Calcium Channel Blockade in Smooth Muscle of The Human Upper Urinary Tract. I. Effects on Depolarization-Induced Activation

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THE JOURNAL OF UROLOGY

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1984 by The V1/illiams & Vlilkins Co.

CALCIUM CHANNEL BLOCKADE IN SIVfOOTH Iv1USCLE HUMAN UPPER URINARY TRACT, L EFFECTS ON DEPOLARIZATION-INDUCED ACTIVATION LOTHAR HERTLE*

AND

THE

HERMANN NAWRATH

From the Department of Urology, University of Bochum, and the Department of Pharmacology, University of Mainz, Federal Republic of Germany

ABSTRACT

The effects of the calcium blockers nifedipine, verapamil, D600 and diltiazem on mechanical activity were studied in isolated preparations of the human upper urinary tract. Two types of activity were used: spontaneous phasic-rhythmic activity in calyceal segments and potassiuminduced depolarization in ureteral muscle strips. Nifedipine (10- 6 mol./1.), verapamil, D600 and diltiazem (all 10-s mol./1.) completely suppressed spontaneous phasic-rhythmic activity. Elevation of extracellular potassium concentration induced contractions concentration-dependently. A loglinear relationship between the extracellular calcium concentration and the 85 mmol./1. potassiuminduced activation was demonstrated. Concentration-response relationships of the compounds were found by activating the muscle strips with 85 mmol./1. potassium in the Tyrode solution. This activation model produced stable and reproducible contractures. The compounds antagonized depolarization-induced activations concentration-dependently, nifedipine being the drug with the lowest EC 50 value; its relative potency with reference to papaverine was about 8,000 to L The order of potency of the other drugs was in the following sequence: D600 > verapamil > diltiazem. It is concluded that processes in the human upper urinary tract which are triggered by depolarization (action potential or high potassium concentrations) are highly sensitive to calcium channel blockers. The tension developed by the contractile proteins of the smooth muscle cell is believed to depend primarily on the concentration of ionized calcium within the cell cytoplasm. The transition from the resting state to maximal activation is thought to occur in the range of approximately 10- 7 to 10- 5 mol./1. cytoplasmic free calcium concentrations, at least according to studies of glycerinated or otherwise chemically skinned smooth muscle. 1' 2 The mobilization of activator calcium by various stimulatory agents can occur by a limited number of mechanisms that include uptake of calcium from the extracellular compartment or associated superficial sites as well as release of the ion from membrane or cellular binding sites or stores. 3 Stimulation by agonists and by depolarization are the 2 most prominent means of activation of smooth muscle, The former acts by binding to specific receptors on the smooth muscle membrane and the latter acts by depolarization of the cell membrane which is not related to any receptm activaand most important tion. The action potential is the mechanism by which a rise in free intracellular calcium is produced in the smooth muscles that normally generate and propagate action potentials. The velocity of propagation of an action potential in a smooth muscle organ such as the upper urinary tract is sufficient to cause a well-synchronized contraction of the smooth muscle cells involved, resulting in a phasic contraction of smooth muscle strips which are often used for in vitro studies. The first direct attempt to examine the role of the action potential in causing contraction was to produce a potassium depolarization. The muscle cell has a resting potential close to potassium equilibrium potential, because the membrane is more permeable to potassium than to any other ion. This potential is intracellularly negative relative to the outside potential, because potassium is low in the extracellular solution and high Accepted for publication August 13, 1984. * Requests for reprints: Urologische Klinik, Ruhr-Universitiit Bochum, Klinikum Marienhospital, Widumer Str, 8, D-4690 Herne 1, Federal Republic of Germany.

in the intracellular solution. Consequently, raising the extracellular potassium would decrease the potassium concentration gradient and depolarize the cell. This -experiment was systematically performed with skeletal muscle by Kuffler4 who found that it produced a sustained contraction, called a contracture. Potassium depolarizations were used in smooth muscle to demonstrate that tension appears to be a graded function of membrane potentiaL 3 ' 5 In the past decade a group of drugs, the so-called calcium channel blockers, have been utilized as pharmacological tools to elucidate and characterize specific mechanisms involved in calcium-requiring processes. The mechanisms of action of these substances, typified by verapamil and nifedipine, have been postulated to be blockade of potential-sensitive calcium channels in the muscle cell membrane which reduces the entry of calcium to the intracellular compartment and causes relaxation. This mechanism has been shown for cardiac muscle, vascular smooth muscle and several other tissues, 6 - 8 The present study was undertaken to investigate the effects of the calcium channel blockers verapamil, nifedipine and dialtiazem on spontaneous phasic-rhythmic and on potassium-induced contracture in muscle strips of human upper urinary tract. The 2nd part of this communication describes the effects of these drugs on an agonist-induced activation. MATERIALS AND METHODS

