obtained by isolating and purify ing plasma membrane vesicles and studying their trans port properties. They possess a vectorial ATP-depen dent Ca transport system capable of accumulating 1000-fold or higher Ca gradients. The properties of this system are described. Plasma membrane ve sicles also possess a Na - Ca exchange system and ATP-independent binding mechanisms. The direction of future research to evaluate the contributions of these systems to control of intra-cellular Ca is discussed. 2+ 2+ Control of intracellular Ca , [Ca ^], in uterine and other smooth muscles is essential for control of tension production. Studies of smooth muscles with plasma membranes damaged by glyce rol (1) or non-ionic detergents (2,3_,4) or of isolated contractile protein_^rom smo_oth muscle (5^,_6, 7^) all suggest that with lejss thgiji 10 M Ca . no active tension is produced while at 10 M Ca . or perhaps less, maximum active tension is produced. 0097-6156/82/0201-0073$06.00/0 © 1982 American Chemical Society 2+
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Rahwan and Witiak; Calcium Regulation by Calcium Antagonists ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
74
CALCIUM
REGULATION
BY CALCIUM
ANTAGONISTS
E l e v a t i o n of f r e e Mg i n c r e a s e s the Ca . c o n c e n t r a t i o n r e q u i r e ment s l i g h t l y (4). Intermediate c o n c e n t r a t i o n s lead to graded changes i n t e n s i o n . £ljie obvious problem i s how the smooth muscle c e l l s can r e g u l a t e Ca ^ over t h i s range. 2+ E l e v a t i o n of [Ca .] Increases i n [Ca ±] a r e , at f i r s t g l a n c e , easy to achieve s i n c e the e x t e r n a l C a * c o n c e n t r a t i o n [Ca i l i s about 10 M, 100 to 10,000-fold higher than i n t e r n a l C a (< 10 M i n relaxed muscle) and the transmembrane e l e c t r i c a l gradient i s -40 to -60mv, i n s i d e negative. Opening of an inward Ca leak down tjj 150 mM and [ K ] i s about 5 mM, tljie e l e c t r o c h e m i c a l e q u i l i b r i u m f o r a membrane passing mostly Κ c u r r e n t s occurs at about -SjO mv. H y p e r p o l a r i z a t i o n a c t i v a t e d by e l e v a t i o n of i n t e r n a l Ca i s a widespread phenomenon i n natur, 1,000-fold gradients of Ca across the v e s i c l e s membrane have been estimated. Since a trgijisvesicle membrane p o t e n t i a l was not knowingly produced, the Ca accumulations may represent the gradients p o s s i b l e with v o l t a g e operated channels open. On the other hand, these channels may ha/^e been damaged o r i n a c t i v a t e d during membrane i s o l a t i o n . Ca accumulated i n s i d e p r o p e r l y o r i e n t e d ves i c l e s i s slowly r e l e a s e d when they are d i l u t e d i n t o Ca-free or Ca-free m ^ i a ; i t i s r a p i d l y , almost instantaneously r e l e a s e d when the Ca - s e l e c t i v e ionophores (A23187, ionomycin, o r X537A) are added (31,35,36,39). In unpublished s t u d i e s we have found the Ca ATP-dependent transport by plasma membrane v e s i c l e s to be enhanced by calmodulin and i n h i b i t e d by phenothiazines and to be stimulated by c-AMP dependent p r o t e i n k i n a s e s . The mechanisms o f a c t i o n of these modulating systems and the p h y s i o l o g i c a l f u n c t i o n s remain to be determined.
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2
Na
+
- Ca^*" Exchange
These plasma membrane v e s i c l e s , a t Iç^st i n tljie two cases examined t o 95%
l 2
Rat Mesenteric A r t e r y :
F
l
Rat Fundus :
F
2
F
l
Rat Vas Deferens:
F
2
Canine T r a c h e a l i s :
F
2
F
3
Rat Mesenteric
Vein:
Canine G a s t r i c Corpus: ( c i r c u l a r muscle)
^ 60%
31, 35 , 46, H-
H
70 - 80%
36
70 - 80%
33,
70 - 80%
38
70 - 80%
33,
70 - 80%
37
- 80%
39
^70
40
40
No f r a c t i o n s of endoplasmic r e t i c u l u m were so f a r obtained. > 50% p u r i t y . I ι Most of the impurity
i n t h i s f r a c t i o n appears to be a t t a c h
ed c o n t r a c t i l e p r o t e i n (31, 46). Table I I I P h y s i c a l P r o p e r t i e s of Rat Myometrium Plasma Membrane References 1.08 - 1.03 g/ml
Density: Size:
Smooth surface v e s i c l e s with c r o s s - s e c t i o n a l d i a meters of 0.05 - 0.5 ym
Trapping Volume:
1 - 3 yl/mg p r o t e i n using sucrose and 4 - 5 yl/mg using i n u l i n (unpublished)
31
47^
Protein: Phospholipid
^ _1 (w/w) Ratio: 20% broken
unpublished 47
Orientation: 40%
sealed
rightside-out
40%
sealed
inside-out
Rahwan and Witiak; Calcium Regulation by Calcium Antagonists ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
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CALCIUM
ANTAGONISTS
