77
Journal of Physiology (1990), 429, pp. 77-94 With 10 figures Printed in Great Britain
MODULATION OF THE ICa2+J SENSITIVITY OF MYOSIN PHOSPHORYLATION IN INTACT SWINE ARTERIAL SMOOTH MUSCLE
BY CHRISTOPHER M. REMBOLD From the Division of Cardiology, Department of Internal Medicine and Physiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA (Received 20 December 1989) SUMMARY
1. The [Ca2"] sensitivity of myosin light chain phosphorylation in vascular smooth muscle is dependent on the form of stimulation. Contractile agonist stimulation, when compared to high-KCl depolarization, is associated with an increase in [Ca2+] sensitivity of phosphorylation. I evaluated potential mechanisms for this stimulusspecific response by measuring aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and isometric stress in swine carotid media. 2. The relative [Ca2"] sensitivity of phosphorylation depended on the type of stimulus (ranked high to low sensitivity): contractile agonists (histamine, phenylephrine) = endothelin (sustained contraction) = combination of histamine and NaF > NaF alone = endothelin (initial contraction) = combination of histamine and depolarization = combination of NaF and depolarization > depolarization = Bay K 8644 = combination of depolarization and low-dose phorbol diester. 3. Activation of L-type Ca2+ channels with Bay K 8644 induced a [Ca2+] sensitivity of phosphorylation similar to depolarization, suggesting that any other effects of high KCI (such as cellular swelling) were not responsible for the low [Ca2+] sensitivity of phosphorylation. 4. The addition of either histamine or NaF (an activator of G proteins) to depolarized tissues produced similar increases in the [Ca2+] sensitivity of phosphorylation, suggesting that NaF (possibly by activation of a G protein) can mimic contractile agonist-induced increases in the [Ca2+] sensitivity of phosphorylation. 5. Phorbol dibutyrate enhanced the contractile effect of depolarization, and this enhancement was primarily caused by increases in [Ca2+] rather than an alteration in the [Ca2+] sensitivity of phosphorylation. 6. These data suggest that the [Ca2+] sensitivity of phosphorylation in smooth muscle may be regulated by agonists (possible by G protein activation); however, the role of protein kinase C activation or depolarization induced Ca2+ compartmentalization requires further study. INTRODUCTION
Myosin light chain phosphorylation appears to be the primary determinant of cross-bridge function in vascular smooth muscle (Rembold & Murphy, 1988 a; Hai & Murphy, 1989; Murphy, 1989). The identity of the second messenger(s) that MS 8155
C M. REMBOLD 78 determine myosin light chain phosphorylation is more controversial. Myoplasmic [Ca2+], through calmodulin-dependent myosin light chain kinase activation, has been proposed as the primary regulator of myosin phosphorylation (Kamm & Stull, 1985, 1989). In support, there is a unique relationship between myoplasmic [Ca2+] and myosin light chain phosphorylation in swine carotid artery stimulated with a variety of agonists (Rembold & Murphy, 1988a; Taylor, Bowman & Stull, 1989). However, the relationship between aequorin-estimated myoplasmic [Ca2+] and force (Morgan & Morgan, 1984) or between 45Ca2+ influx and force development rates (van Breemen, Lukeman, Leijten, Yamamoto & Loutzenhiser, 1986) can vary depending on the form of stimulus employed. Both groups of investigators found that depolarization required higher levels of [Ca2+] or Ca2+ influx to produce force than was observed with a-adrenergic stimulation. Similarly, in the swine carotid media, depolarization was associated with a decreased [Ca2+] sensitivity of phosphorylation when compared with agonist stimulation (Rembold & Murphy, 1988a). The relationship between phosphorylation and steady-state stress was not affected by the type of stimulus. Several mechanisms could affect the [Ca2+] sensitivity of phosphorylation: KCl depolarization per se may alter myosin kinase or phosphatase activity. Replacement of extracellular Na+ with K+ is known to induce intracellular ionic shifts and cellular swelling that potentially could affect these enzymes. Depolarization is felt to primarily induce smooth muscle contraction by opening L-type Ca2+ channels. In contrast, contractile agonist stimulation may activate other regulatory cascades. Specifically, contractile agonists, in addition to their effects on myoplasmic [Ca2+], could, through specific receptor binding, potentially activate G proteins and/or protein kinases C, A, or G. These regulatory systems could alter the [Ca2+] sensitivity of myosin light chain kinase or myosin light chain phosphatase, thereby altering the [Ca2+] sensitivity of phosphorylation. GTP and its non-hydrolysable analogues increased the [Ca2+] sensitivity of force generation in skinned smooth muscle, suggesting a role of G proteins in determining the [Ca2+] sensitivity of phosphorylation (Nishimura, Kolber & van Breemen, 1988; Kitazawa, Kobayashi, Horiuti, Somlyo & Somlyo, 1989). High-dose phorbol diesters have also been reported to induce smooth muscle contraction at lower myoplasmic [Ca2+] (Chatterjee & Tejada, 1986; Jiang & Morgan, 1987: Rembold & Murphy, 1988b). Cyclic adenosine monophosphate (cyclic AMP)-dependent phosphorylation of myosin kinase has been shown to decrease [Ca2+] sensitivity of myosin kinase in vitro (Adelstein, Conti & Hathaway, 1978; de Lanerolle, Nishikawa, Yost & Adelstein, 1984). The goal of this study was to evaluate whether (1) depolarization-induced ionic shifts or (2) agonist-specific actions, such as G protein or protein kinase C activation, could be responsible for the stimulus specificity of the [Ca2+] sensitivity of phosphorylation. METHODS
