complete overlap of caffeine- and k' depolarization-sensitive

THE JOURNAL OF BIOLOGICAL CHEMlSTRY Q 1986 by The American Society of Biological Chemists, Inc.

Val. 261, No. 33, Issue of November 25, pp. 15709-15713,1986 Printed in U.S.A.

Complete Overlap of Caffeine- and K’ Depolarization-sensitive Intracellular Calcium Storage Site in Cultured Rat Arterial Smooth Muscle Cells*

(Received for publication, October 11, 1985, and in revised form July 25, 1986)

Sei Kobayashi, Hideo Kanaide, and Motoomi Nakamura From the Research Institute of Angiocardiology and Cardiovascular Clinic, Faculty of Medicine, Kyushu Uniuersity, 3-1-1 Maidashi, Higashi-ku Fukuoka 81 5 Japan

Changes in the concentration of cytosolic free calcium were recorded microfluorometrically in rat vascular smooth muscle cells in primary culture and loaded with quin-2. The effects of caffeine and high extracellular K+ on the release of calcium from the intracellular storage sites were determined. In the absence of extracellular calcium, both the depolarization of plasma membrane with excess extracellular K+ and the application of caffeine induced a transient and dose-dependent elevation of the cytosolic free calcium concentration, with durations of 4 and 2 min, respectively. Transient elevations of calcium repeatedly appeared in response to both repetitive depolarization (100 mM K”) and caffeine ( lo mM) applications with progressive reductions in peak levels. In either case, the fifth or later treatments induced little or no rise in levels of the cytosolic calcium. The amount of released calcium induced by high K+ depolarization after (n - 1) time applications (1 I n s 5) of caffeine was equal to that induced by the n-th application of caffeine. The amount of released calcium induced by caffeine after (n - 1) time exposures (1 5 n r~ ti) to K” depolarization was equal to that observed during the n-th exposure to K+ depolarization. These results indicate that caffeine- and depolarization-sensitive intracellular calcium storage sites may be identical and that caffeine and K+, in optimal concentrations, will release an equal amount of calcium from the same storage site in cultured arterial smooth muscle cells, irrespective of the amount of stored calcium.

Cytosolic free calcium concentration mediates the contraction of vascular smooth muscle cells (VSMCs’). The release of calcium from intracellular storage sites is an important mechanism in excitation-contraction coupIing (1-3). By measuring the development of tension, pharmacological studies on skinned vascular smooth muscle provided corroborative evidence for the release of calcium from intracellular storage sites (1-4). However, the mechanism whereby calcium is thus released is not well understood, mostly because of difficulties in measuring directly the levels of intracellular calcium. “_________”____I”. “

* This work was supported in part by Grants-in-Aid for Scientific Research 58570395 and 59440044 from the Ministry of the Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: VSMCs, vascular smooth muscle cells; SR, sarcoplasmic reticulum; PSS, physio~ogica1 saline solution; HEPES, 4-(Z-hydroxyethyl)-l-pi~razinee~hanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tet~aac~tic acid.

There i s an established “depolarization”-induced calcium release, an event of great significance in excitation-contraction coupling, in both cardiac and skeletal muscle (5 , 6); depolarization of the surface membrane is conducted to the triad junction through the T tubules and leads to the release of calcium from the junctional sarcoplasmic reticulum (SR). In vascular tissues, since removal of extracellular calcium in high K’ solution resulted in a rapid cessation of potassium- induced contraction without reducing the depo~arization of the surface membrane by high K’, the K‘ depo~arization- induced contraction has been commonly thought to be the result of influx of extracellular calcium and not the release of sequestered calcium (1). However, there are reports that in saponin-treated skinned smooth muscle fibers, depolarization of the internal membrane could produce small, but significant contractions, even in the absence of extracellular calcium (3, 4).

We recently developed a microfluorometric technique using the calcium indicator dye, quin-2, to determine changes in free calcium concentrations in a small area ((1 Mum2) in the cytosol of VSMCs (7, 8). We found that in the absence of extracellular calcium, depolarization of the plasma membrane induces a release of calcium from intracellular storage sites in arterial smooth muscle cells in primary culture (8). With regard to release of cellular calcium, various sources have been considered and the SR may play a central role in this mechanism (1). Caffeine induces a release of calcium from the SR and increases sensitivity of the calcium-induced calcium release mechanism in smooth muscle (1-4), as in the case of cardiac and skeletal muscle (5, 6). In the present study, we used the “quin-2 microfluorometry” method, to characterize the caffeine- and depolarization-induced release of calcium from intracellular storage sites of VSMCs. The relationship between these two mechanisms was given attention.

