Europ. J. clin. Invest. 5, 259-265 (1975)
Force Velocity Characteristics of Oesophageal Muscle: Interaction of Isoproterenol and Calcium Sidney Cohen Gastrointestinal Section, Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pa. , U. S.A. Received: June 25, 1974, and in revised form: October 29, 1974
Abstract. The purpose of this study was to evaluate the interaction of isoproterenol and calcium upon the force velocity characteristics of opossum oesophageal circular muscle, in vitro. Isotonic and isometric recordings were used to determine the velocity of shortening and force, respectively. All muscle was studied at its length of optimal tension development (Lo). To determine the effect of isoproterenol upon muscle contractility, studies were performed at 2.5 mM and 5.0 mM calcium Kreb's solution. The maximal velocity of shortening (V max) and the peak force (Po) of oesophageal muscle were 6.2 f 0.3 mm/s and 16.8 f 1.0 gm, respectively, at 2.5 mM calcium. Isoproterenol M) decreased the V max to 4.9 f 0.6 m/s and the Po to 9.8 ? 1.3 gm (P < .01). Increased calcium (5.0 mM), alone, did not alter either V max or Po. However, at 5.0 mM calcium, isoproterenol reduced the V max to 6.0 f 0.6 mm/s (P > .05) and the Po to 13.9 ? 0.8 gm (P < .05). These studies indicate that isoproterenol significantly reduces the V max and Po of oesophageal smooth muscle during the neural mediated "off response". This effect of isoproterenol is reversed, in part, by an increase in calcium concentration.
sues. The midoesophagus and distal duodenum were ligated, and the upper gastrointestinal tract from midoesophagus to distal duodenum was excised, It has been previously demonstrated that the washed in Kreb's solution with the composition in circular smooth muscle of the oesophagus can be millimoles per litre: N a ' , 138.6; Kt 4.6; Ca++, utilized to evaluate the force-velocity properties 2.5; Mg", 1.2; C1-,0126.2i HC03-, 21.9; PO45, 1.2; of muscle during a neural-mediated contraction glucose, 49.6, at 37 - 38 C, and transferred to (6, 7). The prominent contraction obtained upon an organ bath of Krebs-Ringer solution, bubblgd termination of electrical stimulation, the "off wigh 95 Z 02 and 5 I C02 and maintained at 37 response'', is believed to be mediated through the 38 C. The oesophagus was separated from the non-adrenergic inhibitory nerves (15). The "off stomach at the anatomic gastrooesophageal junction response'' has been reported to serve as the initiating mechanism of oesophageal peristalsis (24). where the narrow oesophagus flares into the stomach. The mucosa from each region was removed We have shown that the calculated maximal velocity to the level of the submucosa. Smooth muscle strips, of muscle shortening (V max) and the peak force 0.5 cm wide and 1.0 cm long, were cut from each (Po) obtained during an "off response" are altered anatomic region. All muscles were lightly blotted by calcium concentration, norepinephrine, acetyland weighed at the termination of the experiment. choline and gastrin I (6, 7). The purpose of this Each muscle was studied using the apparatus study is to evaluate the interaction of isoproterenol and calcium upon the force velocity charac- described previously and diagrammatically illustrated in Figure 1 (6, 7, 19, 20, 21). The muscle teristics of oesophageal smooth muscle, in vitro. was mounted to record tension generated by the circular layer. One end of the muscle was connected to an inflexible wire which was attached to an Methods isometric force transducer (Grass Model FT-03C). The other end was attached to the lever of an isoStudies were performed on 20 adult opossums of tonic transducer (Harvard Model 387). Muscle length both sexes weighing 2.4 5.1 kg. The method of was adjusted by a micrometer. Muscle loading was obtaining muscle, outlined below, has previously accomplished through calibrated balance weights been described in detail (23). All animals were placed on the isotonic lever. A stop allowed the killed by intravenous pentobarbital. The oesophamuscle to receive both a preload and an afterload. gus, 8 cm proximal to the anatomic gastrooesophaEach muscle was stimulated electrically across two geal junction, the entire stomach, and duodenum were mobilized and freed from the surrounding tisplatinum wires adjusted to juxtapose the lateral
Introduction
-
S . Cohen: Force Velocity Characteristics of Oesophageal Muscle
260
stimulation but upon termination of stimulation, a prominent neural mediated contraction ("off response") occurred after a brief latent period (15, 2 3 , 2 4 ) . Stimulus parameters were initially selected to elicit the maximal amplitude of this contraction under pure isometric recording conIsotonic Lever ditions. Maximum amplitude of the "off response" was obtained with a 30-second train of square wave pulses of 1.0 millisecond duration, 10 Hz. Either 50 or 60 volts gave the maximum isometric tension for a given muscle. Force velocity curves were constructed for each muscle under various physiological conditions ::::: ---Af terload using the "off response" as a basis of non-tetanic muscle contraction. As shown in Figure 2 the isoPreload metric force generated prior to shortening, P, was Muscle---measured directly from its respective tracing at the first vertical line. The initial velocity of shortening, V, was obtained from the isotonic PIo t i nu m tracing. The initial velocity of shortening was Electrodes determined by the change in length (dl) per unit change in time (dt). Additionally, velocity of shortening was measured directly using a Beckman velocity coupler ( 9 8 4 1 ) . Measurements obtained by Isometric Force both methods correlated closely. The degree of shortening of the muscle during an afterloaded Tr a n s d u c e r contraction was obtained directly from the isotonic recording ( A L). The load at which shortening first Fig. 1 . Schematic diagram of apparatus used to became zero was denoted as Po. The muscle length measure afterloaded muscle shortening. The muscle at which the maximal Po was achieved was denoted was mounted between an isometric transducer and an as Lo. isotonic transducer. A stop allowed the muscle to Force velocity curves were constructed during receive both a preload and an afterload. The muscle the superfusion of Kreb's solution containing was superfused by heated and oxygenated Kreb's 5.0 mM Ca++ as well as the usual 2.5 mM Ca++. After solution to which isoproterenol was added. The the determination of the force velocity relationmuscle was stimulated electrically across two ship at the different calcium concentrations, isoplatinum wires proterenol (10-7 M, 10-6 M, 10-5 M, 10-4 M) was added to the superfusate. The compounds were evaluated in Kreb's solution containing either 2.5 mM surfaces of the muscle. Stimuli were delivered by Ca++ or 5 . 