121
Journal of Physiology (1992), 448, pp. 121-132 With 5 figures Printed in Great Britain
MECHANISMS OF INTRINSIC TONE IN FERRET VASCULAR SMOOTH MUSCLE BY JOHN PAWLOWSKI AND KATHLEEN G. MORGAN From the Department of Cellular and Molecular Physiology, Cardiovascular Division and Department of Medicine, Harvard Medical School and Harvard-Thorndike Laboratory, Beth Israel Hospital, Boston, MA 02215, USA
(Received 3 April 1991) SUMMARY
1. Circular strips from ferret aorta were used to investigate the mechanism of the intrinsic basal tone. 2. Determinations of stiffness using small sinusoidal length changes showed an abolition of both stiffness and force with cooling, but the temperature dependence of the change in active stiffness did not parallel that of force. At temperatures below 22 °C there appeared to be a relatively large population of attached, non-forcegenerating cross-bridges, indicating that separate mechanisms are involved in regulating cross-bridge attachment and the force per cross-bridge. 3. Active intrinsic tone was not affected by removal of extracellular Ca2" or removal of endothelium. 4. Intracellular ionized Ca2+ concentrations ([Ca2+]1) as measured with the photoprotein aequorin, did not significantly change when intrinsic tone was abolished by cooling. 5. Myosin light chain phosphorylation, as measured by 2-dimensional polyacrylamide gel electrophoresis, significantly decreased on cooling, but the temperature dependence of phosphorylation did not parallel that of force. The change in phosphorylation in the absence of a change in [Caa2+]i suggests the presence of a constitutively active Ca2+-independent form of myosin light chain kinase. 6. Maximal concentrations of staurosporine inhibited but did not eliminate intrinsic tone. 7. Changes in myosin light chain kinase and protein kinase C activities may explain part but not all of the intrinsic tone.
INTRODUCTION
Gaskell (1881) first described a 'state of tonicity' present in vascular smooth muscle that persisted following denervation. Bayliss (1902) further characterized the 'myogenic' state of vascular smooth muscle and observed the phenonemon in denervated and isolated arterial segments. Evidence for an active basal state of isolated arterial segments came from Laher & Beven (1987), who described a level of MS 9266
J. PAWLOWSKI AND K. G. MORGAN tone partially dependent on stretch and extracellular Ca2". In the absence of stretch, however, in vitro preparations of vascular smooth muscle still display active basal tone. Ruegg (1971) described tension produced in the basal state by smooth muscle that involved activation of the muscle contractile apparatus and was dependent on calcium. Winton (1930) described a state of 'Ringer tonus' that is developed on warming the muscle from room to body temperature and which is 'maintained so long as the muscle survives'. Our laboratory has been interested in the state of smooth muscle prior to stimulation and the underlying mechanisms that create tone in the resting state. This laboratory has previously reported that intrinsic tone in ferret aorta amounts to almost 30 % of maximal agonist-induced tone and that the most effective way to abolish active intrinsic tone is to cool the muscle to 0 °C (DeFeo & Morgan, 1985). A number of ions and enzymes regulate the contractile behaviour of vascular smooth muscle. Of these mechanisms, Ca2+-dependent activation of myosin light chain kinase (MLCK) plays a central role although other pathways are thought to exist also (Kamm & Stull, 1989). Of these pathways, the activation of protein kinase C through the endogenous action of diacylglycerol (Rasmussen, Takuwa & Park, 1987; Ruzycky & Morgan, 1989) may play a role in the generation of smooth muscle 122
tone. To investigate the mechanisms by which vascular smooth muscle produces intrinsic tone, we measured force, stiffness, [Ca21]i, and myosin light chain phosphorylation (MLCP) at different temperatures. To assess the role of protein kinase C in the production of intrinsic tone, we used the inhibitor staurosporine. The experiments described in this report indicate that an apparently [Ca21]iindependent increase in MLCP occurs in parallel with an increase in stiffness but that an additional mechanism also regulates the force/stiffness ratio in a Ca2+-independent manner. METHODS
General methods Male ferrets of approximately 1 kg weight were anaesthetized and their aortae removed as previously described (DeFeo & Morgan, 1985). All procedures were performed in accordance with protocols approved by the Institutional Care and Use Committee and in accordance with the Guiding Principles of the American Physiological Society. Briefly, ferrets were placed in a container and exposed to a chloroform overdose, which induced general anaesthesia and death within minutes. The thoracic aorta was taken from the animal and the adventitia and endothelium were removed unless otherwise indicated, and the final strips were 5-6 mm in length and 1-2 mm in width with an average blotted weight of 800 ,ug. Aortic strips were anchored at one end and the other end was attached to a force transducer. A circulating water jacket attached to a temperature-controlled bath circulator (Lauda RM3) maintained the temperature+0 5 'C. Unless otherwise indicated, experiments began at 21 'C and were set at slack length (li) - defined as the maximal length to which muscle could be stretched without a detectable increase in force. Slack length was used for consistency with the stiffness experiments in which it was important to minimize the contribution of passive forces (Brozovich & Morgan, 1989). After 1 h of equilibration, the bath temperature was changed and the muscle was allowed to equilibrate for another hour before measurements were taken. Approximately half the muscles were cooled initially while the other half were warmed and there was no temperature hysteresis noted.
