affect contractions in cardiac tissue (Koch-Weser & Blinks, 1963), and in

Transcription

1 J. Physiol. (1965), 18, pp With 12 text-figures Printed in Great Britain THE RELATION BETWEEN RESPONSE AND THE INTERVAL BETWEEN STIMULI OF THE ISOLATED GUINEA-PIG URETER BY A. W. CUTHBERT From the Department of Pharmacology, University of Cambridge (Received 1 October 1964) Ureteral smooth muscle behaves as a functional syncytium; all-or-none contractions originate at the renal end of the ureter, which is considered to be the pacemaker (Bozler, 1942 a), and pass to its lower end. Thus, there is a superficial resemblance to the heart which also shows decrementless conduction and has pacemaker regions. In addition, both the ureter and cardiac tissue have plateau-type action potentials (Bozler, 1942b; Burnstock & Prosser, 196). The interval between successive stimuli is known to affect contractions in cardiac tissue (Koch-Weser & Blinks, 1963), and in this paper the relation between contractions and the interval between stimuli in the directly stimulated, isolated, guinea-pig ureter has been examined. METHODS Ureters were dissected from freshly killed guinea-pigs and placed in Tyrode's solution. Connective tissue, surface nerves and blood vessels were removed by fine dissection. Ureters prepared in this way were used either suspended in an isolated-organ bath or mounted in a sucrose-gap electrode. Isolated-organ bath The renal end of the ureter was pulled through a shielded platinum-ring electrode held below the surface of the Tyrode's solution in an isolated-organ bath, and the distal end was attached to the recording device. The other stimulating electrode consisted of a silver wire which dipped into the Tyrode's solution. The stimulating electrodes were connected to a Grass stimulator which delivered maximal cathodal shocks to the renal end of the ureter. Three types of recordings were made. First, isotonic shortening by means of a lightly weighted (2 mg approximately) lever moving over a smoked drum. Alternatively, isometric tension changes were measured by a mechano-electronic transducer, using a RCA 5734 valve, records being obtained either on film from an oscilloscope or with a chart recorder (Leeds & Northrup Speedomax H). The organ bath was maintained at 37 C and the Tyrode's solution was gassed with air. Sucrose-gap electrode The method used was similar to that of Bulbring & Burnstock (196) in which provision was made for the simultaneous recording of isometric tension together with changes in membrane potential. A length of ureter of about 2-5 cm was mounted in the apparatus so that the renal end passed between a pair of platinum stimulating electrodes. The preparation was stimulated with maximal cathodal shocks through a radio-frequency isolation unit. 15 Physiol. 18

2 226 A.W.CUTHBERT Changes of membrane potential were measured from a pair of chlorided silver wires, embedded in 2% agar gel containing 3 M-KCI, through a double ended cathode follower. Simultaneous changes of muscle tension were measured from a mechano-electronic transducer. Both the electrical and mechanical changes were recorded photographically from an oscilloscope. The sucrose solution used contained 1% sucrose (w/v) in distilled water, and was passed through a de-ionizing column before use. The solution flowing through the inactive side of the gap was either Tyrode's solution or isotonic K2SO4 solution at room temperature. Tyrode's solution, at C and gassed with air, flowed through the active side of the apparatus. Materials used The Tyrode's solution had the following composition: (mm) NaCl, 137; KCI, 2-7; MgCl2, 1-5; CaCl2, 1-8; NaH2PO4, -4; NaHCO3, 11-9; and glucose, 5-6. RESULTS Guinea-pig ureters have been found to respond well to electrical stimuli for several hours when suspended in Tyrode's solution at 37 C. However, during this time there was a gradual decline in size of the steady-state contractions at a particular frequency, but the ratio of the contractions at two different frequencies remained unchanged throughout the experiment. It is for this reason that changes in contraction heights have been expressed as percentages rather than in absolute terms. Stimuli far in excess of threshold have been used throughout to ensure responses at the higher frequencies (6 and 12/min). High-voltage stimuli of long duration were found to cause visible damage to the ureter in the region of the cathode and this may account, in part, for the gradual reduction in the size of the responses during an experiment. The response to paired stimuli The responses of the ureter were independent of stimulus strength providing the threshold strength was exceeded. Isotonic shortening in response to paired stimuli of increasing strength is shown in Fig. 1. When the stimulus strength was just above threshold the second stimulus was ineffective. When both stimuli were effective there was no increase in the size of the second response or change in the ratio of the two contraction heights with increasing stimulus strength. Therefore, under given conditions, the ureteral response is an all-or-nothing response. Experiments have been made with supramaximal paired stimuli in which the interval between stimuli was varied. Such an experiment is illustrated in Fig. 2, from which it can be seen that changes occur after a ureteral contraction which can reduce or increase the size of a subsequent contraction. The curve in Fig. 3a shows the relation between the percentage reduction in size of the second compared with the first contraction in each pair and the interval between stimuli. Positive values refer to pairs of responses in which the second response was smaller than the first,