EXPERIMENTAL PROCEDURES

Cell Culture-Vascular smooth muscle cells were cultured from the aortic media of male Wistar rats, as described in Ref. 9. Briefly, the aortic media were dispersed into single cells by incubation with collagenase (I mg/mI) and elastase (10 units/ml) and seeded on Lux chamber slides in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co.) containing 10% heat-inactivated fetal bovine serum (GIBCO) and antibiotics. The growth medium was changed every 2 days. We used the primary cell cultures for all observations. Elec- tronmicroscopic observations and direct staining with fluorescein ~sothiocyanate-la~led antibodies against native smooth muscle actin and myosin revealed that these cultured cells had little or no contamination with fibroblasts and endothelial cells (7,9).

Cell Viability-High cell viability (>95%) was maintained throughout the experiments, as assessed by the trypan blue exclusion test. There was no apparent morphological change in the cells, with and without quin-2 loading throughout the experiments, as determined by phase-contrast. microscopy at X 400. These observations indicate

15709

15710 Release of Intracellular Ca2+ from Vascular Smooth Muscle Cells that loading VSMCs with quin-2 may not likely produce cell damage as the result of cell acidification by formaldehyde released on ester hydrolysis (10).

Microfluorometry of Quin-2-On days 5-6, just before reaching confluency, the cells on the culture chamber slides were loaded with quin-2 physiologically, as the acetoxymethyl ester (quin-2/AM) (7, 8). The cells were then washed three times with physiological saline solution (PSS) at 25 “C to remove the dye in the extracellular space and then incubated with normal PSS (1.0 mM Ca”) for a t least 30 min before the optical measurements, in order to compensate for a possible transient chelating effect of quin-2 on cytosolic free calcium, as pointed out by Poggioli et al. (11). Unless otherwise indicated, the experiments were carried out in normal PSS a t 25 “C to prevent leakage of quin-2 from the cells (12). The microfluorometric technique used to measure the fluorescence intensities of quin-2-calcium complex in VSMCs was reported elsewhere (7,8). We used a fluorescence microscope (model Standard 18, Zeiss) equipped with a photon- counting system (Zeiss), a water immersion objective system (Plan- Neofluor 63, Zeiss), an exciter filter with a bandwidth of +50 nm (UV-D35, Toshiba), a chromatic beam splitter (FT395, Zeiss), and barrier filters (LP470 and KP560, Zeiss). With these combinations of filters, the cells can be excited a t 350 nm and analyzed at wave- lengths between 470 and 560 nm. Using a 50-pm pinhole diaphragm (Zeiss) in the light axis, the fluorescence in a small circle area ( 4 pm2) in the cytosol of VSMCs could be measured. For optical measurement, each cell was exposed to the excitation light only once and for not longer than 2 s to avoid photobleaching. An input-output calculator (model 97S, Hewlett-Packard Co.) was used to read the fluorescence intensities. Since the VSMCs appeared as hills and valleys on the culture slide (9), cells in the “valleys” were selected for the microfluorometry to avoid possible fluctuations in the light signals produced by a pileup of the cells in the “hills”. Fluorescence of the quin-2-calcium complex was observed almost exclusively in the cytosol (7,8); the extracellular space, the nuclei, and myofilaments were not stained (Fig. 1). According to Williams et al. (13, 14), quin-2 and fura-2, a new calcium indicator having almost equal affinities for calcium, could be incorporated into the nuclei and the SR after 180 min of incubation, and the fluorescence signals in these areas were higher than that in the cytosol in enzymatically disaggregated smooth muscle cells of the toad stomach. Reasons for the opposite results when the nuclei were stained with the calcium indicator dye are unknown. Differences in species, tissues, and cell preparations between their studies and ours may be pertinent. To avoid possible fluctuations caused by an uneven thickness of the cells, a spot ( 4 pm’) 3 pm from the nonfluorescent nucleus was chosen for the measurements. Relative fluorescence was determined by assuming that the fluorescence intensity in the cytosol of the cells exposed to 135 mM extracellular K+ was 100 and that of the unloaded cells was 0, as described (7, 8).