BASAL VASCUTLAR TONE
123
Solutions For most experiments a modified Krebs solution was used and contained (mM): 120 NaCl, 5 9 KCl, 1 2 MgCl2, 25 NaHCO3, 1-2 NaH2PO4, 2 5 CaCl2 and 11-5 glucose. For the force and stiffness measurements, the calcium-free solution was similar except that no CaCl2 was added and it contained 2 mM-ethylenebis(oxyethylene-nitrilo)tetracetic acid (EGTA). In the aequorin-loaded preparations, the calcium-free solution did not include EGTA in order to minimize aequorin loss. Solutions were bubbled with 95% 02-5% CO2 and the pH adjusted to 7 4.
Stiffness measurement Aortic strips were attached at one end to a length driver (Cambridge series 300) and at the other end to a force transducer (Cambridge series 400; resonant at 1 kHz). Muscles were set at slack length and stiffness was measured as the force response to a small (< 1 % of li) sinusoidal length oscillation. The oscillation frequency (> 100 Hz) was adjusted to assure that the stiffness was independent of the frequency of oscillation. Values of stiffness were recorded on a dual wavelength storage oscilloscope (Tektronix 5113) and were expressed as a percentage of stiffness during a maximal (10-5 M) phenylephrine contraction at 37 'C.
Mleasurement of [Ca2J1i The loading procedure for aequorin into aortic strips was essentially as previously described (Morgan & AMorgan, 1982). Briefly, the muscle strips were incubated in a series of four solutions (A-D) for 30, 90, 30. and 120 min, respectively. The compositions were as follows (mM). Solution A: EGTA, 10; Na2ATP, 5; KCl, 120; MgCl2, 2; N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES), 20. Solution B: EGTA, 01; Na2ATP, 5; KCl, 120; MgCl2, 2; TES, 20; aequorin, 0 5 mg ml-'. Solution C: EGTA, 0 1; Na2ATP, 5; KCl, 120; MgCl2, 10; TES, 20. Solution D: NaCl, 120; KCl, 5-9; dextrose, 11-5; NaHCO3, 25 0; MgCl2, 10; NaH2PO4, 1-4. CaCl2 was added gradually to solution D to a final concentration of 2 5 mm. Within a light collecting apparatus containing ellipsoidal mirrors, light emitted by aequorin was detected with a photomultiplier tube (EMI 9635QA or 9235QA) specially selected for low dark current. Calibration of light signals was performed using the method of fractional luminescence (Allen & Blinks, 1978). To correct for changes in the luminescent activity of aequorin at different temperatures, various time constants for the consumption of aequorin were used. These time constants (Table 1) were interpolated from a plot of various reported values in the literature (Hastings, Mitchell. Mattingly, Blinks & van Leeuwen, 1969; MacKinnon & Morgan, 1986; Jiang & Morgan, 1987). At the end of the experiment, muscle cells were lysed using Triton X-100 to determine maximal luminescence (Lmax). Light levels were expressed as fractional luminescence (L/Lmax) and were then converted to [Ca2+]i using in vitro calibration curves such as that described by Jiang & Morgan (1987).
