Different Release

Pathways

for Ca 2+ Influx

of Ca2 + Mediated in Ileal

Longitudinal

and

Intracellular

by Muscarinic

Receptors

Smooth

Muscle

Xiao-Bing Wang, Takeshi Osugi and Shuji Uchida* Department of Pharmacology I, Osaka University School of Medicine, 2-2 Yamada-Oka, Suita, Osaka 565, Japan Received

October

1, 1991

Accepted

December

27, 1991

ABSTRACT Muscarinic receptor-mediated elevations in intracellular Ca2-1 concen tration ([Ca21]i) in the longitudinal smooth muscle of guinea pig ileum were studied by the use of fura-2 fluorescence. Dose-response analysis indicated a difference in the potencies of carbachol (CCh) to increase [Ca2+]; in the presence and absence of ex tracellular Ca2+. For the increase in [Ca 2+]; due to Ca2+ release from intracellular stores in the absence of extracellular Ca2+' the ED50 value of CCh was 3 X 105 M. On the other hand, in the presence of Ca 2+' the ED50 value was 2.5 X 10-7 M, in dicating that a low concentration of CCh (< 10-7 M) caused influx of extracellular Ca2+ without Ca2+ release. Oxotremorine and pilocarpine induced Ca2+ influx, but were less potent inducers of Ca2+ release. CCh also stimulated the formation of inosi tol trisphosphates (IP3) with an ED50 value of (4.5 X 10-5 M), which was similar to that for Ca2+ release from intracellular stores. Treatment of the smooth muscle with neomycin (1 mM), a phospholipase C inhibitor, abolished both CCh-induced IP3 formation and Ca2+ release from intracellular stores, but did not affect CCh-induced Ca2+ influx. These results suggest that the pathway for muscarinic stimulation of Ca2+ influx through plasma membranes is different from that for Ca 2+ release from intracellular stores, which seems to be coupled with IP3 formation.

Stimulation of smooth muscles with various receptor agonists including muscarinic agonists results in a contractile response by triggering an increase in cytoplasmic free calcium ([Ca2+]i). IP3, a product of receptor-activated phospholipase C, plays an important role in this elevation of [Ca2+]; by inducing the re lease of Ca2+ from intracellular stores (1-4). Many studies have demonstrated that mus

carinic agonists are effective activators of IP3 and diacylglycerol syntheses in smooth muscle (5, 6). However, the dose-response curve for IP3 formation does not coincide with that for muscle contraction, the latter being in a lower concentration range of CCh (7). Moreover, contraction of ileal longitudinal muscle is known mainly to depend on the extracellular Ca 2+ concentration (8). These facts indicate that not only release from intracellular stores, but also influx of extracellular Ca2+ through

the receptor-operated Ca 2+ channels may con tribute to the increase in [Ca2+]; in longitudi nal ileal smooth muscle. The mechanism by which agonist-receptor interaction leads to an increase in [Ca2+]i (i.e., by release of Ca2+ from intracellular stores and/or by influx of external Ca2+ ) has been studied in many tissues and cells, includ ing ileal smooth muscles (9-12). Although the receptor activation can cause both Ca2+ influx and Ca2+ release in the same cell or tis sue, the question of whether the Ca2+ influx and release are mediated by the same pathway or different pathways from Ca 2+ release is still unanswered. Receptor-mediated Ca2+ influx has been demonstrated in many tissues and cells, but in some tissues, agonist-induced Ca 2+ influx is sensitive to voltage-dependent Ca2+ channel blockers (8, 9, 13), whereas in others, it is not (14, 15). These findings suggest that these channels are tissue-specific (16). The existence of another form of agonist-in duced Ca2+ channel, a second messenger operated Ca2+ channel, has been demonstrat ed by the whole cell patch-clamp technique combined with measurement of [Ca2+1i with the Ca 2+ indicator fura-2. Injection of IP3 into mast cells caused an increase in [Ca2+]; by in ducing both the release of Ca2+ from the in tracellular Ca2+ stores and influx of external Ca2+ into the cells (17, 18). In this work, we examined the muscarinic cholinergic regulation of intracellular Ca2+ and IP3 in the longitudinal smooth muscle of the ileum. The present data suggests that the Ca2+ influx through plasma membranes and release from intracellular pools are operated independently by muscarinic receptors. MATERIALSAND METHODS Tissue preparation for Ca2+ measurement Male guinea pigs weighing approximately 300 g were killed by decapitation. The ileum was quickly isolated and the longitudinal smooth muscle was separated from the circular muscles. Thin strips of about 0.8 cm width and