Swine common carotid medial tissues were obtained from a local slaughterhouse, dissected, and the optimal length for stress development was determined (Rembold & Murphy, 1988a). The intimal surface was mechanically rubbed to remove the endothelium. Agonist stimulation was
Ca2` SENSITIVITY OF MYOSIN PHOSPHORYLATION
79
performed by injecting an appropriate volume of 10 mM-stock histamine or phenylephrine into the tissue bath. Stock solutions of agonists were prepared daily. Physiological saline solution (PSS) contained (mM): NaCl, 140; KCl, 5; 3-[N-morpholino]propanesulphonic acid (MOPS), 2; CaCl2, 1-6; MgCl2, 1-2; Na2HPO4, 1-2; D-glucose, 5-6 (pH adjusted to 7-4 at 37 °C). Changes in extracellular [K+] were accomplished by changing the bathing solution to a physiological saline in which KCl was substituted stoichiometrically for NaCl. Changes in extracellular [Ca2+] were performed by replacing the bathing solution twice with Ca2+free PSS which contained 1 mM-ethyleneglycol bis(,8-aminoethylether)NN,N',NV'-tetraacetic acid (EGTA) and no added CaCl2. Tissues that did not produce a stress greater than 1-0 x 10t" N/M2 after 109 mM-KCl depolarization or 10 ,tM-histamine stimulation were discarded (Rembold & Murphy, 1988a). Myoplasmic [Ca2+] was estimated using the photoprotein aequorin which was loaded intracellularly by reversible isosmotic hyperpermeabilization (Rembold & Murphy, 1988a). The aequorin-derived light was collected with a photomultiplier tube and the photon count per second (L) was divided by an estimate of the total [aequorin] (Lmax) which was calculated at each time point to correct for consumption. Data are reported as the change in log L/Lmax in which the resting log L/Lmax was subtracted from all subsequent log L/Lmax values (Rembold & Murphy, 1988a). Aequorin light signals were calibrated in CaEGTA buffers at 37 °C with [Mg2+] = 0-5 mm. The aequorin-loading procedure did not affect the time course of phosphorylation or stress (Rembold & Murphy, 1986, 1989). Because myosin phosphorylation assay is destructive, measurement of [Ca2+] and myosin phosphorylation were performed under identical conditions in different tissues. Myosin light chain phosphorylation in tissues frozen by immersion at -78 °C was determined by the method of Driska et al. (Driska, Aksoy & Murphy, 1981; Aksoy, Mrass, Kamm & Murphy, 1983). Phosphorylation is reported as moles Pi per mole 20 kD smooth muscle specific myosin light chain isoform (mol Pi/mol MLC). Isometric stress was calculated as force per cross-sectional area, which was estimated from measured length, weight, and a density of 1-050 g/cm3. The cyclic AMP assay is also destructive and was performed on the same set of tissues used for determination of phosphorylation levels (McDaniel, Rembold & Murphy, 1988). Cyclic AMP concentration was determined in tissues which were quickly frozen by immersion in a dry ice-acetone slurry (20 g-20 ml) at -78 'C. After warming, the tissues were homogenized in 100 mM-HCl, centrifuged at 14000 g for 20 min, and the resulting supernatant was examined by radioimmunoassay for cyclic AMP and cyclic GMP content on a Gammaflo (Squibb) (Brooker, Teresake & Price, 1976). RESULTS
Depolarization by replacement of extracellular Na+ with K+ induces intracellular ionic shifts and cellular swelling, which potentially could alter the [Ca21] sensitivity of phosphorylation. To evaluate the effect of ionic shifts or cellular swelling, I sought to contract smooth muscle in a manner similar to depolarization except without incubation in very high [K+]. Bay K 8644 is a dihydropyridine Ca21 channel agonist that increases the open probability of L-type Ca21 channels in the presence of only slightly elevated extracellular K+. Stimulation with 1 /aM-Bay K 8644 in 10 mM-KCl induced slow increases in [Ca2+], phosphorylation, and stress (Fig. 1). Subsequent stimulation with 109 mM-KCl markedly increased [Ca2+] and induced a maximal contraction. The [Ca21] sensitivity of phosphorylation observed with Bay K 8644-10 mM-KCl stimulation was similar to that induced by higher levels of depolarization (Fig. 1 B). These results suggest that the [Ca2+] sensitivity of phosphorylation was primarily associated with activation of L-type Ca2+ channels and was not dependent on extracellular [K+]. Therefore, high-KCl-induced ionic shifts or cellular swelling do not appear to be the etiology of depolarization induced decreases in the [Ca2+] sensitivity of phosphorylation.
C. M. REMBOLD
80
A
KCI
ID
1-8 Q 1.5 ~51.2
Bay K 8644 in KCI 500
~~~~~~~~~300
0.9 E 0°6
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_[100
-
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;
0.5
6.---
o
0.32
T
-.
0.0
420.0 0.2 0.4 0.6 0.8 1.0 1.2 log LiLmax change
100
200
300
400
[Ca2+e(nM) Fig. 1. A, the change in log L/Lmai(myoplasmic [Ca2]), myosin phosphorylation and active stress upon stimulation with 1 fSM-Bay K 8644 in 10 mM-KCL-containing PSS at 10 min and 109 mM-KCl at 40 mmn. Data are presented as means (continuous line) +±1 S.E.M. (dotted lines) with n = 4. B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [C!a2+] in tissues stimulated with contractile agonists (O, dotted line), depolarization (@, dashed line), or Bay K 8644 (-, continuous line). Values are means+ S.E.M. with n > 4 (some of the agonist and depolarization data are replotted from Rembold & Murphy, 1988a for comparison). Symbols without error bars in this and succeeding figures reflect means with S.E.M.s less than the size of the symbols.