3 URETERAL CONTRACTIONS 227 whereas negative values refer to pairs of responses in which the second response was greater than the first. When isometric contraction force was used as a measure of paired contractions a curve such as that shown in Fig. 3b was obtained. From this it is seen that reducing the stimulus interval has a less marked effect than when isotonic shortening is used as a measure of contractility. In this preparation the potentiating effect of a single contraction was apparent even after 1 sec V Fig. 1. Isotonic shortening of an isolated guinea-pig ureter in response to pairs of stimuli. The interval between the stimuli was 1 sec, and between the pairs was 2 min. The stimulus voltage is shown below each panel, pulse duration 1 msec Fig. 2. Isotonic shortening in response to pairs of stimuli given at 3 min intervals. The interval (in sec) between the stimuli of each pair is indicated on the figure. The order of the pairs was random but the records have been placed in sequence. The response to trains of stimuli Steady-state contractions are defined as identical responses in response to a train of stimuli at a given frequency. An interval-force curve, relating the interval between stimuli to the isometric contraction force of 15-2

4 228 A.W.CUTHBERT steady-state contractions, is shown in Fig. 4. Figure 5 shows the time course of single steady-state contractions at the various frequencies used. This figure was made by tracing individual contractions on the recording film through an enlarger. It is seen that a reduction in the interval between stimuli to below 5 sec causes a decline in the isometric contraction force, whereas the responses at 5 sec intervals were slightly greater than those at 1 sec intervals. Thus the interval between stimuli affects steady-state 1 a 8 C._4 c) be Ca Interval between stimuli (sec) 5 1 a.._ C) bo Ca 6 r b 4 F I I I Interval between stimuli (sec) Fig. 3. Results from experiments with paired stimuli given at 3 min intervals. Ordinate: percentage reduction in response to the second compared with first stimulus. Abscissa: interval between stimuli. The responses were, in a, isotonic shortening, and, in b, isometric contractions of ureters. I I

5 URETERAL CONTRACTIONS 229 contractions and paired responses in similar ways. The time course of steady-state contractions at different frequencies was similar if the initial slow contraction phase was disregarded. This slow phase is thought to be a local non-propagating response initiated in the region of the stimulating electrode. The time to half-relaxation of each contraction is shown in Fig. 5. Such small changes as do occur are not in a consistent direction with respect to the increase in frequency and are probably due to experimental error. The velocity of isotonic shortening of a lightly loaded ureter decreased with a reduction in the interval between stimuli as shown in Fig b- 5 a 25.1 I 1 I I I Stimulus interval (sec) Fig. 4. An interval-force curve for an isolated guinea-pig ureter. The isometric tension developed by steady-state contractions is plotted against the interval between stimuli. The potentiating effect of a ureteral contraction on a subsequent contraction occurring some 5-1 sec later has already been described. This positive inotropic effect can be demonstrated in other ways. First, responses obtained after a long period of rest (3 min) show a positive staircase as illustrated in Fig. 7. When the stimulus interval was long (1 sec as in Fig. 7a) a gradual increase in size to the steady-state level is seen. At somewhat shorter intervals (5 sec in Fig. 7 b and c) there is an initial reduction in the response followed by a secondary rise, to give a steadystate level greater than the first response. Even shorter intervals (2 sec

6 23 A. W. CUTHBERIT in Fig. 7 d) produce a similar pattern but the final steady-state level is less than that of the first response. The initial reduction seen in Fig. 7 b, c and d is due to the accumulation of negative inotropic effects, whereas the secondary rise is due to an increase in the level of positive inotropic effects. Thus the ureteral response at any instant is the sum of a true rested state contraction (a contraction not influenced by previous activity) I1 I 1 I sec I 1 ~~~~~~~~I/ I II~I I /~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ I' Fig. 5. Time course of isometric tension development of steady-state contractions at various intervals between stimuli. The intervals (in sec) and the time to half relaxation (in sec from the beginning of each extension shown by an interrupted line) are shown respectively above and below each contraction. 5 Fig. 6. The speed of isotonic shortening at various intervals between stimuli. When steady-state conditions were obtained, as shown by the records at low speed, a single contraction was recorded at high speed. The interval between stimuli, in sec, is shown above each high-speed record.

