Calcium regulation in vascular smooth muscle contractility

Calcium Regulation in Vascular Smooth Muscle Contractility

ANTHONY JOHNS, PhD, PAUL LEIJTEN, MD, HIRO YAMAMOTO, MD, KWANG HWANG, MS, and CORNELIS van BREEMEN, DVM, PhD

The contractile activity of vascular smooth muscle is regulated by control over the cytoplasmic calcium concentration. The intracellular calcium receptor is calmodulin, which, through stimulation of myosin light chain kinase, can activate 2 different contractile states. The calcium is supplied from the sarcoplasmic reticulum and the extracellular space; a mi- nor component is supplied from the inner surface of the plasmalemma. The main intracellular messenger responsible for the transduction of receptor occupation and calcium release from the sarcoplasmic re

ticulum is IPS and, to a lesser extent, calcium itself. The superficial location of the sarcoplasmic reticulum in vascular smooth muscle makes it the logical area for control of calcium entry due to calcium leak or through either or both types of calcium channel. The sarcoplasmic reticulum, therefore, acts as a “superficial calcium buffer barrier” and is probably the major system controlling free cytoplasmic calcium concentration in vascular smooth muscle.

(Am J Cardiol 1987;59:18A-23A)

T he most widely accepted theory of smooth muscle activation involves an increase in cytoplasmic free Ca++ leading to formation of the Ca++-calmodulin complex. This complex removes the inhibitory subunit of myosin light chain kinase leading to phosphorylation of the 20,000 D myosin light chain1 The interaction of the phosphorylated myosin with actin con- sists of adenosine triphosphate (ATP) hydrolysis by fast cycling crossbridges and by rapid smooth muscle shortening. The rapid shortening phase may be fol- lowed by phosphatase-mediated partial dephosphorylation and a shift to the latch state,2 which is character- ized by tonic tension during slow crossbridge cycling. Both contractile states are dependent on the ionic calcium concentration in the cytoplasm, although they have different sensitivities.3 Relaxation is brought about by a decrease in the cytoplasmic Ca++ concentration. Although this theory provides the basic con- cepts of smooth muscle tension development, many important questions still need to be addressed, particu- larly regarding the modulating influences of cyclic nucleotides on C-kinase or myofilament Cat+ sensitivity.4J It is now also generally accepted that the systems

From the Department of Pharmacology, University of Miami School of Medicine, Miami, Florida.

Address for reprints: Cornelis van Breemen, DVM, PhD, Department of Pharmacology (R189], University of Miami School of Medicine, P.O. Box 016189, Miami, Florida 33101.

that exert immediate control over smooth muscle contractile activity consist of the cellular membrane Ca++ transport processes.

Mechanisms of Calcium Delivery and Removal During Smooth Muscle Activation/Relaxation

Two membrane systems have control over rapid fluctuations in cytoplasmic Cat+ concentration: the sarcolemma and the sarcoplasmic reticulum. Calcium- binding molecules in the myoplasm also have some control; however, the precise quantitative nature of their calcium-buffering capability is still undefined.

The plasmalemma and sarcoplasmic reticulum serve a dual role as both calcium delivery and calcium removal systems. Delivery of calcium is not an energy- consuming process because calcium is moving from a high to a low electrochemical potential; in contrast, calcium removal is energy consuming through ATP- dependent pumps and, to a lesser extent, through the Na+/Ca++ exchange system. Because these systems have been reviewed extensively,6-*1 this discussion of their activity will be limited to more recent develop- ments in the understanding of how they operate, with an emphasis on work from our laboratory.

Intracellular Calcium Release The release of intracellular calcium from organ-

elles has been measured using different indicators of calcium transients, including contraction,12J3 intracel-

18A

January 23, 1987 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 59 19A

lular calcium indicators,‘-’ electron probe x-ray analy- sis,‘“,“’ measurement of Ca-45 fluxes from intact and chemically skinned cells’Zi.‘7 and calcium electrode in chemically skinned smooth muscle cells.18