1.5 cm length were cut and then transferred to oxygenated PSS containing 127 mM NaCI, 2.7 mM KCI, 1.8 mM CaC12, 1 MM MgC12, 0.5 mM NaH2PO4, 12 mM NaHCO3 and 5 mM glucose. The solution was bubbled with 5% CO2 in 02. Ca2+-free solution was made by addition of 3 mM EGTA to PSS. Under this condition (pH 7.4, 1.8 mM CaC12, 1.0111M MgC12 and 3.0 mM EGTA), the free Ca2+ concentration was calculated to be 5 X 10-8 M, which is practically Ca2+-free for extra cellular conditions (19). The muscle strips were treated with the acetoxymethyl ester of fura-2 (fura-2/AM) at 5 uM for 3 hr at 20°C in PSS containing 0.08% Cremophor EL, a noncytotoxic detergent (20). After fura-2 loading, the strip was spread out and fixed to a holder to minimize contrac tile movement. The fluorescence from a con stant area of the strip, 8 X 5 mm, was meas ured in a quartz cuvette (1 X 1 cm) placed in a thermostatic cuvette-holder with a stirring apparatus with excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm using a two-wavelength fluorometer (Nihon Bunko, CAF-100). The time course of the change in the 380/340 nm ratio was re corded. Measurement of 3H-labeled inositol phosphates After the isolation of longitudinal smooth muscle, slices (0.25 X 0.25 mm) were cut with a Macllwain tissue chopper, and washed 6 times with PSS. All procedures were carried out at 4°C. The assay was based on the method of Zhou et al. (21). Briefly, the slices were incubated with 3H-inositol (5 ,uCi/ml) for 3 hr at 30°C to label cellular phosphoinosi tides, and then washed three times with ice cold PSS containing 10 mM LiC1 and transfer red to test tubes. Stimulation was started by addition of CCh at 37°C and stopped by addi tion of TCA. Then the slices were homoge nized in a Polytron, and the radioactivity in 50-,ul aliquots of homogenate was counted and 20 times the value was taken as the total radioactivity (10000-20000 dpm/mg protein). The homogenate was centrifuged at 1000 X g

for 20 min, and the supernatant was freed of TCA by extraction with diethylether. The mix ture was applied to a column containing 1 ml of 50% (v/v) Dowex 1 X 8 (formate form). The IP3 was separated and its radioactivity was determined (22). Results were expressed as the ratio of 3H-inositol trisphosphates to the total radioactivity to avoid errors due to variations in the samples of slices. Muscarinic agonists were from Sigma Chemi cal Co., fura-2 AM, from Wako Pure Chemi cal Industries; and 3H-inositol, from New Eng land Nuclear. Other compounds were com mercial products of the highest grade avail able. RESULTS Measurement of changes in [Ca2+Jt in longitu dinal smooth muscle by fura-2 fluorescence With the apparatus used, CCh reproducibly caused an increase in the fluorescence from fura-2-loaded strips with excitation at 340 nm, a decrease with excitation at 380 nm and no change with excitation at 360 nm. In this study, we measured the ratio of fluorescence with excitations at 340 nm and 380 nm as an indicator of the change in [Ca 2+]i. In muscle strips not loaded with fura-2, the basal levels of fluorescence with excitations at these wavelengths were very low and were not