Solutions-The millimolar composition of the “normal” PSS (pH 7.4 at 25 “C) was: NaCl 135; KC1 5; CaC12 1; MgC12 1; glucose 5.5;

FIG. 1. A fluorescence photomicrograph of a primary cul- tu re o f VSMCs loaded with quin-2. Fluorescence was observed in the cytosol, but nuclei and myofilaments stained negatively. Since i t was necessary to expose the cells to the excitation light for about 30 s to obtain a photograph with a sharp contrast, the cytosolic fluorescence intensity in some cells is partially reduced.

HEPES 10. A high K+ version of this solution was prepared by replacing NaCl with KCI, isosmotically. Calcium-free PSS always contained 2 mM EGTA. Possible calcium contamination of the calcium-free PSS without EGTA was ruled out by atomic absorption spectrophotometry (model AA-782, Nippon Jarrel-Ash).

Materials-Quin-Z/AM was purchased from DOTITE (Japan). All other reagents were of the highest grade commercially available.

RESULTS

Stability of the Fluorescence Intensities of Quin-2 Trapped within the Celb-In the normal PSS, there was no significant difference in fluorescence intensities of quin-2-loaded cells during 60 min (Fig. 2). When VSMCs were incubated in calcium-free PSS with 2 mM EGTA, cytosolic calcium decreased exponentially with the time constant of 3.20 f 1.32 min (mean & S.D., n = 16 experiments), reaching a steady state in 5 min (Fig. 2). The steady state level remained unchanged for 60 min. It is unlikely that this steady state level in calcium-free PSS was undetectably low or that there were zero calcium levels, since the addition of nitroglycerin to the calcium-free PSS containing EGTA resulted in a much lower steady state level of cytosolic calcium, as reported (7). After 60 min of incubation of quin-2-loaded cells with normal PSS, there was no specific peak of the fluorescence emission spectrum for quin-2 (490 nm) or quin-2/AM (430 nm) in the incubation medium, determined using a fluorescence spectro- photometer (model 650-40, Hitachi). These observations indicate that there is no significant leak of quin-2 from the cells and that the concentration of quin-2 was stable in the cytosol, under the present experimental conditions. The 25 “C tem- perature may be a most important factor to prevent leakage of the dye from the cells, as stated by Berthon et al. (12).

Calcium Release Induced by Caffeine-Fig. 3A depicts one of the most typical responses of fluorescence change observed when VSMCs were exposed to 10 mM caffeine in calcium-free PSS containing 2 mM EGTA. Caffeine induced a transient elevation in cytosolic calcium with a duration of 2 min, but the degree of fluorescence in the cytosol of quin-2-unloaded cells remained unchanged during the above procedure (Fig. 3A). Fig. 3B shows the dose-response relation (p < 0.05 by analysis of variance) of caffeine-induced elevation in the cytosolic fluorescence in calcium-free PSS, but this was not the case in the cytosol of the cells not loaded with quin-2. The minimum and maximum doses of caffeine required to produce the fluorescence changes were 0.05 and 5 mM, respectively, and these values are similar to those obtained in the porcine coronary artery (3). The peak levels of the transient rise in the fluorescence induced by 10 mM caffeine were

normal PSS

k.3 20 2 mM EGTA

I I I I I I I J 0 30 60

FIG. 2. Stability of the fluorescence signal f rom the cytosol in quin-2-loaded cells in the presence and absence of extracellular calcium. Data are means * S.D.; 8 cells were used for each determination. 0, cells incubated in calcium-free PSS containing 2 mM EGTA. Upon the depletion of extracellular calcium, cytosolic free calcium decreased exponentially; the time for quin-2 signal to fall to l /e (= time constant) in calcium-free PSS was determined by the nonlinear least square approximation with a correlation coefficient of at least 0.95. 0, control cells, incubated only in the normal PSS.