0 mM Ca++. Force velocity determinations a stimulator (Grass Model S44 with stimulus isowere made after a minimum of 10 minutes of superlation unit SIU I). The muscles were superfused by a heated and oxygenated Kreb's solution with the fusion of the Kreb's solutions containing different calcium concentrations or isoproterenol. composition noted above. A thermistor was used to monitor temperature at the muscle. The superfusate The maximum isometric force was also determined during muscle depolarization with a Kreb's solution was maintained at 36' - 38OC. A l l recordings were containing 143.6 mM KC1. These determinations were graphed on a rectilinear, ink-miting polygraph made under strict isometric conditions using only (Beckman) the force transducer. After a 30-minute period, the muscle length was Measurements of velocity of shortening in m d s , increased until the passive tension was at zero shortening in mm, load in gm and Po were placed but ready to increase with any further increase in into a computer program. The constants of the Hill length. This length was denoted as the initial length. From this initial lenght, the preload shown equation as determined from the reciprocal plot of the data were used to determine the extrapolated to bring the muscle to its length of optimal tenV max ( I 1 - 13, 16 2 0 ) . The best fit of the line sion development, Lo, was added to the muscle. The for the displaced rectangular hyperbola of the preload was determined previously from forceforce velocity relationship and the standard devivelocity measurements using the "off response'' ( 6 ) ation of that line were directly plotted by the The change in muscle length caused by each preload computer using the least square regression analywas measured directly by the excursion of the isotonic lever as seen on the recorder. Force velocity sis. Statistical analysis was made for paired comparisons using the Student t test. curves were constructed utilizing afterloads in either 0.5 gm or 1.0 gm increments. To obtain the force-velocity relationship of Results the oesophageal muscle, the neurogenic response elicited at the termination of electrical stimuIn Figure 3 are shown the computer plots of the lation was utilized. The muscle, as previously demonstrated, did not develop active tension during force velocity determination and its linear trans-
Kreb's Sojution Superfusion
Q---
/p .
-
.
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
&
26 I
6.0
C
L
I
I
0
2
4
I
I
6 8 Time ( s e c )
I
10
I
12
Fig. 2 . Simultaneous isometric and isotonic recordings for a single strip of oesophageal muscle at 1.5 gm preload (not shown in resting state) and an afterload of 6.0 gm. When isometric force equaled the total load on the muscle (first verti-
cal line), isotonic shortening began. The velocity of shortening was calculated by measuring the change in length (dl) per unit change in time (dt). Total muscle shortening was measured directly (A L). Time in seconds is along the horizontal axis
formation for a single strip of oesophageal muscle studied at Lo, 2 . 0 gm preload, and 2 . 5 mM calcium. The control force velocity determination is above (a) while the result obtained during a superfusion of M isoproterenol is below (b). The linear transformation was based on the Hill equation (p + a) (V + b) = (Po + a) b where P represents load, V the velocity of shortening, Po the maximum load which the muscle was first unable to move, cz a constant with units of force and b a constant with units of velocity ( 1 1 - 13, 17 - 2 0 ) . In both linearized transformations, the points fell along a straight line with excellent correlation Coefficients suggesting that the force velocity relationship was hyperbolic similar to that reported for other types of muscle. From the linearized plot, the slope ('/b) and the Y intercept (ah) provided the constants for the equation. The theoretical maximum velocity of shortening at zero load (v max) was calculated from the equation: V max = In the presence of isoproterenol, the V rnax was reduced from 6 . 8 7 mmfs to 4 . 1 7 m/s. The Po was reduced from 17 gm to 9.0 gm. Table I shows all data obtained on oesophageal smooth muscle taken at three levels of the oesophagus and studied at 2 . 5 mM calcium. Isoproterenol gave a dose-related decrease in V rnax and PO in all oesophageal muscle. The upper oesophageal muscle seemed most sensitive, showing a sig-
nificant reduction in Po at 10-7 M isoproterenol (P < . 0 5 ) and a significant decrease in V max at M isoproterenol (P < . 0 5 ) . The.lower oesophagus required M isoproterenol to significantly reduce the Po (P < .05) and 10-5 M isoproterenol to reduce the V max (P < . 0 5 ) . These data indicate that at 2 . 5 mM calcium, the force velocity parameters, V max and Po, of the oesophageal muscle could be significantly reduced by isoproterenol. The upper oesophagus seemed more sensitive to this effect than music from the distal oesophagus. Table 2 shows the interaction of an increased calcium concentration ( 5 . 0 mM) with 10-5 M isoproterenol. The 5. 0 mM calcium concentration, alone, did not significantly alter the V max or the Po. However, in the presence of 5.0 mM calcium, 10-5 M isoproterenol had no significant effect upon the V rnax of the oesophageal muscle. The Po was significantly reduced at 5 . 0 mM calcium and 10-5 M isoproterenol (P c . 0 5 ) . However, the reduction in Po was less marked than observed at 2 . 5 d calcium. In ten additional studies performed under isometric conditions, K C 1 depolarization gave an active tension of 18.6 2 . 1 gms at a 2 . 5 mM calcium concentration. Thus, at Lo, the maximal isometric force of the muscle to KC1 depolarization was similar to the Po value (Table I ) achieved during the "off response" at the termination of electrical s t imu1ation,.
.