Human tissue was obtained from kidneys removed at operation for renal carcinoma. The age of the patients ranged between 40 and 65 years. Immediately after excision of the kidney the upper third of the ureter was dissected free and removed for further preparation. In a dissection chamber containing warm, oxygenated Tyrode solution the ureter was opened and longitudinal muscle strips approximately 10 by 3 to 4 mm. weighing 50 to 120 mg. were excised. In order to obtain calyceal segments, that part of the kidney unaffected by tumor was dissected to display papillae projecting into minor calyces. The distal halves of the papillae were then removed to fully expose

1265

1266

HERTLE AND NAWRATH

the forniceal region. Strips of calyceal tissue were carefully dissected from around the base of the papillae in a circumferential manner. To be certain of including the most proximal calyceal tissue a narrow border of papilla base was left on the strips. By tying both ends with fine silk sutures (5 X O) the preparations were attached to a tissue pillar and mounted vertically in a muscle chamber containing 5 ml. Tyrode solution. The upper end of the muscle strips was connected to an inductive force displacement transducer (built in our workshop by H. Fleck) by means of a stainless steel wire. Tension was recorded under isometric conditions and registered on a Hellige paper recorder. Preload tension was adjusted to 10 mN. Four preparations were taken from 1 kidney and investigated simultaneously in 4 thermostatically controlled organ baths (37C). Solutions. The Tyrode solution used was prepared with distilled deionized water and had the following composition (mmol./1.): NaCl 136.9, KCl 5.4, MgCl 2, 1.05, NaH 2P0 4 0.42, NaHCOs 11.9, CaCb 1.8 and glucose 5.5. The Tyrode solution in the organ baths was continuously gassed with 95 per cent 0 2 and 5 per cent CO 2 • The pH of the solution was 7.2 to 7.4. The experimental arrangement permitted a rapid exchange (1 to 2 sec.) of solutions. The Tyrode solution used for depolarization-induced contractions contained 85 mmol./1. KCl and 57.3 mmol./1. NaCl. The other constituents remained unchanged. In the experiments with various potassium concentrations (27 to 142 mmol./ 1.) the solutions were made by replacing NaCl in normal Tyrode solution with equimolar amounts of KCl in logarithmic increments. Cumulative concentration-response curves to CaCl2 (0.9 to 9.0 mmol./1.) were obtained at 85 mmol./1. KCl by increasing the calcium concentrations in the Tyrode solution also in logarithmic increments. Stock solutions ofverapamil, D600 and diltiazem were freshly prepared in distilled water. The stock solution of nifedipine was prepared in ethanol. From these stock solutions, desired concentrations of the drugs were prepared in Tyrode solution and added in appropriate volume into the muscle baths to give the desired concentrations. Ethanol in the concentrations used had no effect on the contractions. The pH was maintained at 7.2 ± 0.1. Drugs. The following drugs were used in this investigation: verapamil and D600 (methoxyverapamil) (Knoll AG), diltiazem(Godecke), nifedipine (Bayer AG) and papaverine hydrochloride (Serva). The experiments with nifedipine were carried out under sodium lighting to prevent the breakdown of this substance that occurs at longer wavelengths. Experimental Procedure. All tissues were allowed to equilibrate under preload tension for 1 hour before the beginning of the experiments. Most calyceal segments exhibited spontaneous phasic-rhythmic changes in tension immediately after mounting in the organ bath and application of the preload tension. The frequency of the contractions ranged between 7 to 10 per minute. Those muscle strips which did not show this regularity of contraction were excluded from the study. After the amplitude of the contractions had stabilized, a drug was added in the concentration which had been found in ureteral muscle strips to induce maximal relaxation in potassium-induced activation. The effect of increasing potassium concentrations on resting tension of ureteral segments was studied as follows. After equilibration in normal Tyrode solution the muscle strips were exposed to Tyrode solutions with increased potassium concentrations. The peak tension developed over base line was noted. This process was repeated until the entire concentration-reponse curve was generated. A similar procedure to that described above was employed to study the effects of increasing extracellular calcium concentrations on tension after depolarization with a submaximal potassium concentration of 85 mmol./1.

