The effects of extracellular pH on intracellular pH, Ca2+ and tension of canine tracheal smooth muscle strips

Life Sciences, Vol. 56, No. 8 pp. PL 175-180, 1995 Copyright 0 1995 Ekvier Science Ltd Printed in the USA. All rights reserved 0024-3205/95 $950 t .@I

Pergamon 0024-3205(94)00497.8

PHARhfACOLOGY LETTERS Accelerated Communication

THE EFFECTS OF EXTRACELLULAR pH ON INTRACELLULAR TENSION OF CANINE TRACHEAL SMOOTH MUSCLE

Michiaki

Department

Yamakage,

Shinji

Kohro,

Masanori

Yamauchi

pH, Ca2+ AND STRIPS

and Akiyoshi

Namiki

of Anesthesiology, Sapporo Medical University School of Medicine South 1, West 16, Chuo-ku, Sapporo, Hokkaido 060, Japan (Submitted October 31, 1994; accepted November 17, 1994; received in final form December 1, 1994)

Abstract: The effects of changes in extracellular pH (pH,) on intracellular Ca2+ concentration ([Caz+]i) or intracellular pH (pHi) were measured simultaneously with muscle tension in canine tracheal smooth muscle strips. [Caz+]i and pHi were measured using the fluorescent dyes furaand BCECF, respectively. During high K+-induced contractions (24.2, 36.4 or 72.7 mM) at pH 7.4, pH, was changed to 7.8 or 7.0 with NaOH or HCl, respectively. Induced changes in pHi were equal to - 50 % of the changes in pH,. Alkalinization significantly increased [Caz+]i and enhanced muscle contraction at all concentrations of K+ but did not alter the relationship between muscle tension and [Caz+]i. Acidification significantly decreased [Caz+]i without changing muscle tone; hence, the muscle tension - [Caz+]i relationship was shifted to the left. These results suggest that changes in pH, can alter airway smooth muscle tone by changing [Caz+]i and pHi. Key Words: intracellular Ca2’,

intracellular pH, voltage-dependent calcium channel

Introduction Changes in extracellular pH (pH,) have been shown to affect the contractility of airway smooth muscles in vitro (1) with airway smooth muscle tension increased during alkalosis and decreased during acidosis. Because changes in pH, of airway smooth muscle likely occur in a variety of physiological and pathological situations, a better understanding of the effects of alkalosis and acidosis on the contractility of airway smooth muscles might suggest new ways of preventing or reversing inappropriate bronchoconstriction. Smooth muscle function could be altered directly by pH, and/or indirectly via changes in intracellular pH (pHi), an important regulator of cell function (2). In vascular smooth muscle, changes in pHi alter the contractility (3,4) but the responsiveness of the muscle to changes in pHi is highly variable. Some differences between these studies may be attributable to the methods used to create acidosis and alkalosis or the contractile agonists used to produce precontraction. Twort and Cameron (1) proposed that changes in pH may affect airway smooth muscle contraction by pH-dependent alterations of Ca2+ uptake. However, there has been no direct demonstration that changes in pH, can change intracellular Ca2+ concentration ([Caz+]i), a primary regulator of smooth muscle tone (5). In this study, we sought to clarify the cellular effects of changes in pH, by measuring changes in pHi and [Caz+]i simultaneously with muscle tension during acidosis and alkalosis produced by additions of HCl or NaOH. Address correspondence to: Dr. Michiaki Yamakage, Department of Anesthesiology & Critical Care Medicine, The Johns Hopkins Medical Institutions, Hygiene 7006, 615 N. Wolfe Street, Baltimore, MD 21205, Tel: 410-955-3612, Fax: 410-955-0299