7 URETERAL CONTRACTIONS 231 and any accumulated positive inotropic effect minus any accumulated negative inotropic effect. 5 b 1 a ----J --c flllul Ll] 4 5 c 2 d Fig. 7. Ureteral responses after a prolonged rest period of 3 min (a and b) and 15 min (c and d). Isometric contractions are recorded in a and b and isotonic shortening in c and d. The intervals between stimuli (in sec) are indicated above each trace. Note traces a and b are read from right to left. The stimulus duration was 1 sec in a and b and 1 msec in c and d. The responses shown in Fig. 8 represent isometric contractions in response to stimuli at 1 sec intervals after varying rest periods. When the rest periods were short (-15 min) the responses declined from the initial response due to the accumulation of negative inotropic effect. After longer periods of rest the responses at first increased and then declined, and only in these cases can the initial response be regarded as a true rested state contraction. This would indicate that the changes which occur on contraction to increase the size of subsequent contractions persist for some 2-3 min. If, however, another contraction occurs during this time an extra potentiating effect occurs which is detectable for only some 5-1 sec, as indicated by Fig. 3a. This second, more evanescent, type of effect can be shown to accumulate after a period of rapid stimulation (Fig. 9). In this experiment a ureter was stimulated at a frequency which produced little depression of the responses. When steadystate conditions had been reached the frequency of stimulation was increased for some minutes followed by a return to the original frequency. The period of rapid stimulation caused a considerable decrease in the size of the responses but the depressive effects of this period of rapid stimulation quickly disappeared when the original frequency was resumed,

8 232 A. W. CUTHBERT a b 1-5 g Fig. 8. Isometric contractions of two ureters in response to stimuli at 1 sec intervals after various rest periods. The rest period in min is indicated below each panel. The transition in the configuration of the contraction envelope takes place between 2 and 2 min in a and between 1 and 2 min in b. The atypical shape of the envelope at 9 min in a is thought to have resulted from spontaneous contraction(s) occurring during the rest period. Records to be read from right to left. The stimulus duration throughout was 1 sec Fig. 9. Isotonic shortening in response to stimulation at various frequencies. The intervals between stimuli (in see) are indicated on the figure. An increased response on returning to an interval of 5 sec was seen. The stimulus duration was 1 msec.

9 URETERAL CONTRACTIONS 233 at which time the responses were larger than at the beginning of the experiment. The action potential and the interval between stimuli The action potential of the ureter is of the plateau type and in the case of the guinea-pig has superimposed spikes. The records obtained with the sucrose-gap electrode, in this work, are similar to those recorded by others with intracellular micro-electrodes (Bennett, Burnstock, Holman & Walker, 1962). Action potentials have been recorded from 2 ureters using the sucrose-gap electrode. The stimulus interval was found to affect the amplitude and duration of the action potential, and also the conduction velocity in the ureter. The changes in conduction velocity were measured from the latency between the stimulus artifact and the action potential. Similar observations have been made by Irisawa & Kobayashi (1963). The change in conduction velocity was the most marked effect and occurred in all preparations. Changes in the duration of the action potential were usually present but the effect was more marked in some experiments than in others. Changes in the amplitude of the action potential were the least obvious, and were only marked in one experiment. Changes in the action potential or conduction velocity occurred at stimulus intervals which were consistent with those which caused changes in contraction heights as reported in the previous sections. From Fig. 1 it is seen that at an interval of 5 sec there was no change in the conduction velocity with the first and second responses after a rest, whereas when the interval was reduced to 1 sec the conduction velocity was greatly reduced with the second response following a rest. Subsequent responses showed an even greater delay which finally stabilized with the eighth response. An increase of stimulus interval to 1 sec produced the opposite result, that is, the conduction velocity was increased and remained steady at the value obtained with the second response following a period of rest. In this particular preparation there was no change in the duration of the action potential and only minor changes in its amplitude. For instance, at 1 sec intervals the amplitude of the initial peak was depressed, but the over-all amplitude was increased. The decrease in duration which usually occurred with consecutive stimuli is illustrated in Fig. 11. Each panel is a record obtained after a rest period of 3 min. It is seen that the action potentials of the second and subsequent responses in each panel have shortened plateaux compared with the first re3ponse, also the number of oscillations in potential during the plateau phase was reduced. Changes in the duration of the action potential were associated with changes in the isometric tension response. No change in amplitude of the action potential occurred with the results illustrated