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
262
Esophageal
Circular
Muscle
62.0 gm Preload 2.5mM Ca++
Po-P V
ys.23 x + 2.47 r = .94
I-
0
2
4
6
8
Load
10
12
0
14 16
l
i
6
8
l
l
I
10 12
I
14 16
Load ( g m )
a 1 ( V t b ) = ( P o ta 1 b
Vmax =
= 6.87 m m / s e c . a
Esophageal
::
l
4
(em) (Pt
\
I
2
Circular
Muscle
-
*
3-
V
r =.92
1-
9
E
0
I
I
1
2
I
I
I
I
I
3 4 5 6 L o a d (gm)
7
8
9
0
(P ta1 (Vtb
Vmax =
I
I
I
1
2
3 4 5 6 Load ( g m )
I
I
I
I
I
1
7
6
9
1= (Pota1 b
- = 4.17mm/scc 0
Fig. 3 . Computer plots of two force velocity curves and their linear transformations for a single strip of oesophageal circular muscle studied at 2.0 gm preload and 2 . 5 mM calcium. The control force velocity curve (a) is shown above, while the curve obtained during isoproterenol (10-5 M) superfusion is shown below (b). The constants of the Hill
equation were obtained from the linear plot of (Po P/V) versus P. The slope of the line was l/b and the intercept on the ordinate was a/b. V max was calculated as (Po/a)b. The dotted lines on the linear plot indicate the standard deviations. In the presence of isoproterenol, the V max and the Po were reduced
-
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
263
Table 1 . Effect of isoproterenol on force velocity determinations of oesophageal smooth muscle at 2.5 calcium I - 2 c mt
Control Isoproterenol
3 - 4 c mt
Po
5 - 6 c mt
Po
V max (mml s)
(gm)
V rnax (mml s 1
6.2 t 0.3
16.8 f 1.0
5.9 f 0.5
17.8 f 1.2
6.2 2 0 . 4
Po
V rnax
(gm)
(gm)
(rn/S)
15.8 +- 1.3
10-4 M
4.3
0.2
7.3 f 0.9
4.0 f 0.3
6.8 2 0.6
3.1 f. 0.3
7.0
10-5 M
4.9 f 0.6
9.8 f 1.3
4.7 t 0.6
7.6
& 1.0
3.6 f. 0.3
7.8 f 0.9
10-6 M
6.1 f 0.9
11.7 f 0.7
5.5 2 0.4
10.0 k 0.6
9.2 t 0.8
10-7 M
6.3 f 0.8
14.9 f 1.1
6.3 f 0.2
13.4 f 1.4
4.8 t 0.5 5.6 f 0.7
'Refers
f
2 0.7
10.4 +- 1.0
to distance in cm above the anatomic gastrooesophageal junction.
Table 2 . Effect of isoproterenol on force velocity determinations of oesophageal smooth muscle at 5.0 mM calcium 3 - 4 c mt
1 - 2 c mt
~
~
Control
5 - 6 c mt
~~
7.2 t 0.5
17.9
6.0 f - 0 . 6
13.9 2 0.8
?
1.2
0.9
18.0 f 1.2
7 . 1 f 0.4
17.6 +- 1.0
6.5 t 0.9
12.8 5 1.3
7.5 f. 0. 5
14.4 f 0.8
6.9
5
Isoproterenol 10-5 M
tRefers to distance in cm above the anatomic gastroesophageal junction.
Discussion
that the "off response" or rebound excitation and non-adrenergic inhibition were both mediated by nerves which released adenosine triphosphate (3, The purpose of this study was to evaluate the 4 ) . We have suggested that these nerves mediate interaction of isoproterenol and calcium upon the not only peristalsis but also lower oesophageal force velocity characteristics of opossum circular sphincter relaxation and contraction (23). smooth muscle, in vitro. These studies indicated In previous studies we have shown that acetylthat isoproterenol significantly reduced the V max choline, norepinephrine and gastrin I increased and Po of this muscle during the neural-mediated the V max and Po during the off response ( 6 , 7 ) . "off response". The reduction in force velocity The response to these agents was most marked at parameters associated with isoproterenol was partially reversed by an increased calcium concena calcium concentration of 1.0 mM. The present studies indicated that isoproterenol reduced the trat ion. V rnax and Po o f the oesophageal muscle and that Studies in vitro using the intact opossum oesothis effect was in part reversed by an increased phagus and oesophageal smooth muscle strips decalcium concentration (5.0 mM). The finding that monstrated responses localized to different layers isoproterenol decreased the V m a x of oesophageal of muscle (5). Upon deflation of a balloon in the muscle at 2.5 mM calcium and not at 5.0 mM calcium intact oesophagus, in vitro, a peristaltic consuggested that isoproterenol had exerted its eftraction was observed. This peristaltic contraction corresponded to the "off response" at the ter- fect through an alteration in calcium mobilization. mination- of electrical stimulation as recorded from It is generally accepted that calcium is essential the circular muscle layer. The prominent "off for excitation contraction coupling in muscle of response" was abolished by tetrodotoxin, but not all types (2, 8 10, 14, 16, 20, 22). Furthermore, it has been suggested that adrenergic agents by adrenergic or cholinergic antagonists (15). This response may correspond to rebound excitation may inhibit release of-calciumfrom an intracellular store (the endoplasmic reticulum) or inhibit seen in muscles containing non-adrenergic inhibicalcium entrance into the muscle from the extratory nerves ( I ) . It had been suggested recently
-
264
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
cellular space (10). The effect of a beta adrenergic agonist in this preparation may diminish the availability of calcium for excitation contraction coupling of the muscle. This observation would fit with the previous suggestion that excitatory agents such as gastrin I, acetylcholine and norepinpehrine increased the availability of calcium which in turn increased the V max of the muscle. Thus, it would seem that each agent shown to affect the force velocity properties of oesophageal smooth muscle acted through a common factor, calcium. The validity of this conclusion is dependent upon the interpretation of data obtained during a neural mediated response of smooth muscle. The force velocity relationship was described originally utilizing the tetanic response of skeletal muscle to electrical stimulation ( 1 1 - 13). This was subsequently modified to evaluate the single twitch elicited during direct electrical stimulation of cardiac muscle (17 - 20 ) . The studies described above evaluated the single neurogenically elicited twitch of oesophageal muscle upon the termination of electrical stimulation. The maximal isometric force (Po) during the off response was similar to the maximal tension achieved during muscle depolarization with 143.6 mM KC1. This observation suggested that the maximum isometric force could be achieved during this neural mediated muscle response. The Hill equation was used to derive V max, the theoretical maximum velocity of shortening at zero load. The equation, (P + a) (V + b) = (Po + a)b, expressed the force velocity relationship as a displaced rectangular hyperbola with asymptotes at minus a and minus b ( 1 1 - 13). The constants a and b have dimensions of force and velocity, respectively. These constants have been calculated only for frog sartorius and equal the extra heat liberated with shortening (a) and the rate of heat liberation (b) ( 1 1 - 13). Despite limitations in using the Hill equation to derive the constants from mechanical measurements, other smooth muscles have been evaluated in this manner allowing comparison with these data (16, 21). The observations on oesophageal muscle when evaluated using the Hill equation did form a linear relationship with a high correlation coefficient (all above .90) suggesting that the force velocity relationship could be described as a displaced hyperbola. Thus, the neural mediated contraction of oesophageal muscle appeared to have a force velocity relationship similar to other muscles. Additonally, the Po was similar to the peak tension achieved during maximal KC1 depolarization. The question as to whether isoproterenol and calcium exerted their effect solely upon myogenic rather than neurogenic elements cannot be answered at this time.