It has been suggested that the inner surface of the plasmalemma releases calcium during smooth muscle activation.‘” The calcium from this source is consid- ered to represent only a minor component of total calcium release. The main source of calcium is undoubt- edly the sarcoplasmic reticulum. Electron probe x-ray microanalysis has demonstrated relatively high concentrations of calcium coincident with the superficial and deep sarcoplasmic reticulum; this calcium could be released by either norepinephrine (NE] or caffeine.‘” ‘I1 The calculated concentration of releasable calcium in the 2 smooth muscle preparations translates to a calcium concentration of 4 mmol/liter sarcoplasmic reticulum. In separate experiments the calculated value for caffeine-induced Ca-45 release from rabbit aorta was 5.3 mm01 of Ca++/liter sarcoplasmic reticulum’:+ and 3.1 mmol of Ca/liter sarcoplasmic reticulum for the rabbit mesenteric artery.:!“This high concentration of calcium would suggest that the sarcoplasmic reticulum is the main intracellular calcium source for vascular smooth muscle contraction.

Calcium Release Mechanisms Early investigators claimed th.at agonists were able

to directly release calcium from subcellular organ- elles, but these reports have been subsequently re- futed. The transmitter NE does not affect the intracellular release of calcium in vascular smooth muscle cells chemically skinned with saponin.” When vascular smooth muscle fibers or cultured cells are skinned with saponin for short periods, it is possible to main- tain the integrity and function of the sarcoplasmic reticulum. In isolated smooth muscle fibers of rabbit mescnteric artery, calcium can be loaded into a caffeine-releasable store. This store can also be released by 2 X lo-” M or higher free calcium and this release- measured as contraction [Fig. l]--is potentiated by 10 PM cyclic adenosine monophosphate [AMP). The potentiated contraction is not due to increased sensitivity of the myofilament because similar results can be obtained with Ca-45 flux studies. This role of cyclic AMP is also seen in intact tissues, where the initial response to NE is enhanced. The second effect of cyclic AMP, i.e., to stimulate uptake of Cat+ into the sarcoplasmic reticulum, rapidly overshadows the initial effect, and the net effect is a reduction in the size of the tonic component of contraction17; caffeine and calcium-induced Ca++ release are blocked by procaine. The evidence for existence of calcium-induced calcium release during agonist activation is clear, but not during potassium-induced depolarization of smooth muscle. The sarcoplasmic reticulum actually accumulates calcium during potassium-induced depolarization, while agonist activation of smooth muscle releases calcium from the sarcoplasmic reticulum.‘” This observation could be explained if, during a potassium-induced contraction, the intracellular calcium did not reach sufficient concentration to cause calcium-induced cal-

cium release.z1 It is also possible that unlike agonists, potassium does not cause release of intracellular plasmalemma calcium. In an agonist-induced contraction, however, the increased influx of calcium coupled to release of calcium from the inner surface of the plasmalemma would provide sufficient calcium for calcium-induced calcium release to occur.

Another explanation would be the involvement of the release of sarcoplasmic reticulum calcium by inositol-1,4$trisphosphate (IPs) following receptor occupation but not during membrane depolarization.“’ This new intracellular messenger theory originated in 1953 with the observation by Hokin and HokiS that acetylcholine stimulated phosphate turnover in phos- phatidylinositol and phosphatidic acid of the pancre- as. Berridge24 later found that inositol-1,4,5-trisphosphate releases Ca ++ from the endoplasmic reticulum of pancreatic acinar cells. It has also been demonstrated that IP3 causes release of Ca++ from the sarcoplasmic reticulum of isolated coronary smooth muscle cells2”J6 It is thought that upon occupation of an agonist receptor, the enzyme phospholipase C is activated. This enzyme catalyzes the breakdown of phosphatidy- linositol-4,5-biphosphate (PIP21 to the active IP3, which releases the calcium from the sarcoplasmic reticulum. The PIP2 is then regenerated by the phosphoinositol cycle.27 In cultured aortic smooth muscle cells, it has been shown that the time course for the increase of IPs

Procaine

/*

1 1 1 t

6 5 4

PCs

FIGURE 1. Cat+-induced Cat + release from the sarcoplasmic reticulum (SR) of saponin-skinned mesenteric artery was determined by loading with 10m6 M Ca++ for 3 minutes and then exposing to various concentrations of Ca (pCa) with or without 10m5 M CAMP, 5 mM Mg or 10 mM procaine for 1 minute. The amount of Caf+ remaining in the SR was estimated by the 25 mM caffeine contraction after washing with relaxing solution containing 0.5 mM Ethyl- eneglycol-bis-(8 amino ethyl ether) N,N’-Tetraacetic acid. Repro- duced with permission from J Gen Physiol.‘7