Ca2+ SENSITIVITY OF MYOSIN PHOSPHORYLATION
81 Endothelin is a potent vasoconstrictor peptide recently isolated from endothelial cells (Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Goto & Masaki, 1988). One report suggests that contraction is dependent on extracellular CaCl2 and is not associated with increases in phosphatidylinositol metabolism in rat aortic EGTA, 0 Ca2+ Histamine
t
~~~E
Lo z. Endothelin
0
40
20
60
Time (min)
Fig. 2. The change in active stress upon stimulation with 01 /SM-endothelin-I (bottom tracing) and 10 /M-histamine (both tracings) in the absence of extracellular CaCl2 produced by incubation of tissues in 1 mM-EGTA with no added CaCl2. Similar results were observed in three other preparations and the peak histamine contraction was significantly less in those preparations stimulated with endothelin.
smooth muscle cells (Hirata, Yoshimi, Takata, Watanabe, Kumagai, Nakajima & Sakakibara, 1988). These investigators suggested that endothelin induces smooth muscle contraction primarily by binding to a specific receptor and increasing Ca2+ influx though voltage-dependent Ca2+ channels (i.e. similar to depolarization or Bay K 8644). Others report that endothelin can increase [3H]inositol trisphosphate and release intracellular Ca2+ stores (Marsden, Danthuluri, Brenner, Ballerman & Brock, 1989). Endothelin-I stimulation, after a 2 min lag period, produced a small prolonged contraction in the absence of extracellular CaCl2 in the swine carotid artery (Fig. 2, lower trace). Furthermore, the response to subsequent histamine stimulation was attenuated by prior endothelin stimulation, confirming that endothelin-I partially depleted the intracellular stores (Fig. 2). These results suggest that endothelin-I can release intracellular Ca2+ stores in swine carotid media, and must have effects beyond activation of L-type Ca2+ channels. In the presence of extracellular CaCl2, 041 /M-endothelin-I stimulation was associated with increased aequorin-estimated myoplasmic [Ca2+], myosin light chain phosphorylation, and development of near maximal stress (Fig. 3A). Addition of histamine induced only small increases in [Ca2+], phosphorylation and stress. Replacement of endothelin-I and histamine with 109 mM-KCl produced a large [Ca2+] increase, that was not associated with any further increase in stress. During the sustained endothelin-I contraction, the [Ca2+] sensitivity of phosphorylation was similar to that induced by contractile agonists, suggesting that sustained endothelinI stimulation functions in a manner similar to contractile agonists (Fig. 3B, E). However, during endothelin-I-induced stress development (the 1 and 3 min time points), endothelin induced lower phosphorylation than would be predicted by the
C. M. REMBOLD
82 A
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--Depolarization o Endothelin, sustained 0.7 -* Endothelin, transient 0-02.5
0
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100
200
300
400
[Ca2T1 (nM)
Fig. 3. A, the change in log LiLmax (myoplasmic [Ca2+]), myosin phosphorylation, and active stress upon stimulation with 0-1 /M-endothelin at 1Omin, addition of 102UMhistamine at 40 min, and replacement with 109 mM-KCl at 50 mmn. (Continuous line is the mean and dotted lines are + 1 S.E.M. at 10 s intervals with n = 4.) B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2+] in tissues stimulated with contractile agonists (0, dotted line), depolarization (-, dashed line), initial endothelin (1 and 3 m n, *), or sustained endothelin (10 and 30 mm, O). Values are means+ S.E.M. with n 4.d
C(a2+ SENSITIVITY OF MYOSIN1 PHOSPHOR YLATION
83
[Ca2"] increases. At these early time points, the [Ca2"] sensitivity of phosphorylation was intermediate between agonists and depolarization (Fig. 3B, *). These nonsteady-state results could potentially indicate that during the early phase of contraction, endothelin-I may contract smooth muscle partially by a depolarization1.2
a, 1.0 X 08
Histamine
KCI
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Fig. 4. The change in log LiLmax (myoplasmic [Ca2"]), myosin phosphorylation, and active stress upon stimulation with 10 ,uM-histamine at 10 min and addition of 109 mM-KCl with 10lM-histamine at 40 min. Data are presented as means (continuous line) ± 1 S.E.M. (dotted lines) with n = 4. The response to 109 mM-KCl depolarization only in a second set of tissue is shown as open squares (stimulated at 40 min).
like mechanism. Supporting this interpretation is the slow time course of endothelinI-induced force generation in the absence of extracellular CaCl2 (Fig. 3). EndothelinI-induced transplasmalemmal Ca21 influx via L-type Ca21 channels may precede endothelin dependent intracellular Ca2' release. Contractile agonists could potentially activate G proteins or protein kinase A, C or G, thereby possibly determining the [Ca21] sensitivity of phosphorylation. This possibility was tested by depolarizing tissues that were contracting in response to
C. M. REMBOLD
84 A
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[Ca2T] (nM) Fig. 5. A, the change in log L/Lmax (myoplasmic [Ca2+]), myosin phosphorylation and active stress upon stimulation with 1 /SM-histamine at 10 min and addition of 20 mM-KCl with 1 /SM-histamine at 20 mmn. Data are presented as means (continuous line) + 1 S.E.M. (dotted lines) with n = 4. The response to 1 ,uM-histamine alone in a second set of tissues is shown as open squares (stimulated at 10 min). B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2+] in tissues stimulated with contractile agonists (,0 dotted line), depolarization (A dashed line), or histamine plus depolarization (U, continuous line). Values are means+S.E.M. with n > 4.
Ca2+ SENSITIVITY OF MYOSIN PHOSPHORYLATION A
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mM
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ICa2+J (nM)
Fig. 6. A, the change in log LiLmax (myoplasmic [Ca2+]), myosin phosphorylation, and active stress upon stimulation with 2-5 mM-NaF at 10 min. A second set of tissues was stimulated with 10 mM-NaF at 60 min. Data are presented as means (continuous line) ± 1 S.E.M. (dotted lines) with n = 4. B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2l] in tissues stimulated with contractile agonists (0, dotted line), depolarization (@, dashed line), or NaF (both the 2-5 and 10 mm doses: *, continuous line). The effect of NaF on aequorin light production (see text) was corrected in this correlation by subtracting 0 05 from the 10 mM-NaF-induced log L/Lmax change values.