Time (rnln)

A

Release of I n t r a ~ e l l ~ ~ r Ca2+ from Vascular Smooth

t 2%

h

caffeine 2 rr&l EGTA

i

10 2 mM EGTA

.- c C

I I J 1 10 20

Ccaff eine3 (mM) FIG. 3. Effects of caffeine on fluorescence signal from the

cytosol in quin-2-loaded (a) and unloaded (A) VSMCs in calcium-free PSS containing 2 mM EOTA. A , a representative example of the effect of 10 m M caffeine on fluorescence signal. 0, control cells, incubated only in the normal PSS. Data are means t S.D.; 8 cells were used for each determination. B, the peak levels of the transient rise in fluorescence signal induced by various doses of caffeine. Data are means t S.D. of eight experiments. The hatched horizontal band represent.s mean t S.D. of fluorescence in control cells in normal PSS.

much higher than the levels recorded in the cells in normal PSS (1.0 mM Ca2+, 5 mM Kt). During the measurement, neither contraction nor swelling of cells, with or without quin- 2, was noted, as determined by phase-contrast microscopy a t X 400.

Calcium Release Induced by K' Depolarization-When the cells were exposed to high extracellular K' in calcium-free PSS containing 2 mM EGTA, there was a transient and concentration-dependent (p < 0.05 by analysis of variance) elevation of cytosolic calcium, but such was not the case in the cytosol of cells not loaded with quin-2 (Fig. 4, A and B ) . The minimum and maximum concentration of extracellular K' required to produce the fluorescence changes were 9 and 100 mM, respectively. The duration of this response was 4 min, that is significantly longer than the value obtained in the case of the caffeine-induced calcium release. The peak levels of the transient rise in the fluorescence induced by high extracellular K' (100 mM or higher) were neither more nor less than the levels recorded in the case of cells in normal

A z r .-

Muscle Cells

normal PSS

'2min

15711

u K 2mM EGTA

tL 3

10 K 2mM EGTA

FIG. 4. Effects of K+ depolarization on fluorescence signal from the cytosol in quin-2-loaded (0) and -unloaded (A) VSMCs in calcium-free PSS containing 2 mM EGTA. A , a representative example of the effect of 100 mM extracellular K+ on fluorescence signal. 0, control cells, incubated only in the normal PSS. Data are means f S.D.; 8 cells were used for each determination. B, the peak levels of the transient rise in fluorescence signal induced by various concentrations of extracellular K+. Data are means f. S.D. of eight experiments. The hatched horizontal band represents mean zt S.D. of fluorescence in control cells in normal PSS.

PSS (1.0 mM Ca2+, 5 mM K'). During the measurement, neither contraction nor swelling of cells (wit.h or without quin-2) was noted, as determined by phase contrast microscopy a t x 400.

The finding that neither caffeine nor K' depolarization induces a significant fluorescence change in the cytosol of quin-2-unloaded cells (Figs. 3 and 4) and in the nucleus of quin-2-loaded cells (7, 8) indicates that both treatments and resultant possible changes in cell shape cause no significant light scattering effects which would interfere with the optical measurements. Neither treatment induces any observable morphological change compatible with contract,ion, as mentioned above, although myofilaments are well preserved in the VSMCs in primary culture (7, 9).

Effect of Repetitive Applications of Caffeine on the Subse- quent Release of Calcium Induced by High K' Depolarization- When VSMCs were repeatedly exposed to 10 mM caffeine in calcium-free PSS containing 2 mM EGTA for 2 min with a 1- min interval, transient increases in calcium appeared in re-

15712 Release of ~ n t r a ~ ~ l ~ u l a r Ca2+ from Vascular Smooth Muscle Cells

sponse to each treatment (Fig. 5 A ) . However, the peak level of cytosolic calcium transient was reduced progressively by each application of 10 mM caffeine, and the fifth application produced little or no cellular response by this agent. When VSMCs were exposed to 100 mM KC1 for 4 min after exposure to 10 mM caffeine in calcium-free PSS containing 2 mM EGTA, the peak level of cytosolic calcium transient was reduced, compared to the level obtained without pretreatment with caffeine; the more frequent the application of 10 mM caffeine, the less was the subsequent release of calcium induced by 100 mM KC1 (Fig. 5A, a-f). To estimate the amount of calcium released with each treatment, the area bound by the fluorescence trace and the steady state baseline in calcium-free media was measured using a computerized manip- ulator (Houston Instrument). Thus, the calculated area was expressed as "relative fluorescence (RF) x minute (rnin)," indicating relative amounts of transiently released calcium from intracellular storage sites. As shown in Fig. 5B, the amount of released calcium, as induced by high K' depolari-