ZUA A SYMPOSIUM: THE CALCIUM ION, CARDIAC MYOCYTE AND VASCULAR SMOOTH MUSCLE IN HYPERTENSION AND ITS TREATMENT

on the addition of angiotensin is at best as fast as the release of calcium from the sarcoplasmic reticu- 1um.28~2g Also, in primary cultures of aortic smooth muscle, we have shown that IPB can release the equiv- alent of 200 rmol of calcium/liter cells/l0 s (Fig. 2).26

It has also been demonstrated in chemically skinned vascular smooth muscle that IP3 is capable of inducing a contraction in tissues with intact sarcoplasmic reticulum previously loaded with calcium. In skinned aortic cells, IP3 will cause a release of a greater proportion of calcium [about 90% ATP-dependent calcium uptake] than the maximum dose of caffeine (about 50% ATP-dependent calcium uptake] will pro- duce. (We found this difference to be mainly due to a faster Ca++ release by IPa because it caused a 17-fold increase in the Ca-45 efflux from skinned smooth muscle cells while 25 mM caffeine caused only a four 4- fold increase.) (Fig. 2) In addition to the different maxi- mal rates of Ca++ loss, the IP3 and caffeine-induced mechanisms could be further distinguished by a total insensitivity of the IP3 response to 10 mM procaine (Yamamoto and van Breemen, unpublished observa- tions]. We therefore postulate that smooth muscle sarcoplasmic reticulum, in addition to a nonregulated calcium leak, also has 2 types of excitable Ca++ channels,

3.0

1.0

.3

.I

.03

.Ol 1 10 20 30

Efflux Tlmr (min.)

FIGURE 2. Effects of 10 PM inositol 1,4,5trisphosphate and 25 mM caffeine on Ca-45 elflux from sarcoplasmic reticulum in saponin- skinned primary culture of rat aortic smooth muscle cells. The cells were loaded with Ca-45 in the presence of 3 mM Mg ATP and 1 PM Ca++ for 20 minutes before the start of efflux. Efflux curves obtained after loading in the absence of Mg ATP have been subtract- ed. Each curve is the average of 3 experiments. The so/id lines are computed according to exponential loss of calcium from a single compartment for the control and from 2 parallel compartments for caffeine and IPo.

one activated by IP3 and another activated by either Ca++ or caffeine.

Recycling of Intracellular Calcium In intact smooth muscle, it has been repeatedly

shown that in the absence of calcium entry, exposure to NE or caffeine is sufficient to cause a loss from the cells of all the functional sarcoplasmic reticulum calcium. It has been argued, however, that when there is no calcium entry and when short periods of exposure to NE are used, repeated contractions can be obtained, due to the recycling of calcium within the ce11.15 We have tested this theory by studying the correlation of contraction with Ca-45 efflux stimulation in rabbit mesenteric artery. 2o As shown in Figure 3, regardless of how short the exposure of tissues to NE, there is always a Ca-45 efflux stimulation, indicating that calcium would have to be restored from an extracellular source for the same amount of Ca-45 release to reoccur and to give rise to the same contractions. The data do not exclude the ability of the sarcoplasmic reticulum to directly take up a fraction of the released calcium (but by no means the majority] while calcium cycles between the sarcoplasmic reticulum cytoplasm, the extracellular space and then back to the sarcoplasmic reticulum.