PL-176

Effect of pH on Airway Smooth Muscle

Vol. 56, No. 8, 1995

Methods Muscle Strip Preparation This study was approved by the Sapporo Medical University Committee on Animal Research. Adult mongrel dogs (weighing 9 - 12 kg) were anesthetized with intravenous thiamylal (20 mg/kg). After a surgical level of anesthesia was attained, the trachea was quickly excised and placed in a physiological salts solution (PSS) at room temperature. PSS contained (in mM): NaCl 136.9, KC1 5.4, CaCl2 1.5, MgCl2 1.0, NaHC03 23.9, glucose 5.5 and EDTA 0.01. The solution was aerated continuously with a 95 % 02 / 5 % CO2 gas mixture (pH 7.4). The smooth muscle was dissected free of epithelium, cartilage and connective tissue, and cut into strips - 1 mm width and - 8 mm length. Measurement of intracellular Ca2+ concentration and muscle tension Muscle strips used for measurement of [Caz+]i were pretreated with 5 j.LMacetoxymethyl ester of fura- (fura-2/AM), an indicator of Ca2+, in PSS for 6 - 7 hrs at room temperature (22 - 24 “C). Cremophor EL (0.02 % v/v), a noncytotoxic detergent, was added to increase the solubility of fura2/AM. After fura- loading, the muscle strip was held horizontally in a temperature controlled (37 “C), 5-ml organ bath. One end of the muscle strip was connected to a strain gauge transducer (120T-20B, Kyowa, Tokyo, Japan). The strip was then washed with PSS for 30 min to remove uncleaved fura-2/AM. Experiments were performed within 60 min after the washing. Experiments were performed with a fluorescence spectrometer (CAF- 110, Japan Spectroscopic, Tokyo, Japan) specially designed to measure the surface fluorescence of living tissues. Excitation light obtained from a xenon high-pressure lamp (75 w) was passed through a rotating filter wheel (128 Hz) that contained 340 nm and 380 nm filters. The emitted light from the muscle strip at 500 nm was measured with a photomultiplier. The time constant of the optical measurements was 0.25 sec. The ratio of the fluorescence due to excitation at 340 nm to that at 380 nm (R340/380) was calculated from successive illumination periods and used as an indicator of [Caz+]i. A first contraction evoked by 72.7 mM high K+ solution served as a control. Contractions were then evoked by various concentrations of high K+ solutions (24.2, 36.4 or 72.7 mM). High K+ solutions were made by substituting NaCl of PSS with equimolar KCI. All solutions were aerated with 95 % 02 / 5 % CO2 at 37 “C and pH 7.4. After the muscle tension and [Caz+]i had reached steady state, the high K+ solution was changed to one of similar composition but with pH adjusted to 7.8 or 7.0 using 0.25 N NaOH or 0.2 N HCI. When changes in muscle tension and [Caz+]i had reached steady state, the high K+ solution was again replaced with PSS at pH 7.4. In a preliminary study, we demonstrated that changes in pH, in a range from pH 6.8 to 8.0 had no effect on 500 nmemitted fluorescence of fura- both by 340-nm and 380-nm excitations. Measurement of intracellular pH and muscle tension Muscle strips used for measurement of pHi were treated with 5 pM acetoxymethyl ester of 2’,7’bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF/AM), an indicator of pH, in PSS containing 0.02 % (v/v) cremophor EL for 60 min at room temperature. After BCECF loading, the muscle strip was held horizontally in an organ bath with one end of the muscle strip connected to a strain gauge transducer. It was washed with PSS to remove uncleaved BCECF/AM in the tissue bath at 37 “C for 30 min. Experiments were then performed within 90 min. The pHi experiments were performed with the spectrometer as described above using excitation filter wavelengths of 450 and 500 nm and a 540 nm photomultiplier filter. The ratio of the fluorescence due to excitation at 500 nm to that at 450 nm was calculated from successive illumination periods and referred to as R500/450. At the end of each experiment, R5001450 was calibrated using the high-K+-nigericin technique (6). Briefly, the tissues were exposed to the 10 pM nigericin, a K+-H+ exchanging ionophore, in the presence of elevated external K+ (140 mM) and pH, was varied. There was a linear relationship between pHi and R500/450 over the pH range 6.5 to 7.8.