affected by the addition of CCh. Carbachol-induced changes in [Ca2+J, in ileal longitudinal smooth muscle In the presence of extracellular Cat+, CCh caused an increase in [Ca2+]i at a concentra tion of less than 10-6 M as shown in Fig. 1. A low concentrations of CCh (10-8 M) induced a monophasic increase in [Ca2+],, which reached a plateau in 3 5 min. With 10-8 M CCh, no increase of [Ca2+]i was observed af ter removal of Ca2+ from the extracellular medium with EGTA (Fig. 1c). When ex tracellular Ca2+ was replaced by Mn2+, CCh (10-8 M) caused rapid quenching of the fura-2 fluorescence, indicating Mn2+ influx by CCh (Fig. 1B) (23, 24). Addition of Mn2+ at the plateau phase of increase in [Ca 2+]i by 10-8 M CCh also quenched the fluorescence rapidly (data not shown). These results indicate that the increase in [Ca2+]i by low concentrations of CCh was due to Ca2+ influx from the ex tracellular space. High concentrations of CCh (> 10-6 M) caused a biphasic increase in [Ca2+]i: a transient increase and then a sus tained increase to a plateau (Fig. 2). The ED50 value of CCh was approximately 2.5 X 10-7 M measured at the plateau phase (Fig. 3). In the absence of extracellular Ca2+, CCh caused a small transient increase in [Ca 2+1i, but only at high concentrations. The ED50

Fig. 1. Changes in [Ca2+]; induced by a low concentration of CCh in the presence and absence of extra cellular Ca2+ or Mn2+. Muscle strips loaded with fura-2 were stimulated with a low concentration of CCh (10-8 M) in normal medium containing 1.8 mM calcium (A), in medium in which calcium was replaced by an equivalent concentration of Mn2+ (B), or in Ca2+-free medium (with 3 mM EGTA) (C). Changes in [Ca2+]; are expressed as 340/380 nm ratios. Each recording is representative of at least three separate ex periments with similar results.

Fig. 2. Comparison of patterns of intracellular calcium changes induced by muscarinic agonists. Muscarinic agonists at the indicated concentrations were added to the medium with or without Cat+. Changes in [Cat+]; are expressed as 340/380 nm ratios. Each recording is representative of at least three separate ex periments with similar results.

Fig. 3. Dose-response curves of CCh-inducedchanges in [Ca2+]i in the presence and absence of extracellular Ca2+. Muscle strips loaded with fura-2 were exposed to the indicated concentration of CCh in the presence (0) and absence (0) of extracellular Ca2+. The max imal point of the time course of change in the 340/380 nm ratios was taken as the value of the response at the indicated concentration of CCh. Values (means ± S.E.M. for three separate experiments) are expressed as a percentage of the response to 10-3 M CCh. Points without deviation were taken from a single ex periment.

value (3 X 10-5 M) was approximately 100 fold higher than that in the presence of exter nal Cat+. Other muscarinic agonists, oxotremorine and pilocarpine, increased [Ca21]i monophasi cally with ED50 values of 3 X 10-6 and 1 X 10-6 M, respectively. In the absence of ex tracellular Cat+, neither agonist at concentra tions of up to 10-4 M caused appreciable Cat+ release (Fig. 2). Atropine inhibited the response to muscar inic agonists in both the presence and absence of extracellular Cat+. These results indicate that the increase in [Ca 2+]i in the absence of extracellular Ca2+ (release from intracellular Ca 2+ stores) re quired higher concentrations of agonists than that in the presence of extracellular Ca2+ (both influx of extracellular Cat+ and release from intracellular stores). That is, low concen trations of agonists (< 10-7 M) induced only influx of extracellular Cat+. In the following experiments, an increase in [Cat+]i by 10_8 M