Bibliographg
Acknowledgements. This work was supported by Research Grant 1 R01 AM 16280-01 and Research Career Development Award 1 KO4 AM 70576-01 from the National Institutes of Health. The author wishes to thank Mrs. Fe Green for her expert technical assistance, and Miss Mary Carroll for secretarial assistance.
17.
1 . Benett, M.: Rebound excitation of the smooth
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12. 13.
14.
15. 16.
18.
muscle cells of the guinea-pig taenia coli after stimulation of intramural inhibitory nerves. J. Physiol. 185, 124 (1966) Bockman, E.L., Rubio, R . , Berne, R.M. : Effect of lanthanum on isoproterenol-induced activation of myocardial phosphyorylase. Am. J. Physiol. 225, 438 (1973) Burnstock, G.: Purinergic nerves. Pharmacol. Reviews 24, 509 (1972) Burnstock, G., Campbell, G., Satchell, D., Smythe, A.: Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitdry nerves in the gut. Brit. J. Pharmacol. 40, 668 (1970) Christensen, J., Lund, G.: Esophageal responses to distension and electrical stimulation. J. clin. Invest. 48, 408 (1969) Cohen, S., Green, F.: The mechanics of esophageal muscle contraction: evidence of an inotropic effect of gastrin. J. clin. Invest. 52, 2029 (1973) Cohen, S., Grenn, F.: Force velocity characteristics of esophageal muscle; effect of acetylcholine and norepinephrine. Am. J. Physiol. In press Durbin, R., Jenkonson, D.: The effect of carbachol in the permeability of depolarized muscle to inorganic ions. J. Physiol. 157, 74 (1961) Edman, K., Schild, H.: The need for calcium in the contractile responses induced by acetylcholine and potassium in the rat uterus. J. Physiol. 161, 424 (1962) Edman, K., Schild, H.: Calcium and the stimulant and inhibitory effects of adrenaline in depolarized smooth muscle. J. Physiol. 169, 404 (1963) Hill, A.: The heat of shortening and the dynamic constants of muscle. Proc. roy. SOC. Med. 126, 136 (1939) Hill, A.: A discussion on muscular contraction and relaxation: their physical and chemical basis. Proc. roy. SOC. Med. 137, 40 (1950) Hill, A . : Mechanics of the contractile element of muscle. Nature 166, 415 (1950) Kirby, A., Lindley, B.D., Picken, J.R. : Calcium dependence of potassium contractures in denervated frog muscle. Am. J. Physiol. 225, 166 (1973) Lund, G., Christensen, J.: Electrical stimulation of esophageal smooth muscle and effects of antagonists. Am. J. Physiol. 217, 1369 (1969) Siegman, M., Gordon, A . : Potentiation of contraction: effects of calcium and caffeine on active state. Am. J. Physiol. 222, 1587 (1972) Sonnenblick, E., McCallum, Z.: Active state, force velocity relationships and inotropic mechanisms in mammalian papillary muscle. Fed. Proc. 20, 126 (1961) Sonnenblick, E.: Force-velocity relations in mammalian heart muscle. Am. J. Physiol. 202, 931 (1962)
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle 19. Sonnenblick, E.: Series elastic and contrac-
tile element interactions in generation of myocardial force: effects of changing muscle length, norepinephringe and strophanthidin. Fed. Proc. 23, I I8 (1964) 20. Sonnenblick, E.: Implications of muscle mechanics in the heart. Fed. Proc. 21, 975 (1962) 21. Stephens, N., Kroeger, E., Mehta, J.: Force.velocity characteristics of respiratory airway smooth muscle. J. appl. Physiol. 26, 685 (1969) 22. Sunano, S., Miyazaki, E.: Effects of caffeine on electrical and mechanical activities of guinea pig taenia coli. Am. J. Physiol. 225, 335 (1973)
265
Cohen, S.: The neurogenic basis of lower esophageal relaxation. J. clin. Invest.
23. Tuch, A . ,
52, 14 (1973) 24. Weisbrodt, N., Christensen, J.: Gradients of
contractions in the opossum esophagus. Gastroenterolcgy 62, 1159 (1972) Sidney Cohen, M.D. Hospital of the University of Pennsylvania 3 4 0 0 Spruce Street Philadelphia, Pa. 19104 U.S.A.