The effects of the drugs were studied in ureteral muscle strips using 3 different experimental protocols. In the 1st protocol, concentration-response relationships were constructed in muscle strips previously activated by potassium-depolarization. The experimental protocol was as follows. First, an initial submaximal contraction was produced by using a Tyrode solution containing 85 mmol./1. KCl. The change of the potassium concentration was followed by a marked increase in tension which, following a transient peak, remained stable for several hours after equilibration and varied by less than approximately 5 per cent. After wash-out of the high-potassium Tyrode solution the muscle strip immediately returned to its original resting tension (fig. 1). Then a 2nd contraction was produced with a Tyrode solution containing 85 mmol./1. KCl. After the contraction had stabilized a drug was added cumulatively, allowing time between additions for stabilization of relaxation. Inhibition of depolarization-induced contraction was measured as a function of drug concentration. All data were expressed as percentages of inhibition of equilibrium contraction after depolarization. Only 1 concentration-response curve was obtained from each muscle strip. In a 2nd protocol, muscle strips were contracted by a Tyrode solution containing 85 mmol./1. KCl. Peak tension and steady state tension were noted (control). Then the muscle strips were preincubated for 45 min. with the blockers in concentrations eliciting half maximum relaxation (EC50) in the protocol 1st described. Peak tension and steady state tension were noted after re-exposure to 85 mmol./1. potassium with the blockers still present in the organ baths. In a 3rd protocol, concentration-response curves for calcium (0.9 to 9.0 mmol./1.) after depolarization with a submaximal potassium concentration of 85 mmol./1. (control) were repeated after preincubation of the tissues (45 min.) with and in the presence of the blockers in concentrations producing full relaxation in the 1st protocol. Concentration-response relationships under control and test conditions were determined as functions of tension against log concentration of CaCb by linear regression analysis. Evaluation of results. At the end of each experiment the preparations were blotted with filter paper for 90 sec. under constant pressure (280 gm.) and weighed. Concentrations given are the final concentrations of drugs in the organ bath in moles/ liter. ECso values were determined graphically, taking into account 2 points on the steep portion of each individual con-

10 min

I

-----------------~----5.4

85

5.4

FIG. 1. Effect of 85 mmol./1. potassium on tension of isolated human ureteral muscle strip (original tracing). Depolarization of muscle strip induced rapid increase in tension, which peaked and was followed by partial relaxation and sustained contraction. Steady state tension (double arrow) was reference to test relaxing properties of drugs. After wash-out of high-potassium Tyrode solution muscle strip returned immediately to its original resting tension.

1267

PHARMACOLOGY OF THE HUMAN UPPER URINAR-i TRACT

centration-response curve, and geometric mean values were calculated; 95 per cent confidence limits of means were calculated according to Documenta Geigy, equation 580c. 9 An overall statistical comparison of several independent log ECso values was based on the analysis of variance, Documenta Geigy, equations 623-625; this procedure was followed by modified t statistics according to Bonferroni to identify the sources of differences (suggested by Wallenstein et al.). 10 Linear regression lines were determined and their slopes were tested for differences according to Documenta Geigy, equations 631b and 664a, respectively. 9 Results are expressed as means ± standard error of the means (SEM). A p-value of less than 0.05 was considered significant.

Tension (% of maximum)