85r
C. M. REMBOLD
86 A
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NaF
KCI
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100)
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Fig. 7. For legend
see
facing
400
page.
Ca2+ SENSITIVITY OF MYOSIN PHOSPHOR YLA TION
87
agonist stimulation. If a contractile agonist-dependent mechanism regulated the [Ca2"] sensitivity of phosphorylation, then the contractile agonist should increase the [Ca21] sensitivity of phosphorylation above that observed with depolarization. Depolarization of tissues with 109 mM-KCl that had previously been stimulated with 10 /SM-histamine resulted in a large increase in [Ca2"], and a slight increase in myosin phosphorylation and stress (Fig. 4, continuous line). The [Ca2+] estimates after 5 min of combined histamine and depolarization were similar to that observed when a second set of tissues was depolarized with 109 mM-KCl alone (Fig. 4, LO). Similarly, depolarization of tissues with 20 mM-KCl that had been previously submaximally stimulated with 1 uM-histamine produced a doubling of light with only small slow increases in phosphorylation and stress (Fig. 5A: continuous line compared to control (1 fuM-histamine alone, LO). The 20 mM-KCl-induced increase in light was comparable to that induced by 10 /uM-histamine which produced a larger increase in phosphorylation (Fig. 4). Upon removal of histamine, the light signal remained high for the ensuing phosphorylation and stress values. The [Ca21] sensitivity of phosphorylation observed with the combination of agonists and depolarization was intermediate between agonists and depolarization (Fig. 5B, *). This result suggests that contractile agonists can partially increase the [Ca21] sensitivity of phosphorylation of depolarized tissues. Agonist-dependent release of intracellular Ca21 stores probably involves a G protein in receptor-dependent activation of phospholipase C at the plasma membrane (Litosch & Fain, 1986). The AIF4- ion is a known activator of G proteins (Sternweis & Gilman, 1982). This ion can be generated by the addition of sodium fluoride (NaF) to the bathing solution (since Al3+ is easily leached from the glass chamber). NaF is known to contract and increase Ca2+ influx in smooth muscle (Mirronneau, Mirroneau & Savineau, 1984; Kondo, Kozawa, Takasuki & Oiso, 1989; Zeng, Benishin & Pang, 1989). Additionally, NaF and GTP-y-S have similar effects on spontaneous transient outward currents in rabbit intestinal smooth muscle (Bolton & Lim, 1989). I first evaluated whether NaF affected the [Ca2+] sensitivity of aequorin. Aequorin calibration in CaEGTA buffers with [Mg2+] = 05 mm at [Ca2+] from 100 to 300 nm revealed that 2-5 mM-NaF increased the light signal by 0-018+0-002 units (thereby increasing resting [Ca2+] from 116 to 120 nM) and 10 mM-NaF by 0-049 + 0 004 units (increasing resting [Ca2+] from 116 to 126 nM). At the 2-5 mM-NaF concentration, these changes are very small, and suggest that any Fig. 7. A, the change in log L/Lmax (myoplasmic [Ca2+]), myosin phosphorylation, and active stress upon stimulation with 2-5 mM-NaF at 10 min, and addition of I ,UMhistamine between 30 and 50 min. A second set of tissues was stimulated with 2-5 mmNaF at 90 min, and depolarized with 25 mM-KCI between 110 and 130 min. Data are presented as means (continuous line) ±1 S.E.M. (dotted lines) with n = 4). B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2+] in tissues stimulated with contractile agonists (0, dotted line), depolarization (@, dashed lines), or 2-5 mM-NaF and either 1 or 10 /M-histamine (Ba, *, continuous line). The NaF plus 10 /tM-histamine data was collected 10 and 20 min after histamine stimulation after a 20 min pre-treatment with 2-5 mM-NaF. The bottom panel (b) is the same plot except for the combination of NaF and either 25 or 109 mM-KCl (Bb, *, continuous line). The NaF plus 109 mM-KCl data were collected 10 and 20 min after KCI depolarization after a 20 min pre-treatment with 2-5 mM-NaF. Values are means+ S.E.M. with n > 4.
C. M. REMBOLD NaF-induced changes in aequorin light signals should predominantly represent real changes in [Ca2+]j. Stimulation with 2-5 mM-NaF induced a slow increase in [Ca2+], phosphorylation, and stress (Fig. 6). A larger dose of NaF (10 mM) induced a larger increase in [Ca2+], 88
KCI PDB
Journal of Physiology (1990), 429, pp. 77-94 With 10 figures Printed in Great Britain
MODULATION OF THE ICa2+J SENSITIVITY OF MYOSIN PHOSPHORYLATION IN INTACT SWINE ARTERIAL SMOOTH MUSCLE
BY CHRISTOPHER M. REMBOLD From the Division of Cardiology, Department of Internal Medicine and Physiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA (Received 20 December 1989) SUMMARY