10 234 A.W.CUTHBERT in Fig. 11, but the increase in latency with reduction in the interval between stimuli was marked. In Fig. 12 changes in amplitude of the action potential with concomitant changes in the tension response are recorded. Changes in amplitude of the action potential of the extent illustrated here have only been seen once. Attempts were made to discover a relation between the duration, amplitude and velocity of the action potential and the isometric tension developed. However, no simple relation between these three parameters and the isometric contraction j 1 b -2 sec c j Fig. 1. Action potentials recorded in sucrose-gap electrode in response to stimuli of 12 msec duration. The stimulus interval was 1, 5 and 1 sec in a, b and c, respectively. Each group of action potentials was recorded after a rest period of 2 min. Each action potential is numbered in order and the records were traced so that the stimulus artifacts were superimposed.

11 URETERAL CONTRACTIONS 235 force was found. This was probably due to the fact that the three parameters varied independently. Also the tension measurementwas made from a large piece of tissue compared with the small area from which electrical recordings were obtained, and it cannot be assumed that the extent of the changes in the action potential configuration was constant throughout the preparation. Fig. 11. Each panel shows the electrical and mechanical (isometric) responses of a piece of guinea-pig ureter to stimulation after a 2 min rest period. The interval between stimuli was 3-3 sec in a and b, 5 sec in c and d and 1 sec in e andf. The reduced mechanical response is coincident with a reduction of the action-potential duration and an increase in the action-potential latency. DISCUSSION Discussion of the results presented in this paper can be conveniently arranged under three headings. First, an explanation of the results in terms of those changes, produced by contraction, which reduce and which increase the size of subsequent contractions. Secondly, the equivalence of the observed mechanical changes to changes in configuration of the action potential. Finally, a comparison of these results with the inotropic effects produced by contraction in other muscular tissues will be discussed.

12 236 A.W.CUTHBERT The effects of a given pattern of stimulation on the responses of the ureter may be predicted from rules derived from the experimental results. The negative inotropic effect following a ureteral contraction is large and persists for some 4-5 sec, whereas the positive inotropic effect is small but persists longer, about 1 sec. However, in the case of a true rested state contraction the positive inotropic effect is large and greater than the negative effect and persists for some 2-3 min. This is shown by the increased responses obtained after a true rested state contraction and by the 1 mnv ~~~~~~~~~16-6 _J 5 Fig. 12. Electrical and mechanical (isometric) responses of a guinea-pig ureter recorded with the sucrose-gap electrode. Each panel was recorded after a rest period of two minutes. The interval between stimuli (in sec) is shown below each panel. Reduction in the action-potential amplitude coincides with a reduced tension response. long rest periods necessary before a true rested state contraction can be elicited. The differences between the inotropic effects following a normal contraction and a true rested state contraction can be explained by assuming that inotropic effects from consecutive contractions are additive and that the rate of decay of the positive effect increases with the amount accumulated. A change in the configuration or latency of the action potential will affect both the isometric tension developed and the speed of isotonic