Myosin light chain phosphorylation Muscle protein separation and measurement of the relative amounts of phosphorylated and unphosphorylated myosin light chains were accomplished using 2-dimensional sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) as described by Jiang & Morgan (1989). The first separation employed an isoelectric focusing gel (33% Triton X-100, 15 M-urea, 6 7 % acrylamide, 0 36 % bis-acrylamide, 8-3 % ampholytes (1-7 % pH 3-0-10-0, 6-6 % pH 4 5-5 4)) in a Bio-Rad model 155 apparatus. Proteins were focused at 750-900 V for 12-15 h and then the gels were directly placed onto SDS gel slabs. The gel slabs contained a 3-3 % acrylamide stacking gel and a 123% acrylamide separating layer. The slabs were fixed in 10% glutaraldehyde and silver stained. The gels were scanned using a dual-wavelength scanning densitometer (Shimadzu CS-930) and the areas under the curves measured as the ratio of moles of phosphorylated myosin light chain to moles of total light chain (mol P/mol LC). Staurosporine was purchased from Kyowa Hakko, L-phenylephrine HCI from Sigma. aequorin from J. R. Blinks, Friday Harbor, WA, USA. All other chemical were of reagent grade or better.
J. PAWLOWSKI AND K. G. MORGAN
124
RESULTS
Changes in stiffness and tone Ferret aorta at li and 37 °C demonstrated an intrinsic tone that could be abolished on cooling to 0 °C that averaged 0-039±0-0031 x 105 N m-2 (n = 11). This was paralleled by an intrinsic stiffness of 0091 +0012 x105N m-2 (n= 11). For comparison, the increase in force at li and 37 °C on addition of 10-5 M-phenylephrine 50-
40
S=30 u._ 20-
,
10
00
5
10
15
20
25
30
35
40
Temperature (OC) Fig. 1. Intrinsic stiffness (@) and force (0) of ferret aorta over the temperature range of 0-37 'C. Each interval of temperature represents the steady-state stiffness and force following 45-60 min of equilibrium (see Methods). Data plotted as means + S.E.M. (n = lt strips, 7 ferrets).
was 041± 0'024 x 10-5 N m-2 (n = 11). Stiffness was measured at 1 to minimize interference from passive stiffness. Residual passive stiffness at 0 'C was subtracted from all values. When intrinsic tone at 37 'C was measured at the maximal length for force production (Imax), it measured 0'23 ± 006 x IO' N m2. We have previously reported that the force in response to phenylephrine at Imax is 0x76 x 105 N m-2 in ferret aorta at 37 'C (Ruzycky & Morgan, 1989). Values for force and stiffness both showed non-linear changes to variations in temperature. Figure 1 shows that the curves for force and stiffness over the range of temperatures differ in shape. The approximated Qlo for force (between 10 and 37 'C) was 3-25 + 014 whereas the Ql0 for stiffness was 1P96 + 016. In an effort to address the question of whether changes in passive stiffness contributed to the observed temperature-dependent effects, we attempted to abolish active tone by soaking muscles for 6-48 h in 10 mM-EGTA unoxygenated physiological saline solution, but this did not completely abolish the contraction on addition of 10-5 M-phenylephrine. However, treatment with distilled H20 +10 mM-EGTA for 48 h did abolish any active force development and muscles so treated did not show any change in force or stiffness over the temperature range of 0-37 'C (data not shown). Thus, the changes in force and stiffness in Fig. 1 do not appear to be the response of passive components to changes in temperature.
BASAL VASCULAR TONE
125
-Endothelium
t
t
+Endothelium
t 21 °C
IT
37 °C PE
t CCh
J 100 mg 5 min
Fig. 2. Temperature-induced intrinsic tone in the absence (top) or presence (bottom) of functioning endothelium. The muscle strips were set at slack length and warmed from 21 to 37 'C. The presence of functioning endothelium was confirmed by the change in force in response to 10-5 M-carbachol (CCh) following a 106 M-phenylephrine (PE) contraction. T indicates the initiation of warming.
