Vol.
172,
November
No.
BIOCHEMICAL
3, 1990
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1990
DESENSITIZATION FORCE
AND
AND MYOSIN CYTOPLASMIC
Toshio Department
Received
AND
October
MUSCARINIC
LIGHT
CHAIN
RE-SENSITIZATION PHOSPHORYLATION
CA2+ IN SMOOTH
Kitazawa
and Andrew
OF TO
MUSCLE
P. Somlyo
of Physiology, University of Virginia School Charlottesville, Virginia 22908 2,
1291-1297
of Medicine
1990
Summary: In a-toxin-permeabilized guinea-pig ileum smooth muscle, a step increase in Ca2+ caused a rapid rise in force and myosin light chain (LC20) phosphorylation, followed by their spontaneous decline to a low steady level even though Ca2+ remained constant. Carbachol resensitized the muscles to Ca2+, causing an increase in both the steady state force and LC2o phosphorylation at constant Ca2+. In p-escin permeabilized preparations, calmodulin and okadaic acid converted the phasic responses to Caz+ to more tonic ones. We conclude that Ca2+-sensitivity of force is modulated through changes in LC2o kinaselphosphatase activity ratio by Ca2+ itself (desensitization) and by agonists (sensitization). ‘Q1990Academis PTC55.1:7c. Smooth muscles respond to depolarization with high K+ by a relatively rapid contraction that, in phasic smooth muscles, declines within minutes to an intermediate level, while in tonic smooth muscles it is followed by sustained increases in force for up to 30 minutes (1,2). However, these differences can not be ascribed to the time courses of the cytoplasmic Ca2+ transients that are similar in the two types of smooth muscle; cytoplasmic Ca2+ does not decline any more during the phasic component of contraction in the phasic (ileum) than in the tonic (pulmonary artery) smooth muscle (2). These results suggested that phasic smooth muscles are desensitized to Ca2+ with time, an hypothesis also supported by the rapid (within 30 seconds) dephosphorylation of 20K Da-myosin light chain (LC20) of intact ileum smooth muscle following activation (1). Further evidence indicating that the decline in Ca2+-sensitivity was not an artifact of the fluorescent Ca2+-indicator was provided in preliminary experiments (3) showing that a Abbreviations: LC20, 20K-Da myosin bis( @aminoethyl ether) N,N’-tetraacetic GDPPS, guanosine 5’-0-(P-thiodiphosphate).
light chain; EGTA, ethylene glycol acid; SR, sarcoplasmic reticulum;
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
phenomenon similar to desensitization of force to Ca2+ could also be observed in permeabilized preparations in which cytoplasmic Ca2+ was clamped with high concentrations of EGTA. The purpose of the present study was to further characterize the changes in Ca2+-sensitivity in permeabilized smooth muscle and to determine whether the decline in force could be ascribed to a similar decline in LC20 phosphorylation at constant Ca2+. In addition, we also wished to determine whether the force and LC20 phosphorylation response of such permeabilized smooth muscles can be resensitized to Ca2+ with a muscarinic agonist.
Methods Details of preparations of ileum and pulmonary artery strips, permeabilization with Staphylococcal a-toxin and P-escin to yield permeabilized preparations that retain responses to agonists, depletion of intracellular Ca2+ stores (sarcoplasmic reticulum; SR) with A23187, and measurements of LC20 phosphorylation with colloidal gold staining of 2dimensional isoelectric focusing and sodium dodecyl sulfate gels have been published (4,5,6). Attention was taken to use small (approximately 20-30 ug wet weight) strips of both guinea-pig ileum and rabbit pulmonary artery to assure satisfactory permeabilization. Force and LC20 phosphorylation were measured in the same strips.