A 60

€3

a b C d

e f

- Srnin

FIG. 5. Effects of repeated applications of 10 mM caffeine

by 100 mM K+ ( K ) in calcium-free PSS containing 2 m M (CF) on the subsequent elevation of cytosolic calcium induced

EGTA. A, a representative example of the experiments. After 10 min of calcium depletion, the procedures were initiated. a, caffeine was not applied, caffeine was applied once for 2 min (b) , twice ( c ) , 3-5 times (d-f), prior to K+ depolarization (4 rnin). Interval between exposures was 1 min. B, the mean values of five experiments carried out as in part A. Bars indicate standard deviations. Abscissa; amount of refeased calcium induced by the n-th application of 10 mM caffeine. Ordinate; amount of released calcium induced by 100 mM Kc depolarization after ( n - 1) time applications of 10 mM caffeine. The fine was obtained by linear regression analysis. There i s a positive slope of 1.03 with a correlation coefficient of 0.98.

zation after (n-1) time applications (1 5 n I 5) of caffeine was equal to that induced by the n-th application of caffeine.

Effect of Repeated Exposures to High Extracellular K' on the ~ u b s ~ q u e n t Release of Calcium Induced by Caffeine- When VSMCs were repeatedly exposed to 100 mM KC1 in calcium-free PSS containing 2 mM EGTA for 4 min with an interval of a minute, there were transient increases in calcium in response to each treatment (Fig. 6A). The peak level of cytosolic calcium transient was reduced progressively by each exposure to 100 mM extracellular K', and the fifth exposure produced little or no cellular response. When VSMCs were exposed to 100 mM K' depolarization, the subsequent release

e f

h 5%

B

/ '

(RF x min) FIG. 6. Effects of repeated exposures to 100 mM extracel-

lular K+ (K) on the subsequent elevation of cytosolic calcium induced by 10 mM caffeine (Cm in caicium-free PSS containing 2 mM EGTA. A , a representative example of the experiments. After 10 min of calcium depletion, the procedures were initiated. a, cells were not exposed to high extracellular K'; cells were exposed to high extracellular K' once for 4 min ( b ) , twice (c), 3-5 times (d-f), prior t.o application of caffeine (2 min). B, the mean values of five

tions. Abscissa; amount of released calcium induced by the n-th experiments carried out as in part A. Bars indicate standard devia-

exposure of 100 mM K+ depolarization. Ordinate; amount of released calcium induced by caffeine after ( n - I) time exposures of 100 mM K' depolarization. The line was obtained by linear regression analysis. There is a positive slope of 0.93 with a correlation coefficient of 0.98.

Release of Intracellular Ca2' from Vascular Smooth Muscle Cells 15713

of calcium induced by 10 mM caffeine was reduced, compared to the level obtained without pretreatment; also, the extent of calcium release induced by caffeine was progressively reduced with the increasing number of previous exposures to K' depolarization. As shown in Fig. 6B, the amount of released calcium induced by caffeine after ( n - 1) time exposures (1 s n 5 5) to K' depolarization was equal to that observed during ra-th exposure to K' depolarization.

DISCUSSION

We carried out a quantitative analysis of calcium released from the intracellular storage sites in intact VSMCs, using the quin-2 microfluorometry we developed (7,8); the amount of calcium released was assessed by calculating the integration of the fluorescence change with time (Figs. 5B and GB). As shown in Fig. 3, A and B, the peak levels of caffeine-induced release of calcium in calcium-free PSS were much higher than the levels recorded in the cells in normal PSS (1.0 mM Ca2+, 5 mM K'). However, as shown in Fig. 4, A and B, the peak levels of depolarization-induced release of calcium in calcium- free PSS were equal to those observed in the cells in normal PSS. Therefore, the extent of the possible rise in cytosolic calcium induced by K+ depolarization, as may occur in vascular tissues or strips, might not be sufficiently high to induce muscle contraction. These findings are consistent with observations that contractile responses of various vascular tissues to high K' depolarization, if any, are readily abolished in calcium-free media, but caffeine can induce contractions even in calcium-free media (1-3). However, in the quantitative analyses, the amount of calcium released, as induced by the first exposure to depolarization, was all but equal to that induced by the first application of caffeine, as shown in Figs. 5 and 6. Thus, quin-2 micTofluorometry enables a quantitative evaluation of the release of calcium from intracellular storage sites, especially when the peak response of the calcium release is the subthreshold level required for a contractile response.