1. The [Ca2"] sensitivity of myosin light chain phosphorylation in vascular smooth muscle is dependent on the form of stimulation. Contractile agonist stimulation, when compared to high-KCl depolarization, is associated with an increase in [Ca2+] sensitivity of phosphorylation. I evaluated potential mechanisms for this stimulusspecific response by measuring aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and isometric stress in swine carotid media. 2. The relative [Ca2"] sensitivity of phosphorylation depended on the type of stimulus (ranked high to low sensitivity): contractile agonists (histamine, phenylephrine) = endothelin (sustained contraction) = combination of histamine and NaF > NaF alone = endothelin (initial contraction) = combination of histamine and depolarization = combination of NaF and depolarization > depolarization = Bay K 8644 = combination of depolarization and low-dose phorbol diester. 3. Activation of L-type Ca2+ channels with Bay K 8644 induced a [Ca2+] sensitivity of phosphorylation similar to depolarization, suggesting that any other effects of high KCI (such as cellular swelling) were not responsible for the low [Ca2+] sensitivity of phosphorylation. 4. The addition of either histamine or NaF (an activator of G proteins) to depolarized tissues produced similar increases in the [Ca2+] sensitivity of phosphorylation, suggesting that NaF (possibly by activation of a G protein) can mimic contractile agonist-induced increases in the [Ca2+] sensitivity of phosphorylation. 5. Phorbol dibutyrate enhanced the contractile effect of depolarization, and this enhancement was primarily caused by increases in [Ca2+] rather than an alteration in the [Ca2+] sensitivity of phosphorylation. 6. These data suggest that the [Ca2+] sensitivity of phosphorylation in smooth muscle may be regulated by agonists (possible by G protein activation); however, the role of protein kinase C activation or depolarization induced Ca2+ compartmentalization requires further study. INTRODUCTION
Myosin light chain phosphorylation appears to be the primary determinant of cross-bridge function in vascular smooth muscle (Rembold & Murphy, 1988 a; Hai & Murphy, 1989; Murphy, 1989). The identity of the second messenger(s) that MS 8155
C M. REMBOLD 78 determine myosin light chain phosphorylation is more controversial. Myoplasmic [Ca2+], through calmodulin-dependent myosin light chain kinase activation, has been proposed as the primary regulator of myosin phosphorylation (Kamm & Stull, 1985, 1989). In support, there is a unique relationship between myoplasmic [Ca2+] and myosin light chain phosphorylation in swine carotid artery stimulated with a variety of agonists (Rembold & Murphy, 1988a; Taylor, Bowman & Stull, 1989). However, the relationship between aequorin-estimated myoplasmic [Ca2+] and force (Morgan & Morgan, 1984) or between 45Ca2+ influx and force development rates (van Breemen, Lukeman, Leijten, Yamamoto & Loutzenhiser, 1986) can vary depending on the form of stimulus employed. Both groups of investigators found that depolarization required higher levels of [Ca2+] or Ca2+ influx to produce force than was observed with a-adrenergic stimulation. Similarly, in the swine carotid media, depolarization was associated with a decreased [Ca2+] sensitivity of phosphorylation when compared with agonist stimulation (Rembold & Murphy, 1988a). The relationship between phosphorylation and steady-state stress was not affected by the type of stimulus. Several mechanisms could affect the [Ca2+] sensitivity of phosphorylation: KCl depolarization per se may alter myosin kinase or phosphatase activity. Replacement of extracellular Na+ with K+ is known to induce intracellular ionic shifts and cellular swelling that potentially could affect these enzymes. Depolarization is felt to primarily induce smooth muscle contraction by opening L-type Ca2+ channels. In contrast, contractile agonist stimulation may activate other regulatory cascades. Specifically, contractile agonists, in addition to their effects on myoplasmic [Ca2+], could, through specific receptor binding, potentially activate G proteins and/or protein kinases C, A, or G. These regulatory systems could alter the [Ca2+] sensitivity of myosin light chain kinase or myosin light chain phosphatase, thereby altering the [Ca2+] sensitivity of phosphorylation. GTP and its non-hydrolysable analogues increased the [Ca2+] sensitivity of force generation in skinned smooth muscle, suggesting a role of G proteins in determining the [Ca2+] sensitivity of phosphorylation (Nishimura, Kolber & van Breemen, 1988; Kitazawa, Kobayashi, Horiuti, Somlyo & Somlyo, 1989). High-dose phorbol diesters have also been reported to induce smooth muscle contraction at lower myoplasmic [Ca2+] (Chatterjee & Tejada, 1986; Jiang & Morgan, 1987: Rembold & Murphy, 1988b). Cyclic adenosine monophosphate (cyclic AMP)-dependent phosphorylation of myosin kinase has been shown to decrease [Ca2+] sensitivity of myosin kinase in vitro (Adelstein, Conti & Hathaway, 1978; de Lanerolle, Nishikawa, Yost & Adelstein, 1984). The goal of this study was to evaluate whether (1) depolarization-induced ionic shifts or (2) agonist-specific actions, such as G protein or protein kinase C activation, could be responsible for the stimulus specificity of the [Ca2+] sensitivity of phosphorylation. METHODS
Swine common carotid medial tissues were obtained from a local slaughterhouse, dissected, and the optimal length for stress development was determined (Rembold & Murphy, 1988a). The intimal surface was mechanically rubbed to remove the endothelium. Agonist stimulation was
Ca2` SENSITIVITY OF MYOSIN PHOSPHORYLATION
79