occurred during a 30-min incubation. As shown in Fig. 5, CCh increased the release of [3H]-inositol trisphosphates ([3H]-IP3) concen tration-dependently. Formation of IP3 was observed with a CCh concentration as low as 1 ,uM. Lower concentrations (< 10-7 M), which Effects of neomycin on Ca-2+influx and Ca-2+ elicited Ca2+ influx, caused no detectable formation of IP3 under our experimental con release A muscle strip was treated with 1 mM ditions. Formation of IP3 was maximal (4-6 neomycin, a phospholipase C inhibitor, at times than control) with 1 mM CCh. The ED50 value for IP3 formation was approx 37°C for 15 min. This treatment influenced neither the basal fluorescence nor, as shown in imately 4.5 X 10-5 M, which was similar to Fig. 4A, the Ca2+ influx induced by a low the ED50 value for the stimulation of Ca2+ re concentration of CCh (108 M). lease. Next, a muscle strip was treated with 1 mM Atropine antagonized the effect of CCh in neomycin at 37°C for 12 min, and then EGTA stimulating IP3 formation (data not shown). (final concentration, 3 mM) was added. At 3 min after adding EGTA, a high concentration Effect of neomycin on CCh-induced IP3 forma of CCh (1 mM) was added to stimulate the tion in ileal longitudinal smooth muscle Ca2+ release. Ca2+ release from intracellular Slices of longitudinal smooth muscle were stores was inhibited about 90% by this treat pretreated with 1 mM neomycin for 15 min at ment (Fig. 4B). 37°C. This treatment did not affect the basal formation of IP3. As shown in Fig. 6, neomy Effect of muscarinic receptor activation on cin inhibited IP3 formation induced by a high concentration (10-3 M) of CCh, the percent formation of inositol trisphosphate in longitu dinal smooth muscle inhibition being 89% of the CCh-induced stim In the absence of an agonist, only limited ulation. The value was similar to that of Ca 2t release of inositol phosphates from slices release. CCh in the presence of extracellular Ca2+ was taken as an indication of Ca 2+ influx which was about 20% of the maximal change by 10-4M CCh to avoid the contamination of a fraction of Ca2+ release.

Fig. 4. Effects of neomycin on CCh-induced influx and intracellular release of Ca'+. (A) Muscle strips were loaded with fura-2 and incubated with and without (as a control) 1 mM neomycin in PSS at 37°C for 15 min. Then, 10-g M CCh was added to stimulate Ca2+ influx. For (B), the media were freed from Ca2+ by addition of EGTA (final concentration, 3 mM) during the last 3 min of neomycin treatment. Then the strip was stimulated with 10-3 M CCh to induce Ca 2+ release from intracellular stores.

DISCUSSION

Fig.

5.

tion nal in

Dose-dependence

of 3H-inositol smooth the

slices

Methods.

LiCI.

6.

trisphosphate

for

Effect

value

10 min

muscles.

in the

the

concentra presence

as

release

Values

are

means

Slices with

and

in the presence the

indicated for

neomycin.

on

of

10

ratios

as

± S.E.M.

CCh-induced

in

four

slices

preloaded without

of 10 mM

as described

± S.E.M. without

longitudi

3H-inositol,

indicated

expressed

formation

incubated with

with

the

at 37°C

are

of neomycin

was measured means

loading with

accumula

experiments.

tol

lated

as described

were

in the Methods.

Fig.

15 min

prepared

Slices After

for 3 separate

were

of

Results

described

smooth

in slices

stimulated

of CCh

mM

CCh-induced

muscles.

were

tions

of

trishosphates

longitudinal

with 1 mM LiCI,

3H-inositol neomycin

and

concentrations in the

3H-inosi

of

then of

Methods.

experiments.

*P

for stimu

CCh.

IP3

Values

are

< 0.01

vs.