Calcium Entry Three mechanisms are postulated to lead to calci-

um entry into vascular smooth muscle: the calcium leak, the receptor-operated calcium channels and the potential-operated calcium channel. The calcium leak has been calculated to be sufficient to give rise to a contraction if the calcium-sequestering system in the cells is compromised in any wayS30 The leak is partially blocked by the inorganic calcium antagonists (Co++, Mn++ and La+++) but not by the organic calcium antagonists. In 1979, the existence of the other 2 calcium entry pathways [potential-operated and receptor-operated channels) was proposed.*sg The separate identi- ty of these 2 types of calcium entry systems was suggested by the additivity of maximum responses of agonists and depolarization, and by the observation that maximum responses to different full agonists were not additive on contractility or Ca-45 fluxes.8,g,31

The voltage-operated calcium channels have been investigated more fully using whole cell voltage clamp and isolated patch clamp techniques. This relatively new technique suggests that vascular smooth muscle is heterogenous with respect to population of voltage- sensitive calcium channels. Two types of calcium current have been identified in rat aortic clona1,32 azygous vein33 and mesenteric artery smooth muscle cells.34J5 Worley et a135 describe 2 channels with conductances of 8 pS and 15 pS, which are both sensitive to the calcium-channel blocker nisoldipine. The channels appear to be similar to those reported in canine atria1 cells36 and in the sensory neurones of chick dorsal root ganglia37 where they have been classified as T (transient] and L (long lasting). In rabbit ear artery vascular smooth muscle cells, a sustained calcium current has not been observed3* (P.I. Aaronson, personal commu-

January 23,

nication). Instead, upon depolarization, a calcium current is elicited that inactivates with biexponential kinetics with the deactivation of both components being enhanced by depolarization. The possibility that the functional differences in blood vessels are associated with a different proportion of the different calcium channels remains an intriguing possibility to be investigated.

There are fewer studies on receptor-operated channels using the voltage clamp technique. It has been reported that in the smooth muscle of rabbit jejunum, acetylcholine induces an inward calcium current.3gs40 These calcium channels, however, have been shown to be voltage dependent and are less selective for calcium than the potential-operated channels. The channel is thought to represent a nonselective cation channel because the current appears to be carried by a mixture of ions including K+, Na+ and Ca++. A non- voltage-dependent channel has been demonstrated in voltage-clamped segments of arterioles of the guinea pig small intestine. 41 The channels appear when the perivascular nerves to these arterioles are stimulated [presumably due to release of the neurotransmitter NE or ATP). These channels have been shown to have a reversal potential near 0 mV, suggesting that, like the receptor-operated channels of the rabbit jejunum smooth muscle cells, they represent a nonselective cation channeL41

These receptor-operated channels are thought to be important in hypertension since Mulvany et a142 showed that the resistance vessels of spontaneously hypertensive rats show a greater sensitivity to calcium in the presence of NE than do control animals. This difference in sensitivity to calcium is not seen in the presence of high potassium concentration. Further, it has been shown that NE increases Ca-45 influx in spontaneously hypertensive rat resistance vessels to a greater extent than normotensive control rat resistance vessels,43 and that the calcium antagonist nisoldipine is less potent on spontaneously hypertensive rat resistance vessel Ca-45 fluxes in the presence of NE than in normotensive control rats.44 These data could suggest

FIGURE 3. The effect of decreas- ing the time of norepinephrine ap- plication to small mesenteric arteries on force and stimulated Ca-45 efflux. After 10 minutes in Ca++-free solution, rabbit mesenteric artery rings were exposed to norepinephrine (3.10m5 M) for various periods of time (from 5 to 900 seconds) and the area under the tension transient and stimulated Ca-45 efflux were calculat-

ed. The insets, righf, give the patterns of the 30-second and gOO-

second contractions (fop) and noradrenaline (NA)-stimulated

Ca-45 efflux (boffom).

1987 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 59 21A

that changes in the characteristics of the receptor-operated channel may be associated with spontaneous hypertension.