The present study was undertaken to determine whether the calcium released by depolarization of the plasma membrane is derived from the caffeine-sensitive storage site, sarcoplasmic reticulum, as in the case of cardiac and skeletal muscle (5, 6). We have already reported that K' depolarization of plasma membrane released calcium from the intracellular storage site (8). The present study demonstrated that the amount of calcium released by K' depolarization (100 mM: optimal dose) was exactly the same as that released by application of caffeine (10 mM: optimal dose), irrespective of the amount of calcium present in either caffeine- or depolarization-sensitive storage sites (Figs. 5B and 6B); however, the peak value of the calcium transients induced by K+ depolarization was within the subthreshold range for muscle contraction (Fig. 4). Also, it was shown that K' depolarization released calcium from the caffeine-sensitive intracellular storage site, and application of caffeine released calcium from the K' depolarization-sensitive intracellular storage site. Thus, it was suggested that caffeine- and depolarization-sensitive calcium storage sites overlapped completely. It i s clear that depolarization of the plasma membrane in VSMCs releases calcium from the caffeine-sensitive storage site, which may be comprised of SR.

The mechanism whereby the depoiarization of the surface membrane during K+ contracture causes a calcium release from the SR remains unknown. Itoh et al. ( 3 ) reported that in the saponin-treated porcine coronary artery strips, depolarization of the internal membrane produced contraction, even in the absence of extracellular calcium. A similar result was obtained in skinned smooth muscle strips prepared from

Taenia caecum of guinea pig (4). In addition, although VSMCs have no routes for depolarization waves to travel from the plasma membrane to the internal membrane, such as T tubules found in cardiac and skeletal muscle, electronmicro- scopic observations revealed that the SR is within 12-20 nm of the plasma membrane at the cell periphery and that quas- iperiodic bridging structures are sometimes visualized between the two components; these structures may be involved in the regulation of intracellular calcium homeostasis (15,16). Therefore, depolarization of the plasma membrane by high K' may be conducted to the above-mentioned SR plasma membrane junction and lead to depolarization of SR, which in turn would trigger a release of calcium from the SR. However, our data provide no evidence for an actual depolarization of the membrane of the SR, since electrophysiological measurements were not made. The existence of ~ n c t i o n a l connections between two subcellular organelles remains to be substantiated by studies on muscle morphology and physiol- O D .

For the above reasons, considerable attention has been directed to the mechanism involved in excitation at the plasma membrane and release of intracellular calcium. Ino- sitol-1,4,5-trisphosphate (IPS) mobilizes calcium from intracellular storage sites, presumably the SR, in chemically skinned muscles, including single cells (17) and cultured cells (18) of vascular smooth muscles. Although IP, was assumed to be a possible messenger for pharmaco-mechanical coupling also in VSMCs (17, 18), the role of IP,? in membrane depolarization in vascular smooth muscle has not been defined. Whether or not the depolarization of plasma membrane in VSMCs may induce a production of IP3 and which would trigger the release of calcium from the intracellular storage sites remains to be determined.

In conclusion, our results suggest that depolarization induces a release of calcium from the SR, and this mechanism established in cardiac and skeletal muscle may operate also in VSMCs. However, the mechanism of coupling between the plasma membrane and SR is unknown. The physiological significance of depolarization-induced calcium release in VSMCs also should be evaluated, since our observations were performed in the absence of extracellular calcium, that is, a nonphysiological state.

A c k ~ o ~ l e ~ g ~ e ~ t - W e thank M. Ohara for comments on the man- uscript.

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complete overlap of caffeine- and k' depolarization-sensitive

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