Vol. 56, No. 8, 1995

PL-177

Effect of pH on Ainvay Smooth Muscle

Drugs The following drugs and chemicals were used: acetoxymethyl ester of fura- (fura-2/AM), acetoxymethyl ester of 2’,7’-bis(2-carboxyethyl)-5,6-carboxy-fluorescein (BCECF/AM) (Dojindo Laboratories, Kumamoto, Japan), EDTA (Katayama Chemical, Osaka, Japan), cremophor EL and nigericin (Sigma Chemical, St. Louis, MO). Data analysis Muscle tension data are those obtained with measurements of [Caz+]i. Changes in muscle tension during measurements of pHi were similar (data are not shown). Data are expressed as a percent of the sustained changes in R340/380 or muscle tension induced by 72.7 mM-K+ and given as mean + SD. Mean values were compared with repeated-measures ANOVA and Dunnet’s test with P < 0.05 considered significant. Results Effects of alkalosis on muscle tension, [Caz+]i and pHi Tissues in control PSS (pH, 7.4) had a pHi of 7.17 + 0.14 (n = 6). Typical effects of high K+ (24.2 mM) and alkalosis (pH, 7.8) on the tension, [Caz+]i and pHi of canine tracheal smooth muscle are shown in Fig. 1A. High K+ solution rapidly increased [Caz+]i and contracted the muscle. Both [Caz+]i and muscle tension reached their respective peaks within 5 - 40 sec. pHi exhibited an initial slight decrease with the high K+ solution; but rapidly returned to the original level. Raising pH, from 7.4 to 7.8 significantly increased [Caz+]i and tension at all concentrations of K+ (24.2, 36.4 and 72.7 mu) and alkalinized pHi by 0.20 unit (Fig. 1A and B; Table 1). Steady state levels were attained within 2 min (Fig. 1A). Fig.lB shows the relationship between the tension and [Caz+]i of canine tracheal smooth muscles during administrations of various concentrations of K+ (24.2, 36.4 and 72.7 mM) and alkalosis (pH, 7.8). The muscle tension vs. R340/380 curve was unchanged by alkalosis. Muscle tension, [Caz+]i and pHi returned to their respective baseline values after washout with the PSS at 7.4 (Fig. 1A and Table 1). TABLE 1 EFFECT OF CHANGES IN EXTRACELLULAR pH (pH,) ON INTRACELLULAR PH,

pHi

7.4 7.8

7.17 + 0.14 7.37 f 0.20

7.4

7.18 f 0.18

7.4

7.18 f 0.18

7.0

6.98 f 0.19

7.4

7.16 + 0.12

pH (pHi)

ApHi / ApH,

Alkalinization 0.50 f 0.11 0.45 f 0.09

Acidification 0.45 f 0.08 0.48 + 0.09

Values are means + SD; n = 6 for all values Effects of acidosis on muscle tension, [Caz+]i and pHi The initial pHi of the muscle strips in these experiments was 7.16 f 0.12 (n = 6) in PSS with pH, 7.4 (Fig. 2A and Table 1). Typical effects of high K+ (72.7 mM) and acidosis (pH, 7.0) on the tension, [Caz+]i and pHi of canine tracheal smooth muscle are shown in Fig. 2A. Lowering the pH of high K+ solution from 7.4 to 7.0 significantly decreased R340/380 at all concentrations of K+. Muscle tension showed an initial slight decrease followed by a return to near the previous contracted

PL-178

Vol. 56, No. 8, 1995

Effect of pH on Airway Smooth Muscle

5 min

[Al

LB1

IIO-

0.6

1

R340i3.30 v

IOO-

0.4

c

I

Tension m

SO-

Q e

no-

.P

e

29

?O-

f 60

1

1

7.4

PHi

P&B

6.8

24.2

72.7K+ 7.4

1 50

’

7.6

’

1

40 ! 40

50

60

7.4

70

60

R340/360

Fig.

60

100

I 110

(%)

1

A: Representative effects of extracellular alkalosis (pH, 7.8) on tension, R340/380 (an indicator of intracellular Ca*+ concentration) and intracellular pH (pHi) of a canine tracheal smooth muscle strip. B: Relationship between tension and R3401380 of canine tracheal smooth muscles during contraction with K+ (24.2, 36.4 or 72.7 mM). Paired muscle tension - R340/380 points are mean f SD (n = 6). Arrows connect data from the same tissues obtained at pH, 7.4 (closed circle) and pH, 7.8 (open circle).

5 min

[Al

72.7 mM K+

R340/366 4-r

Tension

60-

5024.2 mM

72.7

72.7K+

P”,

7.4

16.6

’

7.0

’

401 40

7.4

50

60

70 R340/360

Fig.