performed by injecting an appropriate volume of 10 mM-stock histamine or phenylephrine into the tissue bath. Stock solutions of agonists were prepared daily. Physiological saline solution (PSS) contained (mM): NaCl, 140; KCl, 5; 3-[N-morpholino]propanesulphonic acid (MOPS), 2; CaCl2, 1-6; MgCl2, 1-2; Na2HPO4, 1-2; D-glucose, 5-6 (pH adjusted to 7-4 at 37 °C). Changes in extracellular [K+] were accomplished by changing the bathing solution to a physiological saline in which KCl was substituted stoichiometrically for NaCl. Changes in extracellular [Ca2+] were performed by replacing the bathing solution twice with Ca2+free PSS which contained 1 mM-ethyleneglycol bis(,8-aminoethylether)NN,N',NV'-tetraacetic acid (EGTA) and no added CaCl2. Tissues that did not produce a stress greater than 1-0 x 10t" N/M2 after 109 mM-KCl depolarization or 10 ,tM-histamine stimulation were discarded (Rembold & Murphy, 1988a). Myoplasmic [Ca2+] was estimated using the photoprotein aequorin which was loaded intracellularly by reversible isosmotic hyperpermeabilization (Rembold & Murphy, 1988a). The aequorin-derived light was collected with a photomultiplier tube and the photon count per second (L) was divided by an estimate of the total [aequorin] (Lmax) which was calculated at each time point to correct for consumption. Data are reported as the change in log L/Lmax in which the resting log L/Lmax was subtracted from all subsequent log L/Lmax values (Rembold & Murphy, 1988a). Aequorin light signals were calibrated in CaEGTA buffers at 37 °C with [Mg2+] = 0-5 mm. The aequorin-loading procedure did not affect the time course of phosphorylation or stress (Rembold & Murphy, 1986, 1989). Because myosin phosphorylation assay is destructive, measurement of [Ca2+] and myosin phosphorylation were performed under identical conditions in different tissues. Myosin light chain phosphorylation in tissues frozen by immersion at -78 °C was determined by the method of Driska et al. (Driska, Aksoy & Murphy, 1981; Aksoy, Mrass, Kamm & Murphy, 1983). Phosphorylation is reported as moles Pi per mole 20 kD smooth muscle specific myosin light chain isoform (mol Pi/mol MLC). Isometric stress was calculated as force per cross-sectional area, which was estimated from measured length, weight, and a density of 1-050 g/cm3. The cyclic AMP assay is also destructive and was performed on the same set of tissues used for determination of phosphorylation levels (McDaniel, Rembold & Murphy, 1988). Cyclic AMP concentration was determined in tissues which were quickly frozen by immersion in a dry ice-acetone slurry (20 g-20 ml) at -78 'C. After warming, the tissues were homogenized in 100 mM-HCl, centrifuged at 14000 g for 20 min, and the resulting supernatant was examined by radioimmunoassay for cyclic AMP and cyclic GMP content on a Gammaflo (Squibb) (Brooker, Teresake & Price, 1976). RESULTS
Depolarization by replacement of extracellular Na+ with K+ induces intracellular ionic shifts and cellular swelling, which potentially could alter the [Ca21] sensitivity of phosphorylation. To evaluate the effect of ionic shifts or cellular swelling, I sought to contract smooth muscle in a manner similar to depolarization except without incubation in very high [K+]. Bay K 8644 is a dihydropyridine Ca21 channel agonist that increases the open probability of L-type Ca21 channels in the presence of only slightly elevated extracellular K+. Stimulation with 1 /aM-Bay K 8644 in 10 mM-KCl induced slow increases in [Ca2+], phosphorylation, and stress (Fig. 1). Subsequent stimulation with 109 mM-KCl markedly increased [Ca2+] and induced a maximal contraction. The [Ca21] sensitivity of phosphorylation observed with Bay K 8644-10 mM-KCl stimulation was similar to that induced by higher levels of depolarization (Fig. 1 B). These results suggest that the [Ca2+] sensitivity of phosphorylation was primarily associated with activation of L-type Ca2+ channels and was not dependent on extracellular [K+]. Therefore, high-KCl-induced ionic shifts or cellular swelling do not appear to be the etiology of depolarization induced decreases in the [Ca2+] sensitivity of phosphorylation.
C. M. REMBOLD
80
A
KCI
ID
1-8 Q 1.5 ~51.2
Bay K 8644 in KCI 500
~~~~~~~~~300
0.9 E 0°6
l
_[100
-
03
0.6
o
. 0J -0.4 0-62I
0. 2 0
E 2.
a
E: 0.2 NE2-
4-20
10
0--
2 o
0
0
5
0a0
~0. 0-7
_~ Bay K 8644 in 10 mM-KCI
;
0.5
6.---
o
0.32
T
-.
0.0
420.0 0.2 0.4 0.6 0.8 1.0 1.2 log LiLmax change
100
200
300
400
[Ca2+e(nM) Fig. 1. A, the change in log L/Lmai(myoplasmic [Ca2]), myosin phosphorylation and active stress upon stimulation with 1 fSM-Bay K 8644 in 10 mM-KCL-containing PSS at 10 min and 109 mM-KCl at 40 mmn. Data are presented as means (continuous line) +±1 S.E.M. (dotted lines) with n = 4. B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [C!a2+] in tissues stimulated with contractile agonists (O, dotted line), depolarization (@, dashed line), or Bay K 8644 (-, continuous line). Values are means+ S.E.M. with n > 4 (some of the agonist and depolarization data are replotted from Rembold & Murphy, 1988a for comparison). Symbols without error bars in this and succeeding figures reflect means with S.E.M.s less than the size of the symbols.
Ca2+ SENSITIVITY OF MYOSIN PHOSPHORYLATION
81 Endothelin is a potent vasoconstrictor peptide recently isolated from endothelial cells (Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Goto & Masaki, 1988). One report suggests that contraction is dependent on extracellular CaCl2 and is not associated with increases in phosphatidylinositol metabolism in rat aortic EGTA, 0 Ca2+ Histamine
t
~~~E
Lo z. Endothelin
0
40
20
60
Time (min)
Fig. 2. The change in active stress upon stimulation with 01 /SM-endothelin-I (bottom tracing) and 10 /M-histamine (both tracings) in the absence of extracellular CaCl2 produced by incubation of tissues in 1 mM-EGTA with no added CaCl2. Similar results were observed in three other preparations and the peak histamine contraction was significantly less in those preparations stimulated with endothelin.