40r
T
30k
a)
20 K
0 U-
10o
0
V777,,Z,,/7/7/'//Zk/
37 C 21 CC 21 ^C 37 DC -Ca2+ +Ca2+ Fig. 3. Change in level of intrinsic tone between 21 and 37 °C in response to Ca2+containing (+ Ca2+) versus Ca2+-free (- Ca2+) modified Krebs solution. Normal Krebs solution contained 2-5 mM-Ca2 Calcium-free Krebs solution contained 2 mM-EGTA. Data were calculated as a percentage of the maximal response (10-5 M-phenylephrine) at 37 °C and expressed as means + S.E.M. * Significant difference (Student's paired t test) from 21 °C, P
Journal of Physiology (1992), 448, pp. 121-132 With 5 figures Printed in Great Britain
MECHANISMS OF INTRINSIC TONE IN FERRET VASCULAR SMOOTH MUSCLE BY JOHN PAWLOWSKI AND KATHLEEN G. MORGAN From the Department of Cellular and Molecular Physiology, Cardiovascular Division and Department of Medicine, Harvard Medical School and Harvard-Thorndike Laboratory, Beth Israel Hospital, Boston, MA 02215, USA
(Received 3 April 1991) SUMMARY
1. Circular strips from ferret aorta were used to investigate the mechanism of the intrinsic basal tone. 2. Determinations of stiffness using small sinusoidal length changes showed an abolition of both stiffness and force with cooling, but the temperature dependence of the change in active stiffness did not parallel that of force. At temperatures below 22 °C there appeared to be a relatively large population of attached, non-forcegenerating cross-bridges, indicating that separate mechanisms are involved in regulating cross-bridge attachment and the force per cross-bridge. 3. Active intrinsic tone was not affected by removal of extracellular Ca2" or removal of endothelium. 4. Intracellular ionized Ca2+ concentrations ([Ca2+]1) as measured with the photoprotein aequorin, did not significantly change when intrinsic tone was abolished by cooling. 5. Myosin light chain phosphorylation, as measured by 2-dimensional polyacrylamide gel electrophoresis, significantly decreased on cooling, but the temperature dependence of phosphorylation did not parallel that of force. The change in phosphorylation in the absence of a change in [Caa2+]i suggests the presence of a constitutively active Ca2+-independent form of myosin light chain kinase. 6. Maximal concentrations of staurosporine inhibited but did not eliminate intrinsic tone. 7. Changes in myosin light chain kinase and protein kinase C activities may explain part but not all of the intrinsic tone.
INTRODUCTION
Gaskell (1881) first described a 'state of tonicity' present in vascular smooth muscle that persisted following denervation. Bayliss (1902) further characterized the 'myogenic' state of vascular smooth muscle and observed the phenonemon in denervated and isolated arterial segments. Evidence for an active basal state of isolated arterial segments came from Laher & Beven (1987), who described a level of MS 9266
J. PAWLOWSKI AND K. G. MORGAN tone partially dependent on stretch and extracellular Ca2". In the absence of stretch, however, in vitro preparations of vascular smooth muscle still display active basal tone. Ruegg (1971) described tension produced in the basal state by smooth muscle that involved activation of the muscle contractile apparatus and was dependent on calcium. Winton (1930) described a state of 'Ringer tonus' that is developed on warming the muscle from room to body temperature and which is 'maintained so long as the muscle survives'. Our laboratory has been interested in the state of smooth muscle prior to stimulation and the underlying mechanisms that create tone in the resting state. This laboratory has previously reported that intrinsic tone in ferret aorta amounts to almost 30 % of maximal agonist-induced tone and that the most effective way to abolish active intrinsic tone is to cool the muscle to 0 °C (DeFeo & Morgan, 1985). A number of ions and enzymes regulate the contractile behaviour of vascular smooth muscle. Of these mechanisms, Ca2+-dependent activation of myosin light chain kinase (MLCK) plays a central role although other pathways are thought to exist also (Kamm & Stull, 1989). Of these pathways, the activation of protein kinase C through the endogenous action of diacylglycerol (Rasmussen, Takuwa & Park, 1987; Ruzycky & Morgan, 1989) may play a role in the generation of smooth muscle 122
tone. To investigate the mechanisms by which vascular smooth muscle produces intrinsic tone, we measured force, stiffness, [Ca21]i, and myosin light chain phosphorylation (MLCP) at different temperatures. To assess the role of protein kinase C in the production of intrinsic tone, we used the inhibitor staurosporine. The experiments described in this report indicate that an apparently [Ca21]iindependent increase in MLCP occurs in parallel with an increase in stiffness but that an additional mechanism also regulates the force/stiffness ratio in a Ca2+-independent manner. METHODS
General methods Male ferrets of approximately 1 kg weight were anaesthetized and their aortae removed as previously described (DeFeo & Morgan, 1985). All procedures were performed in accordance with protocols approved by the Institutional Care and Use Committee and in accordance with the Guiding Principles of the American Physiological Society. Briefly, ferrets were placed in a container and exposed to a chloroform overdose, which induced general anaesthesia and death within minutes. The thoracic aorta was taken from the animal and the adventitia and endothelium were removed unless otherwise indicated, and the final strips were 5-6 mm in length and 1-2 mm in width with an average blotted weight of 800 ,ug. Aortic strips were anchored at one end and the other end was attached to a force transducer. A circulating water jacket attached to a temperature-controlled bath circulator (Lauda RM3) maintained the temperature+0 5 'C. Unless otherwise indicated, experiments began at 21 'C and were set at slack length (li) - defined as the maximal length to which muscle could be stretched without a detectable increase in force. Slack length was used for consistency with the stiffness experiments in which it was important to minimize the contribution of passive forces (Brozovich & Morgan, 1989). After 1 h of equilibration, the bath temperature was changed and the muscle was allowed to equilibrate for another hour before measurements were taken. Approximately half the muscles were cooled initially while the other half were warmed and there was no temperature hysteresis noted.