60

60

100

110

(%)

2

A: Representative effects of extracellular acidosis (pH, 7.0) on tension, R340/380 (an indicator of intracellular Caz+ concentration) and intracellular pH (pHi) of a canine tracheal smooth muscle strip. B: Relationship between tension and R340/380 of canine tracheal smooth muscles during contraction with K+ (24.2, 36.4 or 72.7 mM). Paired muscle tension - R340/380 points are mean t- SD (n = 6). Arrows connect data from the same tissues obtained at pH, 7.4 (closed circle) and pH, 7.0 (open circle).

Vol. 56, No. 8, 1995

Effect of pH on Airway Smooth Muscle

PL-179

level (Fig. ZA). Lowering pH, of the high K+ solution from 7.4 to 7.0 significantly acidified the intracellular environment by 0.18 unit within 1 min (Fig. 2A and Table 1). Fig. 2B shows the relationship between the tension and R340/380 of canine tracheal smooth muscle during administrations of various concentrations of K+ (24.2, 36.4 and 72.7 mu) and acidosis (pH, 7.0). The muscle tension vs. R340/380 curve was shifted to the left by extracellular acidosis. Muscle tension, [Caz+]i and pHi returned to their respective baseline values after washout with PSS at 7.4 (Fig. 2A and Table 1). Discussion Effects of changes in pH, on pHi The pHi of canine tracheal smooth muscle in PSS with CO2-HC03 buffer was 7.17 f 0.08 (n = 12). This value is comparable to those previously reported values for other smooth muscle types (78). We contracted smooth muscle strips with high K+ solutions to minimize activation of second messenger systems, such as Caz+/phospholipid-dependent protein kinase, which can activate Na+H+ exchange, and alter pHi (89). As expected, high K+ did not change the pHi of canine tracheal smooth muscle in this study, but changes in pH, induced changes in pHi equal to - 50 % of the changes in pH,. Changes in pHi may be reduced by intracellular buffering by various proteins and phosphates. In addition, Na+/H+ (10) and HCO3-/Cl- (11) exchange systems may provide further pHi regulation. Effects of changes in pH, on muscle tension and [Caz+]i Alkalinization of high K+ solutions from 7.4 to 7.8 both increased [Caz+]i, as reflected by the R340/380 ratio, and enhanced the contraction of canine tracheal smooth muscle (Fig. 1A and B). These responses were seen at all K+ concentrations tested. These results are consistent with the increase in airway tone that occurs during hypocapnia and hyperventilation (12). On the other hand, acidification of the high K+ solution from 7.4 to 7.0 decreased [Caa+]i at each K+ concentration tested (Fig. 2A and B). The dependence of [Caz+]i on pH suggests that pH, or pHi may modulate mechanisms for Ca2+ entry or extrusion in airway smooth muscle cells. Under our experimental conditions (high K+) voltage-dependent Ca2+ channels (VDC) are the primary mechanism for Ca2+ entry (13). Changes in pH, could affect CQ+ influx through VDC directly by altering Ca2+ channel properties or indirectly by modifying the membrane potential. Data from other smooth muscles support each of these mechanisms. Aickin (14) and Siegel & Schneider (15) demonstrated that alkalinization of the extracellular solution resulted in depolarization of the guinea-pig vas deferens and carotid arterial smooth muscle, respectively. On the other hand, Kurachi (16) and Kaibara & Kameyama (17) demonstrated that increases in intracellular H+ directly inhibited VDC in guinea-pig ventricular cells. Because high K+-depolarized tissues are resistant to modulation of membrane potential, our data are more consistent with direct modulation of VDC by pH. However, we cannot distinguish effects of pH, and pHi in these experiments. Interestingly, Irisawa and Sato (18) showed that VDCs in guinea-pig ventricular cells were more sensitive to intracellular than extracellular H+. Hence, pHi may be more important than pH, in regulating [Caz+]i of airway smooth muscle. Additional studies to directly measure the inward Ca2+ currents through VDC of airway smooth muscle are needed to confirm these interpretations. It is interesting that during acidification, muscle tension was restored after a transient decrease in spite of a significant, prolonged decrease in [Caz+]i. Twort and Cameron (1) have reported that respiratory acidosis decreased tension of acetylcholine-stimulated rat tracheal smooth muscle. This difference between these studies may attributable to the methods used to create acidosis or to the contractile agonists used to produce precontraction. This result in the present study suggests that acidification may sensitize the contractile elements to Caz+ or activate a Caz+-independent contractile mechanism in canine tracheal smooth muscle. Arheden et al. (19) and Crichton et al. (20), using skinned-fiber techniques, demonstrated that acidification increased the sensitivity of contractile elements to Ca2+ in guinea-pig taenia coli and rat uterine smooth muscle cells, respectively. Our data are consistent with a similar increase in Ca2+ sensitivity during acidosis.