smooth muscle cells (Hirata, Yoshimi, Takata, Watanabe, Kumagai, Nakajima & Sakakibara, 1988). These investigators suggested that endothelin induces smooth muscle contraction primarily by binding to a specific receptor and increasing Ca2+ influx though voltage-dependent Ca2+ channels (i.e. similar to depolarization or Bay K 8644). Others report that endothelin can increase [3H]inositol trisphosphate and release intracellular Ca2+ stores (Marsden, Danthuluri, Brenner, Ballerman & Brock, 1989). Endothelin-I stimulation, after a 2 min lag period, produced a small prolonged contraction in the absence of extracellular CaCl2 in the swine carotid artery (Fig. 2, lower trace). Furthermore, the response to subsequent histamine stimulation was attenuated by prior endothelin stimulation, confirming that endothelin-I partially depleted the intracellular stores (Fig. 2). These results suggest that endothelin-I can release intracellular Ca2+ stores in swine carotid media, and must have effects beyond activation of L-type Ca2+ channels. In the presence of extracellular CaCl2, 041 /M-endothelin-I stimulation was associated with increased aequorin-estimated myoplasmic [Ca2+], myosin light chain phosphorylation, and development of near maximal stress (Fig. 3A). Addition of histamine induced only small increases in [Ca2+], phosphorylation and stress. Replacement of endothelin-I and histamine with 109 mM-KCl produced a large [Ca2+] increase, that was not associated with any further increase in stress. During the sustained endothelin-I contraction, the [Ca2+] sensitivity of phosphorylation was similar to that induced by contractile agonists, suggesting that sustained endothelinI stimulation functions in a manner similar to contractile agonists (Fig. 3B, E). However, during endothelin-I-induced stress development (the 1 and 3 min time points), endothelin induced lower phosphorylation than would be predicted by the
C. M. REMBOLD
82 A
Endothelin
O 0.8 CD 0.6
KCI Histamine
300
0.42 /
-c2 0.2
0)~~~~~~~~~~~~~~~~~~~~~~~~C
o -0. 0-64
00.0-
0
0E0
cQC 0.260
0
B
10
20
o 30 40 Time (min)
° 50
60
o- Agonists
--Depolarization o Endothelin, sustained 0.7 -* Endothelin, transient 0-02.5
0
LO 0.4 o0 0
100
200
300
400
[Ca2T1 (nM)
Fig. 3. A, the change in log LiLmax (myoplasmic [Ca2+]), myosin phosphorylation, and active stress upon stimulation with 0-1 /M-endothelin at 1Omin, addition of 102UMhistamine at 40 min, and replacement with 109 mM-KCl at 50 mmn. (Continuous line is the mean and dotted lines are + 1 S.E.M. at 10 s intervals with n = 4.) B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2+] in tissues stimulated with contractile agonists (0, dotted line), depolarization (-, dashed line), initial endothelin (1 and 3 m n, *), or sustained endothelin (10 and 30 mm, O). Values are means+ S.E.M. with n 4.d
C(a2+ SENSITIVITY OF MYOSIN1 PHOSPHOR YLATION
83
[Ca2"] increases. At these early time points, the [Ca2"] sensitivity of phosphorylation was intermediate between agonists and depolarization (Fig. 3B, *). These nonsteady-state results could potentially indicate that during the early phase of contraction, endothelin-I may contract smooth muscle partially by a depolarization1.2
a, 1.0 X 08
Histamine
KCI
- 400
0
-
3002
x 0.6 - 200
0.2 E 0.0
-
-0.2
_
. 100
0.7 o D- 0.6 .2_0.5 >o 0.4 0 E 0.3 en O o3 0.2 0 E 0-1 _
,
aC
0.0
C;-
2.0
E z LO
0
x
Un
m
n
a) 41
LO
Time (min)
Fig. 4. The change in log LiLmax (myoplasmic [Ca2"]), myosin phosphorylation, and active stress upon stimulation with 10 ,uM-histamine at 10 min and addition of 109 mM-KCl with 10lM-histamine at 40 min. Data are presented as means (continuous line) ± 1 S.E.M. (dotted lines) with n = 4. The response to 109 mM-KCl depolarization only in a second set of tissue is shown as open squares (stimulated at 40 min).
like mechanism. Supporting this interpretation is the slow time course of endothelinI-induced force generation in the absence of extracellular CaCl2 (Fig. 3). EndothelinI-induced transplasmalemmal Ca21 influx via L-type Ca21 channels may precede endothelin dependent intracellular Ca2' release. Contractile agonists could potentially activate G proteins or protein kinase A, C or G, thereby possibly determining the [Ca21] sensitivity of phosphorylation. This possibility was tested by depolarizing tissues that were contracting in response to
C. M. REMBOLD
84 A
Histamine KCI
0.5 C
-J0.1
100 .2 -0 0.10 EX0.3
0
oE 0.2 ~ ~~
0
°
U-
~
~
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10T6 20
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30 40 Time (min)
60
10
12
-
-
T
00.7.0- o Agonists Depolarization c0.6 --.--_-Agonists+KCIi.0 408 . -0.2
lo
-50.4
50
0./@
100
--_
9
200
Tc -
X
300
400
[Ca2T] (nM) Fig. 5. A, the change in log L/Lmax (myoplasmic [Ca2+]), myosin phosphorylation and active stress upon stimulation with 1 /SM-histamine at 10 min and addition of 20 mM-KCl with 1 /SM-histamine at 20 mmn. Data are presented as means (continuous line) + 1 S.E.M. (dotted lines) with n = 4. The response to 1 ,uM-histamine alone in a second set of tissues is shown as open squares (stimulated at 10 min). B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2+] in tissues stimulated with contractile agonists (,0 dotted line), depolarization (A dashed line), or histamine plus depolarization (U, continuous line). Values are means+S.E.M. with n > 4.
Ca2+ SENSITIVITY OF MYOSIN PHOSPHORYLATION A
NaF, 2.5
mM
NaF, 10
mM
, 0.6 0.5 x 0.4X E 0.
~0.2 0.1
x
-
.9 -J
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I
o E 2.0Xa 01
0mN
z
~~
0.3-
0.2-
, 1.00) 0.0 0
*.
W.-
20 30 40 50 60 70 80 90 Time (min)
10
B
0-7 .o Agonists 0.6 - ..-0- Depolarization NaF 0.5 .2CL, E 0.42E 03-o E 0-2 -
.-T *---
4
I/T
sL _
0-5 020-1
T
.