Agonist-induced increase in [Ca2+]i in many cells and tissues is due to both the release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx from the external mediums. An increase in [Ca 2+]i with high concentrations of CCh was observed in the absence of extracellular Ca2+, suggesting that some of the increase was due to mobilization of Ca2+ from intra cellular stores. However, this increase was less than 30% of the maximal increase in [Ca 2+]i, a larger proportion being due to Ca 2+ influx through the plasma membrane in the longitu dinal smooth muscles. Agonist-induced release of Ca2+ from intra cellular stores has been shown to be mediated by activation of phospholipase C and its prod uct, IP3, in many cells and tissues (1, 2, 4, 25 -27) . In longitudinal smooth muscles, muscar inic stimulation causes a rapid increase in 1P3 formation (5) and mobilization of Ca2+ from intracellular Ca 2+ stores (28). Our finding that CCh-induced increase in [Ca2+]i in Ca2+-free solution was parallel with increase in IP3 formation supports these observations. The ED50 values of CCh for IP3 formation (4.5 X 105 M) and Ca2+ release (3 X 10-5 M) were very similar, and neomycin showed similar potencies in inhibiting the two responses. However, the influx of Ca 2+ through plas ma membranes seemed to be mediated by a pathway different from that for the release of Ca 2+ and IP3 formation. The ED50 value of CCh (2.5 X 107 M) for inducing influx was much lower than that (3 X 10-5 M) for induc ing Ca2+ release, low concentrations of CCh (10-8_ 10-7 M) causing only Ca2+ influx. Moreover, oxotremorine and pilocarpine stimulate Ca2+ influx but not Ca2+ release even at high concentration. Furthermore, neomycin at a concentration that caused 90% inhibition of the release of Ca2+ and IP3 formation did not affect the influx. The difference in the ED50 values can be explained by the mechanism of amplification and saturation. That is, a low concentration of CCh might induce a very slight increase in a

product of phospholipase C or [Ca2+]i that was not detectable by our methods, and this may have elicited the opening of Ca2+ chan nels, and a submaximal level of this product may have caused full opening of Ca 2+ chan nels. However, if this were the case, 90% in hibition of phospholipase C by neomycin should have shifted the dose-response curve of CCh for Ca 2+ influx to the right approximate ly 10-fold. However, neomycin did not affect CCh-induced Ca2+ influx. Therefore, muscar inic activation seems to cause Ca 2+ influx and release of intracellular Ca2+ by different path ways with different affinities or efficacies. There are reports that receptor-mediated Ca2+ influx and its intracellular release in the same cells show characteristic differences. Kinetic studies of [Ca2+]i by stopped-flow fluorometry showed that agonist-evoked Ca2+ influx occurred without measurable delay, whereas the release of Ca2+ from intracellular stores was induced with a delay of about 200 msec. Influx of Mn2+ was also found to pre cede intracellular Ca2+ release (24, 29). Moreover, carbachol is reported to have a greater effect on the plateau phase than the peak phase of [Ca 2+]i increase (30). These findings may be due to different pathways for inductions of influx and intracellular release of Ca2+. On the other hand, electrophysiological stu dies have shown that receptor-mediated Ca2+ entry through plasma membranes might be a consequence of IP3 formation. Application of IP3 stimulated a Ca 2+ current in isolated patches of plasma membranes of T lympho cytes (31). Recently, it was demonstrated that injection of IP3 into mast cells induced Ca 2+ influx (17, 18). However, we found that the concentration of CCh that induced Ca2+ in flux was much lower than that for IP3 forma tion, although our results do not disprove the possibility that a high concentration of IP3 can also cause Ca2+ influx. Many studies have shown that Ca 2+ antago nists inhibit the cholinergic contractile re sponse in guinea pig ileum (8, 9, 32). With the fura-2 loading condition used in this study,

CCh-induced Ca2+ influx was not inhibited by nifedipine and verapamil, and CCh did not cause muscle contraction. However, the high K+-induced Ca 2+ influx was sensitive to the Ca 2+ antagonists. When loaded fura-2 was lowered to 1/5, both CCh-induced contraction and inhibition of Ca2+ influx by nifedipine appeared (S. Uchida et al., unpublished observation). This may be explained by chela tion of Ca2+ by fura-2 which prevents eleva tion of [Ca2+]i instead of the change in fura-2 Ca2+ fluorescence. The relationship between fractions of Ca2+ influx sensitive and insensi tive to Ca 2+ antagonists is now under inves tigation. Two different muscarinic receptors, the M2 and M3 subtypes, were present in this tissue (33). The relationship between the subtypes and the responses is also under investigation. Acknowledgments This for

work

Scientific

Science Nakamura

and

was

supported

Research Culture for

in part

from

the

of

Japan.

assistance

in

by

a Grant-in-Aid

Ministry

of Education,

We the

thank preparation

Mrs.

Mieko of

this

manuscript.

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