100

50

RESULTS

T,

•

~/1

Spontaneous phasic-rhythmic activity. The spontaneous mechanical activity of isolated human calyceal segments was characterized by phasic-rhythmic changes in tension with a frequency between 7 to 10 per minute. The amplitude of the contractions ranged between 2 to 6 mN. Addition of nifedipine, verapamil, D600 and diltiazem to the organ bath reduced the amplitude of contractions concentration-dependently. Usually ! the drugs had no effect on the frequency of contractions; 0 however, at maximal concentrations (see below) the drugs 27 48 85 142 occasionally caused brief interruptions of the contraction sequence. The effect of verapamil in the highest concentration Concentration of potassium (m mol/1) tested (10- 5 mol./1.) on spontaneous phasic activity of an isolated minor calyx is illustrated in fig. 2. Addition of verapamil FIG. 3. Concentration-response relationship of increasing potasto the organ bath induced a continuing reduction in amplitude sium concentrations on tension of human ureteral muscle strips. Ordinate tension in per cent of maximal activation; abscissa,;, concenof contractions within a few minutes; after 15 minutes the drug trations,;, of potassium in mmol./1; means ± SEM (no. = 8). Tension suppressed spontaneous activity completely. Similar effects was graded function of extracellular potassium concentration. Activacould be demonstrated with maximal concentrations of nifedi- tion by 85 mmol./1. represented submaximal activation and was therepine (10- 6 mol./1.), D600 (10- 6 mol./1.) and diltiazem (10- 5 mol./ fore chosen as reference activation to test effects of drugs. Mean ± SD value for 100 per cent point: 34.0 ± 6.6 mN/100 mg. wet weight. 1.). After wash-out of the drugs a return of spontaneous phasicrhythmic activity was not observed within 45 minutes. Potassium-induced depolarization. Increase of the potassium Potassiumconcentration in the Tyrode solution from normal (5.4 mmol./ 1.) to 142 mmol./1. in logarithmic increments produced imme- induced tension diate and sustained contractions of isolated ureteral muscle (% of maximum) strips. The threshold for induction of contractions was 27 100 mmol./1. A maximal activation was achieved with a potassium concentration of 142 mmol./1. The concentration-response Y = 16.2 85.1 X curve depicting the effects of high potassium concentrations on r=0.99 tension is shown in fig. 3. A potassium concentration of 85 mmol./1. produced a submaximal activation of the muscle strips and was therefore chosen as reference activation to test the effects of the drugs. To demonstrate the dependence of the potassium-induced activation on extracellular calcium concentration, muscle strips were activated with 85 mmol./1. potassium in the presence of 50 increasing calcium concentrationso The regression line obtained from these experiments is shown in fig. 4. A correlation coef-

+

2 min L___J

m~J 0

0

I

0.9 Verapamil 1

o- 5 mol/1

FIG. 2. Effect of verapamil (10- 5 mol./1.) on spontaneous phasic activity of isolated human minor calyx (original tracing is interrupted because of protracted time course of effect). Addition of drug to organ bath induced continuing reduction in amplitude of contractions without affecting frequency. After 15 minutes drug suppressed spontaneous activity completely.

1.6

2.8

5.1

9.0

Concentration of CaCl 2 (m mol/1) FIG. 4. Graph and function of regression line showing dependence of potassium-induced tension on extracellular calcium concentration in human ureteral muscle strips. Ordinate ,;, tension induced by 85 mmol./1. potassium-containing Tyrode solution in per cent of maximal activation; abscissa ,;, concentration of CaC1 2 in mmol./1., means ± SEM (no. = 8). Regression line demonstrates clear log-linear relationship between extracellular calcium concentration and 85 mmol./1. potassium-induced tension.