Force Velocity Characteristics of Oesophageal Muscle: Interaction of Isoproterenol and Calcium Sidney Cohen Gastrointestinal Section, Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pa. , U. S.A. Received: June 25, 1974, and in revised form: October 29, 1974
Abstract. The purpose of this study was to evaluate the interaction of isoproterenol and calcium upon the force velocity characteristics of opossum oesophageal circular muscle, in vitro. Isotonic and isometric recordings were used to determine the velocity of shortening and force, respectively. All muscle was studied at its length of optimal tension development (Lo). To determine the effect of isoproterenol upon muscle contractility, studies were performed at 2.5 mM and 5.0 mM calcium Kreb's solution. The maximal velocity of shortening (V max) and the peak force (Po) of oesophageal muscle were 6.2 f 0.3 mm/s and 16.8 f 1.0 gm, respectively, at 2.5 mM calcium. Isoproterenol M) decreased the V max to 4.9 f 0.6 m/s and the Po to 9.8 ? 1.3 gm (P < .01). Increased calcium (5.0 mM), alone, did not alter either V max or Po. However, at 5.0 mM calcium, isoproterenol reduced the V max to 6.0 f 0.6 mm/s (P > .05) and the Po to 13.9 ? 0.8 gm (P < .05). These studies indicate that isoproterenol significantly reduces the V max and Po of oesophageal smooth muscle during the neural mediated "off response". This effect of isoproterenol is reversed, in part, by an increase in calcium concentration.
sues. The midoesophagus and distal duodenum were ligated, and the upper gastrointestinal tract from midoesophagus to distal duodenum was excised, It has been previously demonstrated that the washed in Kreb's solution with the composition in circular smooth muscle of the oesophagus can be millimoles per litre: N a ' , 138.6; Kt 4.6; Ca++, utilized to evaluate the force-velocity properties 2.5; Mg", 1.2; C1-,0126.2i HC03-, 21.9; PO45, 1.2; of muscle during a neural-mediated contraction glucose, 49.6, at 37 - 38 C, and transferred to (6, 7). The prominent contraction obtained upon an organ bath of Krebs-Ringer solution, bubblgd termination of electrical stimulation, the "off wigh 95 Z 02 and 5 I C02 and maintained at 37 response'', is believed to be mediated through the 38 C. The oesophagus was separated from the non-adrenergic inhibitory nerves (15). The "off stomach at the anatomic gastrooesophageal junction response'' has been reported to serve as the initiating mechanism of oesophageal peristalsis (24). where the narrow oesophagus flares into the stomach. The mucosa from each region was removed We have shown that the calculated maximal velocity to the level of the submucosa. Smooth muscle strips, of muscle shortening (V max) and the peak force 0.5 cm wide and 1.0 cm long, were cut from each (Po) obtained during an "off response" are altered anatomic region. All muscles were lightly blotted by calcium concentration, norepinephrine, acetyland weighed at the termination of the experiment. choline and gastrin I (6, 7). The purpose of this Each muscle was studied using the apparatus study is to evaluate the interaction of isoproterenol and calcium upon the force velocity charac- described previously and diagrammatically illustrated in Figure 1 (6, 7, 19, 20, 21). The muscle teristics of oesophageal smooth muscle, in vitro. was mounted to record tension generated by the circular layer. One end of the muscle was connected to an inflexible wire which was attached to an Methods isometric force transducer (Grass Model FT-03C). The other end was attached to the lever of an isoStudies were performed on 20 adult opossums of tonic transducer (Harvard Model 387). Muscle length both sexes weighing 2.4 5.1 kg. The method of was adjusted by a micrometer. Muscle loading was obtaining muscle, outlined below, has previously accomplished through calibrated balance weights been described in detail (23). All animals were placed on the isotonic lever. A stop allowed the killed by intravenous pentobarbital. The oesophamuscle to receive both a preload and an afterload. gus, 8 cm proximal to the anatomic gastrooesophaEach muscle was stimulated electrically across two geal junction, the entire stomach, and duodenum were mobilized and freed from the surrounding tisplatinum wires adjusted to juxtapose the lateral
Introduction
-
S . Cohen: Force Velocity Characteristics of Oesophageal Muscle
260
stimulation but upon termination of stimulation, a prominent neural mediated contraction ("off response") occurred after a brief latent period (15, 2 3 , 2 4 ) . Stimulus parameters were initially selected to elicit the maximal amplitude of this contraction under pure isometric recording conIsotonic Lever ditions. Maximum amplitude of the "off response" was obtained with a 30-second train of square wave pulses of 1.0 millisecond duration, 10 Hz. Either 50 or 60 volts gave the maximum isometric tension for a given muscle. Force velocity curves were constructed for each muscle under various physiological conditions ::::: ---Af terload using the "off response" as a basis of non-tetanic muscle contraction. As shown in Figure 2 the isoPreload metric force generated prior to shortening, P, was Muscle---measured directly from its respective tracing at the first vertical line. The initial velocity of shortening, V, was obtained from the isotonic PIo t i nu m tracing. The initial velocity of shortening was Electrodes determined by the change in length (dl) per unit change in time (dt). Additionally, velocity of shortening was measured directly using a Beckman velocity coupler ( 9 8 4 1 ) . Measurements obtained by Isometric Force both methods correlated closely. The degree of shortening of the muscle during an afterloaded Tr a n s d u c e r contraction was obtained directly from the isotonic recording ( A L). The load at which shortening first Fig. 1 . Schematic diagram of apparatus used to became zero was denoted as Po. The muscle length measure afterloaded muscle shortening. The muscle at which the maximal Po was achieved was denoted was mounted between an isometric transducer and an as Lo. isotonic transducer. A stop allowed the muscle to Force velocity curves were constructed during receive both a preload and an afterload. The muscle the superfusion of Kreb's solution containing was superfused by heated and oxygenated Kreb's 5.0 mM Ca++ as well as the usual 2.5 mM Ca++. After solution to which isoproterenol was added. The the determination of the force velocity relationmuscle was stimulated electrically across two ship at the different calcium concentrations, isoplatinum wires proterenol (10-7 M, 10-6 M, 10-5 M, 10-4 M) was added to the superfusate. The compounds were evaluated in Kreb's solution containing either 2.5 mM surfaces of the muscle. Stimuli were delivered by Ca++ or 5 . 