Results
and
Discussion
Desensitization of permeabilized ileum smooth muscle to Ca2 + A step increase in Ca2+ to pCa 6.3 produces different contractile responses in the different types of smooth muscle permeabilized with Staphylococcal a-toxin: a rapid rise in force, followed by a spontaneous decline to a low steady state level, in the very phasic ileum smooth muscle (Fig. lA), and a monotonic increase in force in the (tonic) pulmonary artery smooth muscle (not shown, see 1,2). Since Ca2+ was buffered with 1OmM EGTA and all sources of intracellular Ca2+ were removed with A23187, the phasic contraction in ileum could not be due to a transient increase in cytoplasmic Ca2+. Furthermore, ryanodine (10 PM) or inositol 1,4,5-trisphosphate (100 PM), agents that release Ca 2+ from the SR, did not affect the time course of this phasic contraction (not shown). The size of the pores created by atoxin in the plasma membrane is so small that intrinsic, important soluble proteins, such as calmodulin, can not leak out of the cells, although CaEGTA penetrates into cytosol and can clamp free cytoplasmic Ca2+ (4,7). Preincubation of the strips in a very low concentration (0.1 mM) of EGTA, instead of 10 mM, prior to the pCa6.3 (10mM EGTA step), increased the peak of the phasic contraction, but not the steady state level (not shown). 1292
Vol.
172,
No.
BIOCHEMICAL
3, 1990
PCs
>a-,-6.3+>8-,-7-H
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
8+6.7+6.5~.4+6.3->8~6.3-f>8
A2
PG.2 6.3
10
I I 1'
15'
15'
5
Free
Ca
ion
(M)
Fig. 1. A. Properties of the phasic contractile response of permeabilized ileum smooth muscle to constant Ca*+. a-toxin-permeabilized, A23187treated strips from ileum were first incubated in the relaxing solution (pCa HI) for IO min and activated with C$+(pCa 6.3)-containing solution. Ca2+ concentrations were buffered with 10 mM EGTA. Note the transient contractile response of the phasic smooth muscle to a constant Ca2+ stimulus. B. Ca2+-sensitivity of the peak (0) and steady state (*) component of phasic contractions in permeabilized ileum smooth muscle. Before addition of Ca2+, the strips were first incubated in the relaxing solution containing 10 mM EGTA for 8 min and then 0.1 mM EGTA for 2 min. Force was recorded until Ca*+-activated contraction reached a steady state tonic level. Force was normalized to the peak force at pCa 4.5. Error was expressed as +S.D. n=4.
When Ca2+ was cumulatively increased (Fig. ZAl) or repeatedly applied at shorter intervals (Fig. lA2), the peak of the phasic contraction was greatly
reduced, but the tonic level was not significantly
half time
affected.
The
of the spontaneous decline in force at pCa 6.3 was 2-3 min. The decline in the phasic component of contractions induced by Ca2+ was not due to “run-down” of the preparations, as the decline was reversible (Fig. 1A) and recovered in Ca2+-free solution with a half time of about 1 min; 1293
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
complete recovery time was about 10 min (Fig. lA2). Phasic contractions were seen even at pCa 4.5. Fig. 1B shows the comparison between the Ca2+sensitivities of the peak and the steady state of phasic contractions in the ileum smooth muscle. The steady state Ca2+- sensitivity was similar to that when Ca2+ was cumulatively increased. In the tonic pulmonary artery smooth muscle, repeated applications of Ca2+ at shorter intervals or cumulative increases in Ca2+ resulted in little or no decrease in contractility. To exclude the possibility that phasic contractions at constant Ca2+ are due to nonlinear end compliance or inhomogeneity of contractile regions, in some experiments small particles of carbon were attached to the surface of the muscle strips to detect whether inhomogeneous shortening occurs during phasic contractions: there was no evidence of such inhomogeneity. Changing the muscle length from 0.8 to 1.9 times slack length also did not significantly affect the time course of phasic contractions. Therefore, we believe that the phasic contractions are due to the intrinsically phasic reactions of the regulatory/contractile apparatus in phasic type of smooth muscles. We used P-escin-permeabilized smooth muscle to change the on- and off-rates of LC20 phosphorylation by introducing calmodulin or a phosphatase inhibitor, okadaic acid (8). p-escin, a saponin ester, permeabilizes cell membranes to much higher molecular weight solutes than does o-toxin, yet, similarly to a-toxin and unlike crude saponin, retains receptor-coupled responses, such as Ca release from the SR and Ca2+-sensitization by agonists (5). The response of /3-escin-permeabilized ileum to Ca2+ was also very phasic, like that of the a-toxin-permeabilized preparations, and it was greatly affected by extrinsic calmodulin and by okadaic acid; both activation of LC20 kinase with calmodulin and inhibition of protein phosphatase with okadaic acid converted the phasic responses to Ca2+ to more tonic ones and increased their amplitude (Fig. 2). These results suggested that rapid LC20 dephosphorylation is a potential mechanism responsible for the phasic contractile properties of some smooth muscles.