provide a complete c o n t r o l (49,5()) but may p a r t i c i p a t e i n regu l a t i n g i t by a f f e c t i n g Ca f l u x e s (30,51). Iiji the i s o l a t e d plasma membrane v e s i c l e s which we s t u d i e d , the Na -pump was not a c t i v a t e d (no ATP present) and the Na gradients were s h o r t l i v e d (1 to 3 minutes). A l s o , these i s o l a t e d v e s i c l e s may have no transmembrane p o t e n t i a l d i f f e r e n c e u n l i k e the i n t a c t c e l l ; so the t o t a l e l e c t r o c h e m i c a l gradient was le_ss. Furthermore, they probably have an increased leak of both Na and Ca through t h e i r membrane. The absence of ATP may have reduced the a f f i n i t y of exchange s i t e s f o r Ca . A l l these u n c e r t a i n t i e s l i m i t the conclusions about the p o t e n t i a l r o l e of Na - Ca ex change that should be drawn^at t h i s stage. Nevertheless, the maximum i n i t i a ^ r a t e of Ca transport (from a medium c o n t a i n i n g 40 μΜ f r e e Ca ) d r i v e n by the Na gradient was comparable to that e f f e c t e d by the^^TP-dependent Ca pump (from a medium con t a i n i n g 1 μΜ f r e e Ca ) (34). T h i s r a t e was obtained with a Na gradient of about 2 0 - f o l d . In the i n t a c t c e l ^ the Na -gradient i s probably about 10-fold and the i n t e r n a l C a concentration during r e l a x a t i o n i s 0.1 μΜ; so t h i s Na ~2§. exchange system may operate r a p i d l y only where i n t e r n a l Ca concentrations are elevated. 2+ 2 +
a
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A f i n a l judgment cannot be made u n t i l Na - Ca exchange has been studied under optimal c o n d i t i o n s i n the presence of a maintained Na gradient i n i s o l a t e d plasma mem^r_anes and i n t a c t cells. Tljie stoichiometry, e l e c t r o g e n i c i t y , Ca -gradient depen dence, Na -gradient dependence, as w e l l as i n f l u e n c e of ATP and v a r i o u s r_egulat^.ng f a c t o r s need to be determined. V a r i a t i o n be tween Na - Ca exchange i n plasma membranes of d i f f e r e n t smooth muscles i s to be expected, adding to present u n c e r t a i n t y . A s i m ^ a r suggestion has been made that i n t e r n a l s e q u e s t r a t i o n of Ca by mitochondria occurs onj-y at high i n t e r n a l Ca concentrations, based on t h e i r low Ca content i n s t u d i e s using x-ray d i f f r a c t i o n a n a l y s i s of s e c t i o n s r a p i d l y £r_ozen to minimize t r a n s l o c a t i o n s (52,53) and on s t u d i e s of the Ca dependence f o r Ca transport (54,55). In humgiji myometrium, however, i s o l a t e d mitochondria could transport Ca from concentrations l e s s than 1 μΜ (56). Endoplasmic Reticulum
2+ and Regulation of Ca .
Based l a r g e l y on analogy to seriated muscle, i t has become a dogma that the major s i t e of Ca s e q u e s t r a t i o n and transport i n smooth muscle i s i n the endoplasmic r e t i c u l u m (ER). The best p o s i t i v e evidence f o r t h i s c o n s i s t s of s t u d i e s by e l e c t r o n probe of r a p i d l y frozen muscles (52,53,57) ; so f a r only large_ blood v e s s e l s of guinea p i g have been studied and i n them Ca was r e ported to be accumulate^ i n ER under c o n d i t i o n s when i n t r a c e l l u l a r Ca was increased by Κ - d e p o l a r i z a t i o n and even i n relaxed muscle. This method has important l i m i t a t i o n s ; i t would not
Rahwan and Witiak; Calcium Regulation by Calcium Antagonists ACS Symposium Series; American Chemical Society: Washington, DC, 1982.
4.
DANIEL E T A L .
Intracellular
Calcium
in Smooth
Muscle
85
2+ de£e_ct a Ca transport f u n c t i o n by plasma membrane s i n c e the Ca i s t r a n s p o r t e ^ i n t o the e x t r a c e l l u l a r space. Also i t s a b i l i t y to r e s o l v e Ca bound to one surface of a b i o l o g i c a l mem-^^ brane i s not e s t a b l i s h e d . F i n a l l y no q u a n t i t a t i v e study of Ca t r a n s l o c a t i o n s during preparations o f specimens i s a v a i l a b l e . Other problems e x i s t f o r a p p l i c a t i o n of t h i s evidence to provide general support f o r the dogma. F i r s t , many smooth muscle c e l l s , e s p e c i a l l y small a r t e r i e s , v e i n s , and some gut muscles c o n t a i n a small volume o£ ER, i n s u f f i c i e n t to provide f o r accumulation of the necessary Ca . Second, t o f u n c t i o n as a major f u n c t i o n a l s i t e f o r Ca accumulâtio^ during r e p e t i t i v e c o n t r a c t i o n c y c l e s , ER must e i t h e r r e l e a s e Ca again a f t e r accumulation or i n some way t r a n s l o c a t e i t to the e x t e r i o r . No d i r e c t evidence f o r e i t h e r of these processes e x i s t s i n f u l l y studied smooth muscles (see above). T h i r d , no one has been able t o ^ ^ s o l a t e a h i g h l y p u r i f i e d ER f r a c t i o n with the appropriate Ca transport properties. Instead, crude microsomal f r a c t i o n s c o n t a i n i n g a l a r g e amount (probably i t s major £