0 mM Ca++. Force velocity determinations a stimulator (Grass Model S44 with stimulus isowere made after a minimum of 10 minutes of superlation unit SIU I). The muscles were superfused by a heated and oxygenated Kreb's solution with the fusion of the Kreb's solutions containing different calcium concentrations or isoproterenol. composition noted above. A thermistor was used to monitor temperature at the muscle. The superfusate The maximum isometric force was also determined during muscle depolarization with a Kreb's solution was maintained at 36' - 38OC. A l l recordings were containing 143.6 mM KC1. These determinations were graphed on a rectilinear, ink-miting polygraph made under strict isometric conditions using only (Beckman) the force transducer. After a 30-minute period, the muscle length was Measurements of velocity of shortening in m d s , increased until the passive tension was at zero shortening in mm, load in gm and Po were placed but ready to increase with any further increase in into a computer program. The constants of the Hill length. This length was denoted as the initial length. From this initial lenght, the preload shown equation as determined from the reciprocal plot of the data were used to determine the extrapolated to bring the muscle to its length of optimal tenV max ( I 1 - 13, 16 2 0 ) . The best fit of the line sion development, Lo, was added to the muscle. The for the displaced rectangular hyperbola of the preload was determined previously from forceforce velocity relationship and the standard devivelocity measurements using the "off response'' ( 6 ) ation of that line were directly plotted by the The change in muscle length caused by each preload computer using the least square regression analywas measured directly by the excursion of the isotonic lever as seen on the recorder. Force velocity sis. Statistical analysis was made for paired comparisons using the Student t test. curves were constructed utilizing afterloads in either 0.5 gm or 1.0 gm increments. To obtain the force-velocity relationship of Results the oesophageal muscle, the neurogenic response elicited at the termination of electrical stimuIn Figure 3 are shown the computer plots of the lation was utilized. The muscle, as previously demonstrated, did not develop active tension during force velocity determination and its linear trans-
Kreb's Sojution Superfusion
Q---
/p .
-
.
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
&
26 I
6.0
C
L
I
I
0
2
4
I
I
6 8 Time ( s e c )
I
10
I
12
Fig. 2 . Simultaneous isometric and isotonic recordings for a single strip of oesophageal muscle at 1.5 gm preload (not shown in resting state) and an afterload of 6.0 gm. When isometric force equaled the total load on the muscle (first verti-
cal line), isotonic shortening began. The velocity of shortening was calculated by measuring the change in length (dl) per unit change in time (dt). Total muscle shortening was measured directly (A L). Time in seconds is along the horizontal axis
formation for a single strip of oesophageal muscle studied at Lo, 2 . 0 gm preload, and 2 . 5 mM calcium. The control force velocity determination is above (a) while the result obtained during a superfusion of M isoproterenol is below (b). The linear transformation was based on the Hill equation (p + a) (V + b) = (Po + a) b where P represents load, V the velocity of shortening, Po the maximum load which the muscle was first unable to move, cz a constant with units of force and b a constant with units of velocity ( 1 1 - 13, 17 - 2 0 ) . In both linearized transformations, the points fell along a straight line with excellent correlation Coefficients suggesting that the force velocity relationship was hyperbolic similar to that reported for other types of muscle. From the linearized plot, the slope ('/b) and the Y intercept (ah) provided the constants for the equation. The theoretical maximum velocity of shortening at zero load (v max) was calculated from the equation: V max = In the presence of isoproterenol, the V rnax was reduced from 6 . 8 7 mmfs to 4 . 1 7 m/s. The Po was reduced from 17 gm to 9.0 gm. Table I shows all data obtained on oesophageal smooth muscle taken at three levels of the oesophagus and studied at 2 . 5 mM calcium. Isoproterenol gave a dose-related decrease in V rnax and PO in all oesophageal muscle. The upper oesophageal muscle seemed most sensitive, showing a sig-
nificant reduction in Po at 10-7 M isoproterenol (P < . 0 5 ) and a significant decrease in V max at M isoproterenol (P < . 0 5 ) . The.lower oesophagus required M isoproterenol to significantly reduce the Po (P < .05) and 10-5 M isoproterenol to reduce the V max (P < . 0 5 ) . These data indicate that at 2 . 5 mM calcium, the force velocity parameters, V max and Po, of the oesophageal muscle could be significantly reduced by isoproterenol. The upper oesophagus seemed more sensitive to this effect than music from the distal oesophagus. Table 2 shows the interaction of an increased calcium concentration ( 5 . 0 mM) with 10-5 M isoproterenol. The 5. 0 mM calcium concentration, alone, did not significantly alter the V max or the Po. However, in the presence of 5.0 mM calcium, 10-5 M isoproterenol had no significant effect upon the V rnax of the oesophageal muscle. The Po was significantly reduced at 5 . 0 mM calcium and 10-5 M isoproterenol (P c . 0 5 ) . However, the reduction in Po was less marked than observed at 2 . 5 d calcium. In ten additional studies performed under isometric conditions, K C 1 depolarization gave an active tension of 18.6 2 . 1 gms at a 2 . 5 mM calcium concentration. Thus, at Lo, the maximal isometric force of the muscle to KC1 depolarization was similar to the Po value (Table I ) achieved during the "off response" at the termination of electrical s t imu1ation,.
.