Re-sensitization
of force
to Ca 2+ by carbachol
and by histamine
The contractile response of a-toxin permeabilized vascular smooth muscle to constant Ca2+ is markedly enhanced by al -adrenergic agonists (4,9) and by a thromboxane analogue, U46619 (10). GDPbS blocks both phenylephrineand U466 19-induced potentiation of contraction, suggesting that both responses are coupled by G-protein (4,lO). Fig. 3 shows that both a muscarinic agonist, carbachol, and histamine can “resensitize” contraction at constant CaZ+, even when the 1294
Vol.
BIOCHEMICAL
172, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
calmodulin I
I 8 FM
pCa
6.3
t
okadaic ~PM
acid
4 5 min
-
Fig. 2. Effects of calmodulin and okadaic acid on the time course of contraction activated by pCa6.3 in P-escin-permeabilized ileum smooth muscle. Muscles permeabilized with P-escin and treated with A23187 were incubated in the 1 mM EGTA-containing relaxing solution, foIlowed by a step increase in Ca2+ to pCa 6.3, buffered with 10 mM EGTA . Note that the initial contractile response is phasic, like in the a-toxin permeabilized preparations. Following incubation with calmodulin and okadaic acid, to, respectively, increase LC20 kinase and inhibit LC20 phosphatase, the amplitude of the contractile response to pCa 6.3 is increased, and the rapid phase of contraction is followed by a high, sustained level of force. regulatory/contractile apparatus is desensitized to Ca2+ by prolonged incubation in Ca2+-containing solution. U46619 had minimal and phenylephrine no potentiating effect on ileum, reflecting the absence of the respective excitatory receptors to these agonists. GDPPS (1mM) reversibly inhibited carbachol-induced potentiation, but not the Ca2+activated contraction (not shown). The agonist-potentiated contractions were somewhat phasic, but less so than the initial responses to Ca2+, and the amplitude of the phasic component varied from preparation to preparation. Desensitization and re-sensitization of LC20 phosphorylation to Ca2+ The primary mechanism regulating the contraction/relaxation cycle in smooth muscle is phosphorylation/dephosphorylation of LC20 by, respectively, LC20 kinase and LC20 phosphatases (11,12), although the possibility of thin filament regulated mechanisms involving proteins, such as caldesmon and calponin, has been considered (13). Therefore, it was necessary to determine whether or not the desensitization and resensitization of force to Ca2+ are correlated, respectively, with dephosphorylation and re-phosphorylation of LC20. Table 1 shows the summary of simultaneous measurements of LC20 phosphorylation and force in permeabilized ileum smooth muscle under 1295
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
U46619
PE
Carb
IOOlrM
IO@M
’
L
I
t>8.
Fie. 3. Re-sensitization of desensitized force by agonists and different efficacies of various agonists on sensitization in the a-toxin permeabilized ileum.
various conditions. At resting state (pCa >S), llC2.0 % of total LC20 kS.E.M. of LC20 was phosphorylated, although no significant force was detectable. At the peak of contraction induced by pCa 6.3, LC20 phosphorylation was significantly increased to 33k2.8 %, and then significantly decreased to 25k1.8 % of total LC20 during the steady state contraction (10 min after Ca2+ was added), while force first increased to 36&2.7% and then decreased to 5+0.9% of the maximum force at pCa 5. The muscarinic agonist, carbachol (100pM) significantly increased the amount of phosphorylated LC20 from the desensitized control value (25+1.8%) at pCa 6.3, to 34*2.5% of total LC20, while force was increased from the desensitized sustained contractions (5*0.9%) to 33+5.4% of the maximum force at pCa 5. In conclusion, contractile desensitization to Ca2+ by prolonged incubation in the Ca2+-containing solution and re-sensitization by agonist
Table 1. Ca2+-induced desensitization and agonist-induced sensitization of LC20 phosphorylation and force to Ca2+. Phosphorylation is expressed as % of total LC20 +S.E.M. and force as % of maximum at pCa 5 fS.E.M. Conditions
Force (%)
Phosphorylation (%)
Rest (pCa>8; n=13)
0
11 f 2.0
Peak (pCa 6.3; n=7)
36 f 2.7
33 + 2.8
5 f 0.9
25+ 1.8
33 + 5.4
34 f 2.5
P
172,
November
No.