BASAL VASCUTLAR TONE
123
Solutions For most experiments a modified Krebs solution was used and contained (mM): 120 NaCl, 5 9 KCl, 1 2 MgCl2, 25 NaHCO3, 1-2 NaH2PO4, 2 5 CaCl2 and 11-5 glucose. For the force and stiffness measurements, the calcium-free solution was similar except that no CaCl2 was added and it contained 2 mM-ethylenebis(oxyethylene-nitrilo)tetracetic acid (EGTA). In the aequorin-loaded preparations, the calcium-free solution did not include EGTA in order to minimize aequorin loss. Solutions were bubbled with 95% 02-5% CO2 and the pH adjusted to 7 4.
Stiffness measurement Aortic strips were attached at one end to a length driver (Cambridge series 300) and at the other end to a force transducer (Cambridge series 400; resonant at 1 kHz). Muscles were set at slack length and stiffness was measured as the force response to a small (< 1 % of li) sinusoidal length oscillation. The oscillation frequency (> 100 Hz) was adjusted to assure that the stiffness was independent of the frequency of oscillation. Values of stiffness were recorded on a dual wavelength storage oscilloscope (Tektronix 5113) and were expressed as a percentage of stiffness during a maximal (10-5 M) phenylephrine contraction at 37 'C.
Mleasurement of [Ca2J1i The loading procedure for aequorin into aortic strips was essentially as previously described (Morgan & AMorgan, 1982). Briefly, the muscle strips were incubated in a series of four solutions (A-D) for 30, 90, 30. and 120 min, respectively. The compositions were as follows (mM). Solution A: EGTA, 10; Na2ATP, 5; KCl, 120; MgCl2, 2; N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES), 20. Solution B: EGTA, 01; Na2ATP, 5; KCl, 120; MgCl2, 2; TES, 20; aequorin, 0 5 mg ml-'. Solution C: EGTA, 0 1; Na2ATP, 5; KCl, 120; MgCl2, 10; TES, 20. Solution D: NaCl, 120; KCl, 5-9; dextrose, 11-5; NaHCO3, 25 0; MgCl2, 10; NaH2PO4, 1-4. CaCl2 was added gradually to solution D to a final concentration of 2 5 mm. Within a light collecting apparatus containing ellipsoidal mirrors, light emitted by aequorin was detected with a photomultiplier tube (EMI 9635QA or 9235QA) specially selected for low dark current. Calibration of light signals was performed using the method of fractional luminescence (Allen & Blinks, 1978). To correct for changes in the luminescent activity of aequorin at different temperatures, various time constants for the consumption of aequorin were used. These time constants (Table 1) were interpolated from a plot of various reported values in the literature (Hastings, Mitchell. Mattingly, Blinks & van Leeuwen, 1969; MacKinnon & Morgan, 1986; Jiang & Morgan, 1987). At the end of the experiment, muscle cells were lysed using Triton X-100 to determine maximal luminescence (Lmax). Light levels were expressed as fractional luminescence (L/Lmax) and were then converted to [Ca2+]i using in vitro calibration curves such as that described by Jiang & Morgan (1987).