1268

HERTLE AND NAWRATH

ficient of r = 0.99 demonstrates a clear log-linear relationship between the extracellular calcium concentrations and the 85 mmol./1. potassium-induced muscle tension. The contractile response induced by high potassium concentrations was not affected in the presence of either tetrodotoxin (2 X 10-5 mol./ 1.) or phentolamine (10- 5 mol./1.). Concentration-response relationships of the compounds were obtained by activating ureteral muscle strips with a submaximal concentration of 85 mmol./1. potassium in the Tyrode solution. Immediately after exposure, the muscle strips showed an initial rapid increase in tension followed by partial relaxation and sustained contracture (fig. 1). Only during the development of the initial phase were rapid phasic-rhythmic contractions often observed, which disappeared completely within 3 min. There were several reasons for using potassium-induced contractions as a reference to find concentration-response relationships: the responses were stable, reproducible and appeared to provide a good approximation of the amount of smooth muscle present in the ureteral wall. The peak tension development of ureteral muscle strips after activation with 85 mmol./1. extracellular potassium amounted to 32.2 ± 16.3 mN/100 mg. wet weight (mean ± SD, no. = 60). Figure 5 demonstrates an example of the concentrationdependent suppression of tension by methoxyverapamil (D600) in a ureteral muscle strip previously activated by 85 mmol./1. extracellular potassium concentration. The reference activation in this experiment was about 50 mN; a concentration of 10-9 mol./1. was the threshold for relaxing activity, and the potassium-induced activation was completely antagonized at a concentration of 10-6 mol./1. The time between additions of the drugs to the organ baths and stabilization of relaxation for all drugs and concentrations ranged between 20 and 30 minutes. Eight experiments were performed with each drug. Figure 6 shows the concentration-response relationships of nifedipine, verapamil, D600 and diltiazem indicating qualitatively similar actions of the drugs. Table 1 summarizes the EC 50 values with the 95 per cent confidence limits of the tested compounds obtained in this activation model. The EC5o values and the relative potencies with reference to papaverine (the EC5o value of papaverine, a classical smooth muscle relaxant drug, was defined to be 1) indicate marked differences in potency in the following sequence: nifedipine > D600 > verapamil > diltiazem. All indicated EC 50 values were significantly (p < 0.05) different from each other except D600 versus verapamil. Table 2 shows the effects of the blockers on muscle tension when given 45 min. before the addition of 85 mmol./1. potassium. The drugs were applied in concentrations which elicited half maximum relaxation in muscle strips previously activated with potassium, according to the 1st experimental protocol. Under these experimental conditions a weaker effect of the drugs on peak tension than on steady state tension was observed. Preincubation of the tissues with the blockers in concentrations which produced full relaxation in the 1st experimental protocol induced a strong suppression of contractions induced

Potassium-induced tension (% of control)

100

50

• Nifedipine o D 600

. g'

• Papaverine

\~:~.."'\ '\.j,, .

0

-8

-9

-10

-7

-6

1. EC50 values with 95 per cent confidence limits of tested drugs and relative potencies with reference to papaverine

TABLE

EC50 Values with 95% Confidence Limits Papaverine Nifedipine D600 Verapamil Diltiazem

2.5 2.8 3.6 6.3 1.7

Relative Potency

(1.61-3.23) X 10-• mol./1. (1.91-3.45) X 10-9 mol./l. (2.94-4.82) x 10-• mol./1. (3.28-11.8) X 10-• mol./1. (1.45-1.87) x 10-7 mol./1.

1

8 214 605 365 135

All indicated EC 50 values are significantly (p < 0.05) different from each other except D600 versus verapamil. Means ± SEM, no. = 8.

Effects of drugs on peak tension and on steady state tension induced by depolarization with Tyrode solution containing 85 mmol./1. potassium after preincubation and in presence of drugs (no.= 4 for all drugs)

TABLE 2.

Steady State Tension (% of Control)

Peak Tension (% of Control) Papaverine Nifedipine D600 Verapamil Diltiazem

3 x 10-• mol./1. 3 X 10-9 mol./1. 3 X 10-• mol./1. 10-7 mol./1. 1.5 x 10- 1 mol./1.

40.4 60.2 81.4 78.5 72.7

± ± ± ± ±

22.9 ± 14.8 ± 50.0 ± 61.7 ± 62.8 ±

3.8 5.5 5.2 2.1 1.7

...

10-9

10-8

10-7

... 10-6 mol/1

0600 [K+] 0 85 m mol/1 FIG. 5. Effect of D600 on tension of human ureteral muscle strip previously activated by 85 mmol./1. potassium. Control activation is marked by double arrow. Drug antagonized high-potassium-induced activation concentration-dependently (original recording).

3.8 7.4 4.3 3.3 1.1

TABLE 3. Functions of regression lines of calcium concentrationresponse relationships (0.9-9.0 mmol./1.) in depolarized (85 mmol./l. K+J ureteral muscle strips before (control) and after (test) incubation (45 min.) of tissues with blockers (no. = 4 for all drugs)

Control

...

-4

Log concentration (mol/1)

Papaverine Nifedipine D600 Verapamil Diltiazem

...

-5

FIG. 6. Concentration-response relationships of nifedipine, D600, verapamil, diltiazem and papaverine on tension of human ureteral muscle strips previously activated by high potassium depolarization. Ordinate ,;, tension induced by 85 mmol./1. potassium containing Tyrode solution in per cent of control (control was steady state tension after depolarization); abscissa,;, logarithmic concentrations of drugs in mol./1., means ± SEM (no. = 8).