BIOCHEMICAL
3, 1990
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1990
DESENSITIZATION FORCE
AND
AND MYOSIN CYTOPLASMIC
Toshio Department
Received
AND
October
MUSCARINIC
LIGHT
CHAIN
RE-SENSITIZATION PHOSPHORYLATION
CA2+ IN SMOOTH
Kitazawa
and Andrew
OF TO
MUSCLE
P. Somlyo
of Physiology, University of Virginia School Charlottesville, Virginia 22908 2,
1291-1297
of Medicine
1990
Summary: In a-toxin-permeabilized guinea-pig ileum smooth muscle, a step increase in Ca2+ caused a rapid rise in force and myosin light chain (LC20) phosphorylation, followed by their spontaneous decline to a low steady level even though Ca2+ remained constant. Carbachol resensitized the muscles to Ca2+, causing an increase in both the steady state force and LC2o phosphorylation at constant Ca2+. In p-escin permeabilized preparations, calmodulin and okadaic acid converted the phasic responses to Caz+ to more tonic ones. We conclude that Ca2+-sensitivity of force is modulated through changes in LC2o kinaselphosphatase activity ratio by Ca2+ itself (desensitization) and by agonists (sensitization). ‘Q1990Academis PTC55.1:7c. Smooth muscles respond to depolarization with high K+ by a relatively rapid contraction that, in phasic smooth muscles, declines within minutes to an intermediate level, while in tonic smooth muscles it is followed by sustained increases in force for up to 30 minutes (1,2). However, these differences can not be ascribed to the time courses of the cytoplasmic Ca2+ transients that are similar in the two types of smooth muscle; cytoplasmic Ca2+ does not decline any more during the phasic component of contraction in the phasic (ileum) than in the tonic (pulmonary artery) smooth muscle (2). These results suggested that phasic smooth muscles are desensitized to Ca2+ with time, an hypothesis also supported by the rapid (within 30 seconds) dephosphorylation of 20K Da-myosin light chain (LC20) of intact ileum smooth muscle following activation (1). Further evidence indicating that the decline in Ca2+-sensitivity was not an artifact of the fluorescent Ca2+-indicator was provided in preliminary experiments (3) showing that a Abbreviations: LC20, 20K-Da myosin bis( @aminoethyl ether) N,N’-tetraacetic GDPPS, guanosine 5’-0-(P-thiodiphosphate).
light chain; EGTA, ethylene glycol acid; SR, sarcoplasmic reticulum;
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
phenomenon similar to desensitization of force to Ca2+ could also be observed in permeabilized preparations in which cytoplasmic Ca2+ was clamped with high concentrations of EGTA. The purpose of the present study was to further characterize the changes in Ca2+-sensitivity in permeabilized smooth muscle and to determine whether the decline in force could be ascribed to a similar decline in LC20 phosphorylation at constant Ca2+. In addition, we also wished to determine whether the force and LC20 phosphorylation response of such permeabilized smooth muscles can be resensitized to Ca2+ with a muscarinic agonist.
Methods Details of preparations of ileum and pulmonary artery strips, permeabilization with Staphylococcal a-toxin and P-escin to yield permeabilized preparations that retain responses to agonists, depletion of intracellular Ca2+ stores (sarcoplasmic reticulum; SR) with A23187, and measurements of LC20 phosphorylation with colloidal gold staining of 2dimensional isoelectric focusing and sodium dodecyl sulfate gels have been published (4,5,6). Attention was taken to use small (approximately 20-30 ug wet weight) strips of both guinea-pig ileum and rabbit pulmonary artery to assure satisfactory permeabilization. Force and LC20 phosphorylation were measured in the same strips.