Sarcoplasmic Reticulum Sequestration of Calcium Entry

The first indication that the sarcoplasmic reticulum could compete with the contractile machinery for the calcium entering the smooth muscle cell came from the observation that tension development resulting from net entry of calcium varied with the rate of net uptakee7 A similar observation was noted when the tension development and calcium influx were measured in smooth muscle in response to the calcium agonist Bay K8644. In the rabbit aorta, Bay K8644 results in appreciable calcium entry with minimal tension development. It was also found that the calcium gained was in a caffeine-releasable store.45 It therefore appears that moderate increases in calcium influx can be sequestered by the sarcoplasmic reticulum before the Ca++ concentration is increased in the cell interior. Figure 4 shows the relation between Ca-45 influx and tonic tension (after the initial phase possibly involving Ca++ release is finished] under 3 different activating conditions. When Ca++ influx is stimulated by high K+ depolarization and inhibited by graded doses of calcium antagonists, the threshold for Cat+ influx-related tension development is determined by calcium accumulation into the superficial sarcoplasmic reticulum as explained previously. If the uptake into the sarcoplasmic reticulum is compromised by NE, with various lanthanum concentrations used to inhibit the calcium leak, it can be seen that the threshold calcium influx required to cause a contraction is much lower [Fig. 4). In contrast, if the uptake into the sarcoplasmic reticulum is enhanced by increasing cyclic AMP concentration in the tissue with forskolin, the calcium influx is increased to a much higher level for the same tension production (Fig. 4). It could be argued that the agonist and the cyclic AMP could directly modulate myofilament sensitivity to calcium,21 thus explaining the observed results (Fig. 4). However,

0 -900 i

1’ 1’

0’ /'

420 &..I ./'

30

45Ca efflux (rel. area)

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t NA(xsec)

A SYMPOSIUM: THE CALCIUM ION, CARDIAC MYOCYTE AND VASCULAR SMOOTH MUSCLE IN HYPERTENSION AND ITS TREATMENT 22A

NE+La

; 15 30 46

.%a InfhJx qJmoh/kg/nlh)

FIGURE 4. Relation between tonic tension and Ca-45 influx during stimulation by 0 X lop5 M noreplnephrlne (NE), 80 mM KCI and 80 mM potassium (K) with W5 M forskolin in the presence of variable concentrations of Ca++ entry blockers. Rings of rabbit aorta were exposed to high K+ or NE in the absence and presence of various concentrations of DBOO or La. The maintained contractlons were measured at 5 minutes. At this time point parallel aortic rings from the same animals were exposed for 90 seconds to Ca-45 to measure calcium Influx. 0 = D600 (10 -s - lOmE M) -I 80 mM KCI. l = D600 (10-s - lo-* M) -I NE. l = La3+ (3. 10e5 - 3. 10m3 M) + NE. W = 80 mM KCI + M forskolin i- D600 lo-* - 1g8 M). Verf/ca/ dashed line represents basal Ca-45 Influx through the intrinsic Ca++ “leak.”

/ C Kinase med

FIGURE 5. Summary of known and putative mechanisms maklng up the Ca++ control system of vascular smooth muscle (see text). Activation of contraction is initiated by calcium-calmodulln activation of myosin light chain kinase (MLCK), which phosphorylates myosin to initiate rapid shortening. After dephosphorylation, the actomyosin passes through a “latch” state back to rest. Cyclic nucleotides can modify the affinity of MLCK for calcium calmodulin. Cytoplasmlc Ca ++ is supplied from the sarcoplasmic reticulum (SR) through InsP, and Ca++-sensitive Ca++ channels, from the extracellular space through a slow uncontrolled leak and several types of voltage-sensitive Ca++ channels (with single channel conductances of 8 pS and 15 pS, and In some arteries through receptor-operated Ca++ channels (ROC), all of which can lead to a pharmacologic response. Activation of receptors leads to stimulation of phospholipase C through transduction by a G protein and yields the intracellular messengers diacyl-glycerol D (DG) and inositol 1,4,5-trisphosphate (InsP,), which respectively stimulate C kinase in the plasmalemma and InsP, receptors in the SR, and may be involved in opentng of receptor-operated channels. Besides supplying Ca ++, the SR takes up Ca++ during the fast phase of relaxation and buffers a fraction of the Ca++, which crosses the plasmalemma before it activates the myofilaments. This latter superficial buffering function is regulated by IP3 and cyclic nucleotldes. CAMP is thought to mainly stimulate Ca++ uptake into the SR whereas cGMP is thought to stimulate mainly the plasmalemmal ATPase. The Na/Ca exchange carrier contributes to different degrees to the Caf+ extrusion process. Two functionally different portions of the SR are postulated, one functioning mainly in uptake and relaxation and the other functioning both in uptake and release of Cat+.

January 23, 1987 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 59 23A

indirect evidence for the modulating role of the sarco- mediutrtl arteriul smooth muscle contraction. J Cen Physiol 1984;84:307-318.

plasmic reticulum has also been obtained in the ab- 18. Somlyo AV. Bond M, Somlyo AP. Sarpa A. Inositol trlsphosphate-induced calcium release and contraction in vasculur smooth muscle. Proc Nat Acad

sence of agonist activation of contraction.46 SC1 LISA i!JR4;Rl’:5231-5235.