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
262
Esophageal
Circular
Muscle
62.0 gm Preload 2.5mM Ca++
Po-P V
ys.23 x + 2.47 r = .94
I-
0
2
4
6
8
Load
10
12
0
14 16
l
i
6
8
l
l
I
10 12
I
14 16
Load ( g m )
a 1 ( V t b ) = ( P o ta 1 b
Vmax =
= 6.87 m m / s e c . a
Esophageal
::
l
4
(em) (Pt
\
I
2
Circular
Muscle
-
*
3-
V
r =.92
1-
9
E
0
I
I
1
2
I
I
I
I
I
3 4 5 6 L o a d (gm)
7
8
9
0
(P ta1 (Vtb
Vmax =
I
I
I
1
2
3 4 5 6 Load ( g m )
I
I
I
I
I
1
7
6
9
1= (Pota1 b
- = 4.17mm/scc 0
Fig. 3 . Computer plots of two force velocity curves and their linear transformations for a single strip of oesophageal circular muscle studied at 2.0 gm preload and 2 . 5 mM calcium. The control force velocity curve (a) is shown above, while the curve obtained during isoproterenol (10-5 M) superfusion is shown below (b). The constants of the Hill
equation were obtained from the linear plot of (Po P/V) versus P. The slope of the line was l/b and the intercept on the ordinate was a/b. V max was calculated as (Po/a)b. The dotted lines on the linear plot indicate the standard deviations. In the presence of isoproterenol, the V max and the Po were reduced
-
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
263
Table 1 . Effect of isoproterenol on force velocity determinations of oesophageal smooth muscle at 2.5 calcium I - 2 c mt
Control Isoproterenol
3 - 4 c mt
Po
5 - 6 c mt
Po
V max (mml s)
(gm)
V rnax (mml s 1
6.2 t 0.3
16.8 f 1.0
5.9 f 0.5
17.8 f 1.2
6.2 2 0 . 4
Po
V rnax
(gm)
(gm)
(rn/S)
15.8 +- 1.3
10-4 M
4.3
0.2
7.3 f 0.9
4.0 f 0.3
6.8 2 0.6
3.1 f. 0.3
7.0
10-5 M
4.9 f 0.6
9.8 f 1.3
4.7 t 0.6
7.6
& 1.0
3.6 f. 0.3
7.8 f 0.9
10-6 M
6.1 f 0.9
11.7 f 0.7
5.5 2 0.4
10.0 k 0.6
9.2 t 0.8
10-7 M
6.3 f 0.8
14.9 f 1.1
6.3 f 0.2
13.4 f 1.4
4.8 t 0.5 5.6 f 0.7
'Refers
f
2 0.7
10.4 +- 1.0
to distance in cm above the anatomic gastrooesophageal junction.
Table 2 . Effect of isoproterenol on force velocity determinations of oesophageal smooth muscle at 5.0 mM calcium 3 - 4 c mt
1 - 2 c mt
~
~
Control
5 - 6 c mt
~~
7.2 t 0.5
17.9
6.0 f - 0 . 6
13.9 2 0.8
?
1.2
0.9
18.0 f 1.2
7 . 1 f 0.4
17.6 +- 1.0
6.5 t 0.9
12.8 5 1.3
7.5 f. 0. 5
14.4 f 0.8
6.9
5
Isoproterenol 10-5 M
tRefers to distance in cm above the anatomic gastroesophageal junction.
Discussion
that the "off response" or rebound excitation and non-adrenergic inhibition were both mediated by nerves which released adenosine triphosphate (3, The purpose of this study was to evaluate the 4 ) . We have suggested that these nerves mediate interaction of isoproterenol and calcium upon the not only peristalsis but also lower oesophageal force velocity characteristics of opossum circular sphincter relaxation and contraction (23). smooth muscle, in vitro. These studies indicated In previous studies we have shown that acetylthat isoproterenol significantly reduced the V max choline, norepinephrine and gastrin I increased and Po of this muscle during the neural-mediated the V max and Po during the off response ( 6 , 7 ) . "off response". The reduction in force velocity The response to these agents was most marked at parameters associated with isoproterenol was partially reversed by an increased calcium concena calcium concentration of 1.0 mM. The present studies indicated that isoproterenol reduced the trat ion. V rnax and Po o f the oesophageal muscle and that Studies in vitro using the intact opossum oesothis effect was in part reversed by an increased phagus and oesophageal smooth muscle strips decalcium concentration (5.0 mM). The finding that monstrated responses localized to different layers isoproterenol decreased the V m a x of oesophageal of muscle (5). Upon deflation of a balloon in the muscle at 2.5 mM calcium and not at 5.0 mM calcium intact oesophagus, in vitro, a peristaltic consuggested that isoproterenol had exerted its eftraction was observed. This peristaltic contraction corresponded to the "off response" at the ter- fect through an alteration in calcium mobilization. mination- of electrical stimulation as recorded from It is generally accepted that calcium is essential the circular muscle layer. The prominent "off for excitation contraction coupling in muscle of response" was abolished by tetrodotoxin, but not all types (2, 8 10, 14, 16, 20, 22). Furthermore, it has been suggested that adrenergic agents by adrenergic or cholinergic antagonists (15). This response may correspond to rebound excitation may inhibit release of-calciumfrom an intracellular store (the endoplasmic reticulum) or inhibit seen in muscles containing non-adrenergic inhibicalcium entrance into the muscle from the extratory nerves ( I ) . It had been suggested recently
-
264
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle
cellular space (10). The effect of a beta adrenergic agonist in this preparation may diminish the availability of calcium for excitation contraction coupling of the muscle. This observation would fit with the previous suggestion that excitatory agents such as gastrin I, acetylcholine and norepinpehrine increased the availability of calcium which in turn increased the V max of the muscle. Thus, it would seem that each agent shown to affect the force velocity properties of oesophageal smooth muscle acted through a common factor, calcium. The validity of this conclusion is dependent upon the interpretation of data obtained during a neural mediated response of smooth muscle. The force velocity relationship was described originally utilizing the tetanic response of skeletal muscle to electrical stimulation ( 1 1 - 13). This was subsequently modified to evaluate the single twitch elicited during direct electrical stimulation of cardiac muscle (17 - 20 ) . The studies described above evaluated the single neurogenically elicited twitch of oesophageal muscle upon the termination of electrical stimulation. The maximal isometric force (Po) during the off response was similar to the maximal tension achieved during muscle depolarization with 143.6 mM KC1. This observation suggested that the maximum isometric force could be achieved during this neural mediated muscle response. The Hill equation was used to derive V max, the theoretical maximum velocity of shortening at zero load. The equation, (P + a) (V + b) = (Po + a)b, expressed the force velocity relationship as a displaced rectangular hyperbola with asymptotes at minus a and minus b ( 1 1 - 13). The constants a and b have dimensions of force and velocity, respectively. These constants have been calculated only for frog sartorius and equal the extra heat liberated with shortening (a) and the rate of heat liberation (b) ( 1 1 - 13). Despite limitations in using the Hill equation to derive the constants from mechanical measurements, other smooth muscles have been evaluated in this manner allowing comparison with these data (16, 21). The observations on oesophageal muscle when evaluated using the Hill equation did form a linear relationship with a high correlation coefficient (all above .90) suggesting that the force velocity relationship could be described as a displaced hyperbola. Thus, the neural mediated contraction of oesophageal muscle appeared to have a force velocity relationship similar to other muscles. Additonally, the Po was similar to the peak tension achieved during maximal KC1 depolarization. The question as to whether isoproterenol and calcium exerted their effect solely upon myogenic rather than neurogenic elements cannot be answered at this time.