Myosin light chain phosphorylation Muscle protein separation and measurement of the relative amounts of phosphorylated and unphosphorylated myosin light chains were accomplished using 2-dimensional sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) as described by Jiang & Morgan (1989). The first separation employed an isoelectric focusing gel (33% Triton X-100, 15 M-urea, 6 7 % acrylamide, 0 36 % bis-acrylamide, 8-3 % ampholytes (1-7 % pH 3-0-10-0, 6-6 % pH 4 5-5 4)) in a Bio-Rad model 155 apparatus. Proteins were focused at 750-900 V for 12-15 h and then the gels were directly placed onto SDS gel slabs. The gel slabs contained a 3-3 % acrylamide stacking gel and a 123% acrylamide separating layer. The slabs were fixed in 10% glutaraldehyde and silver stained. The gels were scanned using a dual-wavelength scanning densitometer (Shimadzu CS-930) and the areas under the curves measured as the ratio of moles of phosphorylated myosin light chain to moles of total light chain (mol P/mol LC). Staurosporine was purchased from Kyowa Hakko, L-phenylephrine HCI from Sigma. aequorin from J. R. Blinks, Friday Harbor, WA, USA. All other chemical were of reagent grade or better.
J. PAWLOWSKI AND K. G. MORGAN
124
RESULTS
Changes in stiffness and tone Ferret aorta at li and 37 °C demonstrated an intrinsic tone that could be abolished on cooling to 0 °C that averaged 0-039±0-0031 x 105 N m-2 (n = 11). This was paralleled by an intrinsic stiffness of 0091 +0012 x105N m-2 (n= 11). For comparison, the increase in force at li and 37 °C on addition of 10-5 M-phenylephrine 50-
40
S=30 u._ 20-
,
10
00
5
10
15
20
25
30
35
40
Temperature (OC) Fig. 1. Intrinsic stiffness (@) and force (0) of ferret aorta over the temperature range of 0-37 'C. Each interval of temperature represents the steady-state stiffness and force following 45-60 min of equilibrium (see Methods). Data plotted as means + S.E.M. (n = lt strips, 7 ferrets).
was 041± 0'024 x 10-5 N m-2 (n = 11). Stiffness was measured at 1 to minimize interference from passive stiffness. Residual passive stiffness at 0 'C was subtracted from all values. When intrinsic tone at 37 'C was measured at the maximal length for force production (Imax), it measured 0'23 ± 006 x IO' N m2. We have previously reported that the force in response to phenylephrine at Imax is 0x76 x 105 N m-2 in ferret aorta at 37 'C (Ruzycky & Morgan, 1989). Values for force and stiffness both showed non-linear changes to variations in temperature. Figure 1 shows that the curves for force and stiffness over the range of temperatures differ in shape. The approximated Qlo for force (between 10 and 37 'C) was 3-25 + 014 whereas the Ql0 for stiffness was 1P96 + 016. In an effort to address the question of whether changes in passive stiffness contributed to the observed temperature-dependent effects, we attempted to abolish active tone by soaking muscles for 6-48 h in 10 mM-EGTA unoxygenated physiological saline solution, but this did not completely abolish the contraction on addition of 10-5 M-phenylephrine. However, treatment with distilled H20 +10 mM-EGTA for 48 h did abolish any active force development and muscles so treated did not show any change in force or stiffness over the temperature range of 0-37 'C (data not shown). Thus, the changes in force and stiffness in Fig. 1 do not appear to be the response of passive components to changes in temperature.
BASAL VASCULAR TONE
125
-Endothelium
t
t
+Endothelium
t 21 °C
IT
37 °C PE
t CCh
J 100 mg 5 min
Fig. 2. Temperature-induced intrinsic tone in the absence (top) or presence (bottom) of functioning endothelium. The muscle strips were set at slack length and warmed from 21 to 37 'C. The presence of functioning endothelium was confirmed by the change in force in response to 10-5 M-carbachol (CCh) following a 106 M-phenylephrine (PE) contraction. T indicates the initiation of warming.
40r
T
30k
a)
20 K
0 U-
10o
0
V777,,Z,,/7/7/'//Zk/
37 C 21 CC 21 ^C 37 DC -Ca2+ +Ca2+ Fig. 3. Change in level of intrinsic tone between 21 and 37 °C in response to Ca2+containing (+ Ca2+) versus Ca2+-free (- Ca2+) modified Krebs solution. Normal Krebs solution contained 2-5 mM-Ca2 Calcium-free Krebs solution contained 2 mM-EGTA. Data were calculated as a percentage of the maximal response (10-5 M-phenylephrine) at 37 °C and expressed as means + S.E.M. * Significant difference (Student's paired t test) from 21 °C, P