It is therefore proposed that the sarcoplasmic retic- 19. Saida K. van Breemen C. Inhibiting effect of diltiazem on introceliular

ulum of smooth muscle, due to its partially superficial Ca++ release rn vascular smooth muscle. Blood Vessels 1983;20:105-108. 20. Leijten P.4A. van Breemen C. The relationship between noradrenuline-

location, is the logical area to control the delivery of induced tc,nsion development und stimulated 45Ca efflux in rabbit mrsenteric

calcium from the extracellular space to the contractile smull artery. Br J Pharmacol 1986 in press.

machinery. This “superficial calcium buffer barrier” 21. Morgan JP. Morgan KG. Stimulus-specific patterns of intrucellular (;a++ levels in smooth muscle of ferret portal vein. J Physiol 1984:351:156-167.

may be a major controller of the activating Ca++ con- 22. Akhtar RA. Abdel-Latif AA. Carbachol causes rapid phosphodiesterase

centrations in small cells with a large surface to vol- cleavage of phosphotidylinositol-1,4,5&phosphate and accumulation of inosi-

ume ratio and, hence, a substantial calcium leak. The tol phosphates in rabbit iris smooth muscle; xxxxx inhibits norudrenaline- and ionophore .&3187-stimulated accumulation of inositol phosphates. fjlochem J

maintenance of this sequestration barrier, however, 1984;224:291-300.

requires that under normal resting conditions, the sys- 23. Hokin MR. Hokin LE. Enzyme secretion and the incorporation of 321’ into phospholipids of pancreus slices. J Biol Chem 1953;2(Ki:978--977.

tern never becomes overloaded and that excessive cal- 24. Berridge Mj. Inositol trisphosphate and diacylglycerol as second messen-

cium in this system is discharged to the outside of the gem. Biochem J 1984;212:849-858.

cell, thus maintaining the ability of the sarcoplasmic 25. Suematsu E. Hirata M, Hashimoto?‘, Kuriyama H Inosltol 1,4$trisphosphate releases (:a++ from intracellular store sites in skinned single cells of

reticulum to continually modulate cytoplasmic calci- porcine coronary artery. Biochem fliophys Res Comm 1984;120:481-485.

urn concentration. This continual sarcoplasmic reticu- 26. Yamamoto H. van Brcemen C. lnositol-l,4.5-trisphosphute releases colci-

lum Ca++ discharge may take place through the spe- urn from skinnetl cultured smooth muscle cells. Biochem Biophys Hrs Comm 1wm0:270-274.

cial junctional areas between the plasmalemma and 27. Williamson JR, Cooper RtI, Suresh JK, Thomas Al’. lnositol trlsphosphute

sarcoplasmic reticulum. It is conceivable that the basal and diacylglycerol as intracellular second messengers in liver. Am J Physiol 1965:248:C203-C216.

IP3 production is able to preferentially permeabilize 28. Alexander RW. Brock TA, Gimbrone MA, Rittenhouse SE. Angmtensin

the sarcoplasmic reticulum in these areas because the increases inositol trisphosphate und calcium in vascular smooth muscle. Hy-

IP:,-phosphatase would hydrolyze it before it could pertension 1985;7:447-451. 29. Smith IB, Smith L. Brown ER. Barnes D, Sabir MA, Davis IS. Farese RV.

diffuse away any further. Angiotensin II rapidly increases phosphatidate-phosphoinositide synthesis

The Ca++ regulatory mechanisms presently known und phosphninositidr hydrolysis and mobilizes intracellular calcium in cultured arterial muscle cells. Proc Nat Acad Sci USA 1984;81:7812-7816.

and hypothesized for vascular smooth muscle are 30. Loutzenhiser R, Leijten P, Saida K. van Breemen C. Calcium compart-

shown in Figure 5. men& and mobilization during contraction of smooth muscle. In: Grover AK, Daniel EE, eds. Calcium and Contractility. Clifton, NJ: I lumana Press.

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Calcium regulation in vascular smooth muscle contractility