PL-180

Effect of pH on Airway Smooth Muscle

Vol. 56, No. 8, 1995

In conclusion, although canine tracheal smooth muscle has intrinsic buffering power, intracellular pH was significantly altered by changes in extracellular pH. In tracheal smooth muscle strips precontracted by high K+ solutions, alkalinization increased both [Caz+]i and muscle tension while acidification decreased [Caz+]i without changing the muscle tension. We conclude that pH regulates [Caz+]i of tracheal smooth muscle but that a decrease in [Caz+]i during acidosis can occur without relaxation. Hence, acidification may sensitize the contractile elements of tracheal smooth muscle to Ca2+. Acknowledgments The first author thanks Drs. Carol A. Hirshman and Thomas L. Croxton, for comments and suggestions concerning this manuscript and the Japan Society for the Promotion of Science (Postdoctoral Fellowship for Research Abroad) for support. REFERENCES

2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

C.H.C. TWORT and I.R. CAMERON, Respir. Physiol. 66 259-267 (1986). S. GRINSTEIN, S. COHEN and A. ROTHSTEIN, J. Gen. Physiol. Q 341-369 (1984). H. NIELSEN, C. AALKJ/ER and M.J. MULVANY, Pflugers Arch. 419 51-56 (1991). N.C. SPURWAY and S. WRAY, J. Physiol. (Lond.) 393 57-71 (1987). R.S. ADELSTEIN, M.D. PAT0 and M.A. CONTI, Muscle Contraction: Its Reeulatorv Mechanisms, S. Ebashi, K. Maruyama and M. Endo (Eds), 303-313, Japanese Scientific Society Press, Tokyo (1980). J.A. THOMAS, R.N. BUCHSBAUM, A. ZIMNIAK and E. RACKER, Biochemistry 18 2210-2218 (1979). C. AALKJWR and E.J. CRAGOE JR, J. Physiol (Lond.) &2 391-410 (1988). R. BOSE, J. YU, E.J. CRAGOE and J. DELAIVE, Frontiers in Smooth Muscle Research, N. Sperelakis and J.D. Wood (Eds), 695-702, Alan R. Liss Press, New York (1989). Y. NISHIZUKA, Science 233 305-3 12 (1986). P.L. WEISSBERG, P.J. LITTLE, E.J. CRAGOE JR and A. BOBIK, Am. J. Physioi. 253 Cl93-Cl98 (1987). B.F. BECKER and J. DUHM, J. Physiol. (Lond.) 282 149-168 (1978). D.R. STIRLING, D.J. COTTON, B.L. GRAHAM, W.C. HODGSON, D.W. COCKROFT and J.A. DOSMAN, J. Appl. Physiol. 54 934-942 (1983). H. KARAKI and G.B. WEISS, Gastroenterology HI 960-970 (1984). CC. AICKIN, J. Physiol. (Lond.) 349 571-585 (1984). G. SIEGEL and W. SCHNEIDER, Vasodilation, P.M. Vanhoutte and I. Leusen (Eds), 285298, Raven Press, New York (1981). Y. KURACHI, Pfliigers Arch. 394 264-270 (1982). M. KAIBARA and M. KAMEYAMA, J. Physiol. (Lond.) 403 621-640 (1988). H. IRISAWA and R. SATO, Circ. Res. 59 348-355 (1986). H. ARHEDEN, A. ARNER and P. HELLSTRAND, Pfltigers Arch. 413 476-48 1 (1989). C.A. CRICHTON, M.J. TAGGART, S. WRAY, and G.L. SMITH, J. Physiol. (Lond.) 465 629-645 (1993).