-06
0-0 4
-0-2
0.0 100)
0.2
0.4 0.6 0-8 1-0 log L/Lmax change
200
300
1.2
400
ICa2+J (nM)
Fig. 6. A, the change in log LiLmax (myoplasmic [Ca2+]), myosin phosphorylation, and active stress upon stimulation with 2-5 mM-NaF at 10 min. A second set of tissues was stimulated with 10 mM-NaF at 60 min. Data are presented as means (continuous line) ± 1 S.E.M. (dotted lines) with n = 4. B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2l] in tissues stimulated with contractile agonists (0, dotted line), depolarization (@, dashed line), or NaF (both the 2-5 and 10 mm doses: *, continuous line). The effect of NaF on aequorin light production (see text) was corrected in this correlation by subtracting 0 05 from the 10 mM-NaF-induced log L/Lmax change values.
85r
C. M. REMBOLD
86 A
NaF
NaF
KCI
His
c, 0.5= 0.4
200
i C4 +
0-0-1 0.0 100
C , 0.40
X-
2 0-3 -
o
E 0.2-
L
QC0-01
/0
/0
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(N 2.0..........
-Z
1-
o
'r.
x
......
1-0
1-
0.0
cn
20
0
80 ioo 120 140 160 Time (min)
60
40
B
0*7 a 0.6-
o0.
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- se-4
If--
06 -
I / lK
T
i
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o E 0.70
A
-.^
0- Agonists -*- Depolarization A NaF, 2.5 mM --- NaF + histamine
-, ,' .t
b 04
a-0-c E 0.5 -
0
0-3 0-2 0.1 -
°0" Agonists -.- Depolarization A NaF, 2.5 mM -_- NaF + KCI
_
nn
-0.2! 0.0 0.2 0.4 0.6 0.8 1.0
1-2
log L/Lmax change
100)
300 [Ca2+J (nM) 200
Fig. 7. For legend
see
facing
400
page.
Ca2+ SENSITIVITY OF MYOSIN PHOSPHOR YLA TION
87
agonist stimulation. If a contractile agonist-dependent mechanism regulated the [Ca2"] sensitivity of phosphorylation, then the contractile agonist should increase the [Ca21] sensitivity of phosphorylation above that observed with depolarization. Depolarization of tissues with 109 mM-KCl that had previously been stimulated with 10 /SM-histamine resulted in a large increase in [Ca2"], and a slight increase in myosin phosphorylation and stress (Fig. 4, continuous line). The [Ca2+] estimates after 5 min of combined histamine and depolarization were similar to that observed when a second set of tissues was depolarized with 109 mM-KCl alone (Fig. 4, LO). Similarly, depolarization of tissues with 20 mM-KCl that had been previously submaximally stimulated with 1 uM-histamine produced a doubling of light with only small slow increases in phosphorylation and stress (Fig. 5A: continuous line compared to control (1 fuM-histamine alone, LO). The 20 mM-KCl-induced increase in light was comparable to that induced by 10 /uM-histamine which produced a larger increase in phosphorylation (Fig. 4). Upon removal of histamine, the light signal remained high for the ensuing phosphorylation and stress values. The [Ca21] sensitivity of phosphorylation observed with the combination of agonists and depolarization was intermediate between agonists and depolarization (Fig. 5B, *). This result suggests that contractile agonists can partially increase the [Ca21] sensitivity of phosphorylation of depolarized tissues. Agonist-dependent release of intracellular Ca21 stores probably involves a G protein in receptor-dependent activation of phospholipase C at the plasma membrane (Litosch & Fain, 1986). The AIF4- ion is a known activator of G proteins (Sternweis & Gilman, 1982). This ion can be generated by the addition of sodium fluoride (NaF) to the bathing solution (since Al3+ is easily leached from the glass chamber). NaF is known to contract and increase Ca2+ influx in smooth muscle (Mirronneau, Mirroneau & Savineau, 1984; Kondo, Kozawa, Takasuki & Oiso, 1989; Zeng, Benishin & Pang, 1989). Additionally, NaF and GTP-y-S have similar effects on spontaneous transient outward currents in rabbit intestinal smooth muscle (Bolton & Lim, 1989). I first evaluated whether NaF affected the [Ca2+] sensitivity of aequorin. Aequorin calibration in CaEGTA buffers with [Mg2+] = 05 mm at [Ca2+] from 100 to 300 nm revealed that 2-5 mM-NaF increased the light signal by 0-018+0-002 units (thereby increasing resting [Ca2+] from 116 to 120 nM) and 10 mM-NaF by 0-049 + 0 004 units (increasing resting [Ca2+] from 116 to 126 nM). At the 2-5 mM-NaF concentration, these changes are very small, and suggest that any Fig. 7. A, the change in log L/Lmax (myoplasmic [Ca2+]), myosin phosphorylation, and active stress upon stimulation with 2-5 mM-NaF at 10 min, and addition of I ,UMhistamine between 30 and 50 min. A second set of tissues was stimulated with 2-5 mmNaF at 90 min, and depolarized with 25 mM-KCI between 110 and 130 min. Data are presented as means (continuous line) ±1 S.E.M. (dotted lines) with n = 4). B, the dependence of myosin phosphorylation on the aequorin-estimated myoplasmic [Ca2+] in tissues stimulated with contractile agonists (0, dotted line), depolarization (@, dashed lines), or 2-5 mM-NaF and either 1 or 10 /M-histamine (Ba, *, continuous line). The NaF plus 10 /tM-histamine data was collected 10 and 20 min after histamine stimulation after a 20 min pre-treatment with 2-5 mM-NaF. The bottom panel (b) is the same plot except for the combination of NaF and either 25 or 109 mM-KCl (Bb, *, continuous line). The NaF plus 109 mM-KCl data were collected 10 and 20 min after KCI depolarization after a 20 min pre-treatment with 2-5 mM-NaF. Values are means+ S.E.M. with n > 4.
C. M. REMBOLD NaF-induced changes in aequorin light signals should predominantly represent real changes in [Ca2+]j. Stimulation with 2-5 mM-NaF induced a slow increase in [Ca2+], phosphorylation, and stress (Fig. 6). A larger dose of NaF (10 mM) induced a larger increase in [Ca2+], 88
KCI PDB