Bibliographg
Acknowledgements. This work was supported by Research Grant 1 R01 AM 16280-01 and Research Career Development Award 1 KO4 AM 70576-01 from the National Institutes of Health. The author wishes to thank Mrs. Fe Green for her expert technical assistance, and Miss Mary Carroll for secretarial assistance.
17.
1 . Benett, M.: Rebound excitation of the smooth
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12. 13.
14.
15. 16.
18.
muscle cells of the guinea-pig taenia coli after stimulation of intramural inhibitory nerves. J. Physiol. 185, 124 (1966) Bockman, E.L., Rubio, R . , Berne, R.M. : Effect of lanthanum on isoproterenol-induced activation of myocardial phosphyorylase. Am. J. Physiol. 225, 438 (1973) Burnstock, G.: Purinergic nerves. Pharmacol. Reviews 24, 509 (1972) Burnstock, G., Campbell, G., Satchell, D., Smythe, A.: Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitdry nerves in the gut. Brit. J. Pharmacol. 40, 668 (1970) Christensen, J., Lund, G.: Esophageal responses to distension and electrical stimulation. J. clin. Invest. 48, 408 (1969) Cohen, S., Green, F.: The mechanics of esophageal muscle contraction: evidence of an inotropic effect of gastrin. J. clin. Invest. 52, 2029 (1973) Cohen, S., Grenn, F.: Force velocity characteristics of esophageal muscle; effect of acetylcholine and norepinephrine. Am. J. Physiol. In press Durbin, R., Jenkonson, D.: The effect of carbachol in the permeability of depolarized muscle to inorganic ions. J. Physiol. 157, 74 (1961) Edman, K., Schild, H.: The need for calcium in the contractile responses induced by acetylcholine and potassium in the rat uterus. J. Physiol. 161, 424 (1962) Edman, K., Schild, H.: Calcium and the stimulant and inhibitory effects of adrenaline in depolarized smooth muscle. J. Physiol. 169, 404 (1963) Hill, A.: The heat of shortening and the dynamic constants of muscle. Proc. roy. SOC. Med. 126, 136 (1939) Hill, A.: A discussion on muscular contraction and relaxation: their physical and chemical basis. Proc. roy. SOC. Med. 137, 40 (1950) Hill, A . : Mechanics of the contractile element of muscle. Nature 166, 415 (1950) Kirby, A., Lindley, B.D., Picken, J.R. : Calcium dependence of potassium contractures in denervated frog muscle. Am. J. Physiol. 225, 166 (1973) Lund, G., Christensen, J.: Electrical stimulation of esophageal smooth muscle and effects of antagonists. Am. J. Physiol. 217, 1369 (1969) Siegman, M., Gordon, A . : Potentiation of contraction: effects of calcium and caffeine on active state. Am. J. Physiol. 222, 1587 (1972) Sonnenblick, E., McCallum, Z.: Active state, force velocity relationships and inotropic mechanisms in mammalian papillary muscle. Fed. Proc. 20, 126 (1961) Sonnenblick, E.: Force-velocity relations in mammalian heart muscle. Am. J. Physiol. 202, 931 (1962)
S. Cohen: Force Velocity Characteristics of Oesophageal Muscle 19. Sonnenblick, E.: Series elastic and contrac-
tile element interactions in generation of myocardial force: effects of changing muscle length, norepinephringe and strophanthidin. Fed. Proc. 23, I I8 (1964) 20. Sonnenblick, E.: Implications of muscle mechanics in the heart. Fed. Proc. 21, 975 (1962) 21. Stephens, N., Kroeger, E., Mehta, J.: Force.velocity characteristics of respiratory airway smooth muscle. J. appl. Physiol. 26, 685 (1969) 22. Sunano, S., Miyazaki, E.: Effects of caffeine on electrical and mechanical activities of guinea pig taenia coli. Am. J. Physiol. 225, 335 (1973)
265
Cohen, S.: The neurogenic basis of lower esophageal relaxation. J. clin. Invest.
23. Tuch, A . ,
52, 14 (1973) 24. Weisbrodt, N., Christensen, J.: Gradients of
contractions in the opossum esophagus. Gastroenterolcgy 62, 1159 (1972) Sidney Cohen, M.D. Hospital of the University of Pennsylvania 3 4 0 0 Spruce Street Philadelphia, Pa. 19104 U.S.A.