13 URETERAL CONTRACTIONS 237 shortening. If a contraction wave moves along a ureter with a velocity, V, and if the action potential has a duration, t, then the length of ureter depolarized at any one time will be Vt. Using typical values of 3 cm/sec for V and of 1 sec for t this length is of the order of 3 cm. It is reasonable to assume that depolarization and contraction are coupled in smooth muscle (Csapo, 196), so that the length depolarized is related to the length of the ureter which is in a contracted state. A reduction in either V or t will reduce the number of cells activated in a wave of contraction, and as a consequence the isometric tension developed and the velocity of shortening will be reduced. In the ureter changes of conduction velocity with stimulus interval have always been seen and can partially account for changes in contractions on the basis of synchronization as outlined above. The reduced tension response which sometimes occurred together with a reduction in amplitude of the action potential is explained by supposing that at short stimulus intervals some of the muscle fibres fail to be activated. This may be due to conduction failure at the myo-myo junctions. On this basis the potentiating effect is explained by the recruitment of additional fibres. Proof of these explanations would require the simultaneous recording of intracellular potentials from many muscle fibres. The strength of ureteral contractions will depend upon the degree of activation of the muscular elements and the duration of the active state, and the latter will be related to the duration of the action potential. In those preparations in which the duration of the action potential was reduced, the reduction in the mechanical response can be explained, in part, by a reduction in duration of the active state. Not all preparations showed a change in duration of the action potential, and furthermore the time course of steady-state contractions was little affected by increasing frequency. It appears that changes in the amplitude and velocity of the action potential cause changes in the degree of activation in the ureter, whereas changes in the actionpotential duration affect the duration of the active state. From these results it is not possible to state the relative contributions that changes in activation and in active state have on ureteral contractions. Neither is it possible to know whether other changes in the degree of activation occur with changes in the interval between stimuli which are not reflected by changes in the action potential. Interval strength phenomena have been most closely studied in heart muscle and it is of interest to compare results for the heart with those of the ureter. Using the atria of several mammals and with rat papillary muscle Blinks & Koch-Wester (1961) found that each beat produced a large negative inotropic effect and a small positive inotropic effect. The former was found to decay rapidly while the latter decayed more slowly

14 238 A.W.CUTHBERT and at a rate proportional to the amount present. Similar results were reported in this paper for the ureter. Most of the interval-dependent changes in heart muscle result from changes in the degree of activation but in mammalian ventricular muscle (Koch-Wester, 1963) and frog-heart muscle (Niedergerke, 1956), changes in the duration of the active state can play a large part in determining the effect of the interval between stimuli on contractions. Similarly, in the guinea-pig ureter, changes in active state and the degree of activation contribute to the changes in the contractile response. SUMMARY 1. Isometric contractions and isotonic shortening of isolated guinea-pig ureters, stimulated directly, have been measured under various conditions. 2. The interval between stimuli was found to affect the contractions of the guinea-pig ureter. The negative inotropic effect due to a single contraction was large and decayed to zero in about 4 sec. The positive inotropic effect due to a single contraction was small and persisted for 5-1 sec. Both the positive and negative effects were shown to accumulate under some conditions. 3. A true rested state contraction, that is, one uninfluenced by previous activity, was obtained only after rest periods of 2-3 min. 4. Simultaneous recording of the ureteral action potential anld the isometric tension showed that interval-dependent changes in the tension response coincided with changes in the configuration of the action potential and its latency. REFERENCES BENNETT, M. R., BURNSTOCK, G., HOLMAN, M. E. & WALKER, J. W. (1962). The effect of Ca2+ on plateau-type action potentials in smooth muscle. J. Physiol. 161, P. BLINKS, J. R. & KOCH-WESER, J. (1961). Analysis of the effects of changes in rate and rhythm upon myocardial contractility. J. Pharmacol. 134, BOZLER, E. (1942a). The activity of the pacemaker previous to the discharge of a muscular impulse. Amer. J. Physiol. 136, BOZLER, E. (1942b). The action potentials accompanying conducted responses in visceral smooth muscles. Amer. J. Physiol. 136, BULBRING, E. & BURNSTOCK, G. (196). Membrane potential changes associated with tachyphylaxis and potentiation of the response to stimulating drugs in smooth muscle. Brit. J. Pharmacol. 15, BuRNSTOCK, G. & PROSSER, C. L. (196). Conduction in smooth muscles: comparative electrical properties. Amer. J. Physiol. 199, CsAPo, A. (196). Molecular structure and function of smooth muscle. In Muscle, Structure and Function, 1, New York and London: Academic Press. IRISAWA, H. & KOBAYASHI, M. (1963). Effects of repetitive stimuli and temperature on ureter action potentials. Jap. J. Physiol. 13, KoCH-WESER, J. (1963). Effect of rate changes on strength and time course of contraction of papillary muscle. Amer. J. Physiol. 24, KOCH-WESER, J. & BLINKS, J. R. (1963). The influence of the interval between beats on myocardial contractility. Pharmacol. Rev. 15, NIEDERGERKE, R. (1956). The 'staircase' phenomenon and the action of calcium on the heart. J. Physiol. 134,