10 min L.__J

• Verapamil

° Diltiazem

10-• mol./l. 10-• mol./l. 10-• mol./1. 10-• mol./1. 10-• mol./1.

y = 25.1 + 76.lx y = 10.3 + 91.9x y = 27.2 + 78.3x y = 49.2 + 55.4x y = 23.9 + 79.8x

Test y

= O*

y= O*

y = 1.5 + 23.4x* y = 1.0 + 5.4x* y = 1.0 + ll.7x*

Correlation coefficients >0.99 for all regression lines. Slopes of regression lines (control versus test) were tested for difference (*p < 0.001).

by increasing calcium concentrations (0.9 to 9.0 mmol./1.) in depolarized muscle strips (85 mmol./1. K+). The functions of the regression lines under control and test conditions are given in table 3. DISCUSSION

The availability of specific antagonists of a number of stimulants of living cells has contributed much to the analysis of

PHARMACOLOGY OF THE HUMAN UPPER URINARY TRACT

physiological and biochemical processes. Antagonists to specific effects of inorganic ions have been notably lacking until quite recently. The importance of such agents is illustrated by the contributions of tetrodotoxin and local anesthetics to studies of the conductivity of cell membranes. By analogy to the role of receptor antagonists, a group of drugs with broad structural diversity has in recent years been found to have calcium antagonistic properties. These compounds include verapamil, D600, nifedipine and diltiazem. Electrophysiologic evidence, derived largely from cardiac tissue, indicates that they act on calciuminduced responses by inhibiting the calcium influx through voltage-operated calcium channels. The literature reports only little on the effects of calcium antagonists on smooth muscle of the upper urinary tract. Golenhofen and Lammel11 showed that verapamil (10- 5 mol./1.) produced an almost complete block of the spike component and a reduction of plateau duration of the action potential of the guinea-pig ureter. Furthermore, they observed a complete suppression of the mechanical activity with large changes of the action potential at a concentration of 10-4 mol./1. In the study of Vereecken et al., 12 D600 abolished all electrical and mechanical activity of guinea-pig ureter, an action similar to that of removal of extracellular calcium. The same authors demonstrated a relaxing effect of D600 on potassium-induced activation of dog ureter. Golenhofen and Hannappel1 3 and Forman et al. 14 reported that nifedipine completely inhibited spontaneous rhythmic activity as well as potassium- and barium-chloride-induced activity in several species and in man. The action potential, a sequence of depolarization and repolarization, functions as the physiological trigger for contraction. In several smooth muscle cells action potentials occur spontaneously and induce contractions described as a phasic type of contraction. The action potential of diverse smooth muscle cells is characterized by spike discharges which are superimposed on slower changes of membrane potential.11 The spikes are usually regarded as calcium spikes and could increase the cytoplasmic calcium directly because of the inward calcium current that occurs during the rising phase of the action potential, or they could elicit a release of calcium from an intracellular store. 15 Ureteral peristalsis in vivo as well as spontaneous phasic rhythmic activity of isolated human calyceal segments used in this investigation are both regarded to be induced by action potentials generated from pacemaker cells located in the utmost distal part of the collecting system. Our results confirm that these bioelectrically induced mechanical events are highly sen sitive to calcium antagonists. 16 The drugs acted qualitatively in the same way, but marked differences in the degree of potency could be observed, nifedipine being the most potent drug. Potassium-induced activation is a method of depolarization different from that occurring in the action potential. Increasing the external potassium concentration results in an increased spike frequency and, at higher potassium values, in a sustained depolarization of the membrane without action potentials.5 During application of solutions with high potassium concentrations, an increased influx of 45 Ca++ has been detected and total tissue calcium has been described to be increased. 17 This stimulation of the calcium influx induced by potassium depolarization can be assumed to depend on the opening of voltageoperated channels through which external calcium can flow into the cell. 15 In our experiments we could demonstrate a clear relationship between extracellular potassium concentration and tension. A threshold for tension was found at 27 mmol./1. potassium when outside c;:i.lcium was 1.8 mmol./1. and a maximal activation was achieved with 142 mmol./1. external potassium concentration. Furthermore, responses to potassium activation appeared to be a log-linear function of the extracellular calcium concentration over a considerable range. These findings suggest that potassium-induced activation mainly utilizes calcium from a labile