Results
and
Discussion
Desensitization of permeabilized ileum smooth muscle to Ca2 + A step increase in Ca2+ to pCa 6.3 produces different contractile responses in the different types of smooth muscle permeabilized with Staphylococcal a-toxin: a rapid rise in force, followed by a spontaneous decline to a low steady state level, in the very phasic ileum smooth muscle (Fig. lA), and a monotonic increase in force in the (tonic) pulmonary artery smooth muscle (not shown, see 1,2). Since Ca2+ was buffered with 1OmM EGTA and all sources of intracellular Ca2+ were removed with A23187, the phasic contraction in ileum could not be due to a transient increase in cytoplasmic Ca2+. Furthermore, ryanodine (10 PM) or inositol 1,4,5-trisphosphate (100 PM), agents that release Ca 2+ from the SR, did not affect the time course of this phasic contraction (not shown). The size of the pores created by atoxin in the plasma membrane is so small that intrinsic, important soluble proteins, such as calmodulin, can not leak out of the cells, although CaEGTA penetrates into cytosol and can clamp free cytoplasmic Ca2+ (4,7). Preincubation of the strips in a very low concentration (0.1 mM) of EGTA, instead of 10 mM, prior to the pCa6.3 (10mM EGTA step), increased the peak of the phasic contraction, but not the steady state level (not shown). 1292
Vol.
172,
No.
BIOCHEMICAL
3, 1990
PCs
>a-,-6.3+>8-,-7-H
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
8+6.7+6.5~.4+6.3->8~6.3-f>8
A2
PG.2 6.3
10
I I 1'
15'
15'
5
Free
Ca
ion
(M)
Fig. 1. A. Properties of the phasic contractile response of permeabilized ileum smooth muscle to constant Ca*+. a-toxin-permeabilized, A23187treated strips from ileum were first incubated in the relaxing solution (pCa HI) for IO min and activated with C$+(pCa 6.3)-containing solution. Ca2+ concentrations were buffered with 10 mM EGTA. Note the transient contractile response of the phasic smooth muscle to a constant Ca2+ stimulus. B. Ca2+-sensitivity of the peak (0) and steady state (*) component of phasic contractions in permeabilized ileum smooth muscle. Before addition of Ca2+, the strips were first incubated in the relaxing solution containing 10 mM EGTA for 8 min and then 0.1 mM EGTA for 2 min. Force was recorded until Ca*+-activated contraction reached a steady state tonic level. Force was normalized to the peak force at pCa 4.5. Error was expressed as +S.D. n=4.
When Ca2+ was cumulatively increased (Fig. ZAl) or repeatedly applied at shorter intervals (Fig. lA2), the peak of the phasic contraction was greatly
reduced, but the tonic level was not significantly
half time
affected.
The
of the spontaneous decline in force at pCa 6.3 was 2-3 min. The decline in the phasic component of contractions induced by Ca2+ was not due to “run-down” of the preparations, as the decline was reversible (Fig. 1A) and recovered in Ca2+-free solution with a half time of about 1 min; 1293
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
complete recovery time was about 10 min (Fig. lA2). Phasic contractions were seen even at pCa 4.5. Fig. 1B shows the comparison between the Ca2+sensitivities of the peak and the steady state of phasic contractions in the ileum smooth muscle. The steady state Ca2+- sensitivity was similar to that when Ca2+ was cumulatively increased. In the tonic pulmonary artery smooth muscle, repeated applications of Ca2+ at shorter intervals or cumulative increases in Ca2+ resulted in little or no decrease in contractility. To exclude the possibility that phasic contractions at constant Ca2+ are due to nonlinear end compliance or inhomogeneity of contractile regions, in some experiments small particles of carbon were attached to the surface of the muscle strips to detect whether inhomogeneous shortening occurs during phasic contractions: there was no evidence of such inhomogeneity. Changing the muscle length from 0.8 to 1.9 times slack length also did not significantly affect the time course of phasic contractions. Therefore, we believe that the phasic contractions are due to the intrinsically phasic reactions of the regulatory/contractile apparatus in phasic type of smooth muscles. We used P-escin-permeabilized smooth muscle to change the on- and off-rates of LC20 phosphorylation by introducing calmodulin or a phosphatase inhibitor, okadaic acid (8). p-escin, a saponin ester, permeabilizes cell membranes to much higher molecular weight solutes than does o-toxin, yet, similarly to a-toxin and unlike crude saponin, retains receptor-coupled responses, such as Ca release from the SR and Ca2+-sensitization by agonists (5). The response of /3-escin-permeabilized ileum to Ca2+ was also very phasic, like that of the a-toxin-permeabilized preparations, and it was greatly affected by extrinsic calmodulin and by okadaic acid; both activation of LC20 kinase with calmodulin and inhibition of protein phosphatase with okadaic acid converted the phasic responses to Ca2+ to more tonic ones and increased their amplitude (Fig. 2). These results suggested that rapid LC20 dephosphorylation is a potential mechanism responsible for the phasic contractile properties of some smooth muscles.