1269

site (extracellular and/or superficially bound calcium). The present results clearly show that all the drugs effectively inhibited potassium-evoked contractions in isolated human ureteral smooth muscle. Nifedipine was the drug with the lowest EC 50 value (2.8 x 10-9 mol./1.) and its relative potency with reference to papaverine was about 8,000 to 1. It can be concluded that in this activation model and in spontaneous phasic-rhythmic activity nifedipine is of high specificity in blocking the underlying mechanism of calcium activation. The present observations appear to be fully compatible with the assumption that nifedipine, verapamil, D600 and diltiazem specifically block some step in the process by which extracellular and/or loosely bound calcium moves to the contractile elements of smooth muscle cells. Acknowledgements. We thank Prof. Dr. R. Hohenfellner, Chairman, Department of Urology, University of Mainz (FRG) for generously supplying human tissues and Prof. Dr. K. Golenhofen, Department of Physiology, University of Marburg (FRG) for helpful discussions and suggestions. REFERENCES 1. Gordon, A. R.: Contraction of detergent-treated smooth muscle. Proc. Natl. Acad. Sci. USA, 75: 3527, 1978. 2. Endo, M., Kitazawa, T., Yagi, S., Iino, M. and Kakuta, Y.: Some properties of chemically skinned smooth muscle fibers. In: Excitation-Contraction Coupling in Smooth Muscle. Edited by R. Casteels, T. Godfraind and J. Riiegg. Amsterdam, Elsevier, pp. 199-209, 1977. 3. Kuriyama, H.: Excitation-contraction coupling in various visceral smooth muscles. In: Smooth Muscle: An Assessment of Current Knowledge. Edited by E. Biilbring, A. F. Brading, A. W. Jones and T. Tomita. London, Edward Arnold, pp. 171-197, 1981. 4. Kuffler, S. W.: The relation of electrical potential changes to contracture in skeletal muscle. J. Neurophysiol., 9: 367, 1946. 5. Golenhofen, K., Hermstein, N. and Lammel, E.: Membrane potential and contraction of vascular smooth muscle (portal vein) during application of noradrenaline and high potassium, and selective inhibitory effects of iproveratril (verapamil). Microvasc. Res., 5: 73, 1973. 6. Fleckenstein, A.: Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Ann. Rev. Pharmacol. Toxicol., 17: 149, 1977. 7. Triggle, D. J. and Swamy, V. C.: Pharmacology of agents that affect calcium. Agonists and antagonists. Chest (Suppl), 78: 174, 1980. 8. Bolton, T. B.: Mechanism of action of transmitters and other substances on smooth muscle. Physiol. Rev., 59: 606, 1979. 9. Documenta Geigy, Wissenschaftliche Tabellen. Edited by D. Diem and C. Lentner, 7th Ed., Basel, Geigy, 1968. 10. Wallenstein, S., Zucker, C. L. and Fleiss, J. L.: Some statistical methods useful in circulation research. Circ. Res., 47: 1, 1980. 11. Golenhofen, K. and Lammel, E.: Selective suppression of some components of spontaneous activity in various types of smooth muscle by iproveratril (verapamil). Pfliigers Arch., 331: 233, 1972. 12. Vereecken, R. L., Hendricks, H. and Casteels, R.: The influence of calcium on the electrical and mechanical activity of the guinea pig ureter. Urol. Res., 3: 149, 1975. 13. Golenhofen, K. and Hannappel, J.: A tonic component in the motility of the upper urinary tract (renal pelvis-ureter). Experientia, 34: 64, 1977. 14. Forman, A., Anderson, K.-E., Henniksson, L., Rud, T. and Ulmsten, U.: Effects ofnifedipine on the smooth muscle of the human urinary tract in vitro and in vivo. Acta Pharmacol. Toxicol., 43: 111, 1978. 15. Casteels, R. and Droogmans, G.: Membrane potential and excitation-contraction coupling in smooth muscle. Fed. Proc., 41: 2879, 1982. 16. Golenhofen, K.: Differentiation of calcium activation processes in smooth muscle using selective antagonists. In: Smooth Muscle: An Assessment of Current Knowledge. Edited by E. Biilbring, A. F. Brading; A. W. Jones and T. Tomita. London, Edward Arnold, pp. 157-170, 1981. 17. Breemen, C. van, Farinas, B. R., Gerba, P. and McNaughton, E. D.: Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx. Circ. Res., 30: 44, 1972.