Re-sensitization
of force
to Ca 2+ by carbachol
and by histamine
The contractile response of a-toxin permeabilized vascular smooth muscle to constant Ca2+ is markedly enhanced by al -adrenergic agonists (4,9) and by a thromboxane analogue, U46619 (10). GDPbS blocks both phenylephrineand U466 19-induced potentiation of contraction, suggesting that both responses are coupled by G-protein (4,lO). Fig. 3 shows that both a muscarinic agonist, carbachol, and histamine can “resensitize” contraction at constant CaZ+, even when the 1294
Vol.
BIOCHEMICAL
172, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
calmodulin I
I 8 FM
pCa
6.3
t
okadaic ~PM
acid
4 5 min
-
Fig. 2. Effects of calmodulin and okadaic acid on the time course of contraction activated by pCa6.3 in P-escin-permeabilized ileum smooth muscle. Muscles permeabilized with P-escin and treated with A23187 were incubated in the 1 mM EGTA-containing relaxing solution, foIlowed by a step increase in Ca2+ to pCa 6.3, buffered with 10 mM EGTA . Note that the initial contractile response is phasic, like in the a-toxin permeabilized preparations. Following incubation with calmodulin and okadaic acid, to, respectively, increase LC20 kinase and inhibit LC20 phosphatase, the amplitude of the contractile response to pCa 6.3 is increased, and the rapid phase of contraction is followed by a high, sustained level of force. regulatory/contractile apparatus is desensitized to Ca2+ by prolonged incubation in Ca2+-containing solution. U46619 had minimal and phenylephrine no potentiating effect on ileum, reflecting the absence of the respective excitatory receptors to these agonists. GDPPS (1mM) reversibly inhibited carbachol-induced potentiation, but not the Ca2+activated contraction (not shown). The agonist-potentiated contractions were somewhat phasic, but less so than the initial responses to Ca2+, and the amplitude of the phasic component varied from preparation to preparation. Desensitization and re-sensitization of LC20 phosphorylation to Ca2+ The primary mechanism regulating the contraction/relaxation cycle in smooth muscle is phosphorylation/dephosphorylation of LC20 by, respectively, LC20 kinase and LC20 phosphatases (11,12), although the possibility of thin filament regulated mechanisms involving proteins, such as caldesmon and calponin, has been considered (13). Therefore, it was necessary to determine whether or not the desensitization and resensitization of force to Ca2+ are correlated, respectively, with dephosphorylation and re-phosphorylation of LC20. Table 1 shows the summary of simultaneous measurements of LC20 phosphorylation and force in permeabilized ileum smooth muscle under 1295
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
U46619
PE
Carb
IOOlrM
IO@M
’
L
I
t>8.
Fie. 3. Re-sensitization of desensitized force by agonists and different efficacies of various agonists on sensitization in the a-toxin permeabilized ileum.
various conditions. At resting state (pCa >S), llC2.0 % of total LC20 kS.E.M. of LC20 was phosphorylated, although no significant force was detectable. At the peak of contraction induced by pCa 6.3, LC20 phosphorylation was significantly increased to 33k2.8 %, and then significantly decreased to 25k1.8 % of total LC20 during the steady state contraction (10 min after Ca2+ was added), while force first increased to 36&2.7% and then decreased to 5+0.9% of the maximum force at pCa 5. The muscarinic agonist, carbachol (100pM) significantly increased the amount of phosphorylated LC20 from the desensitized control value (25+1.8%) at pCa 6.3, to 34*2.5% of total LC20, while force was increased from the desensitized sustained contractions (5*0.9%) to 33+5.4% of the maximum force at pCa 5. In conclusion, contractile desensitization to Ca2+ by prolonged incubation in the Ca2+-containing solution and re-sensitization by agonist
Table 1. Ca2+-induced desensitization and agonist-induced sensitization of LC20 phosphorylation and force to Ca2+. Phosphorylation is expressed as % of total LC20 +S.E.M. and force as % of maximum at pCa 5 fS.E.M. Conditions
Force (%)
Phosphorylation (%)
Rest (pCa>8; n=13)
0
11 f 2.0
Peak (pCa 6.3; n=7)
36 f 2.7
33 + 2.8
5 f 0.9
25+ 1.8
33 + 5.4
34 f 2.5
P
