Original Paper Pharmacology 1992;45:216-230

Department of Pharmacology, ICI Pharmaceuticals Group, ICI Americas Inc., Wilmington, Del., USA

KeyWords Guinea pig detrusor strip Guinea pig portal vein strip K+ channel openers K+ channel blockers Spontaneous myogenic activity KCl-evoked myogenic activity

Comparison of the in vitro Effects of K +Channel Modulators on Detrusor and Portal Vein Strips from Guinea Pigs

Abstract The effects of K+channel openers and blockers on smooth mus­ cles of vascular and nonvascular origin from guinea pigs have been investigated. Cromakalim, pinacidil, nicorandil and mi­ noxidil sulfate all abolished the spontaneous myogenic activity of the guinea pig portal vein and the KCl-evoked activity of detrusor strips with the same rank order of potency. Whereas both apamin and charybdotoxin stimulated myogenic activity of the detrusor strips, they produced insignificant effects on spontaneously active portal vein strips and failed to antagonize the mechanoinhibitory effects of cromakalim in the two tis­ sues. Glibenclamide, on the other hand, only stimulated the myogenic activity of portal vein strips but antagonized the mechanoinhibitory effects of cromakalim, pinacidil, nicoran­ dil and minoxidil sulfate in both tissues. Rubidium, at millimolar concentrations, stimulated the myogenic activity, and an­ tagonized the actions of cromakalim in both tissues. The data indicate that there are definite functional dissimilarities as exhibited by the differential response of the two tissues to K+ channel modulators. These findings may be exploited in the design of new drugs with tissue selectivity.

Introduction The actions of a chemically diverse group of compounds in promoting 86Rb+ and/or 42K+efflux from preloaded tissues, such as the

Received: December 10, 1991 Accepted: January 7, 1992

guinea pig portal vein [1, 2] or the guinea pig detrusor [3-5], and in abolishing the tissues’ spontaneous or evoked contractile myogenic activity, have generated an enormous interest in the field of K+ channel physiology and

Dr. Jack H. Li Department of Pharmacology ICI Pharmaceuticals Group, ICI Americas Inc. Wilmington, DE 19897 (USA)

© 1992 S. Karger AG, Basel 0031 -7012/92/ 0454-0216$ 2.7 5/0

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Panagiotis Zografos Jack H. Li Sen T. Kau

Materials and Methods Adult male Hanley guinea pigs (450-500 g) were stunned by a blow to the head and quickly exsangui­ nated. In the portal vein assay, the abdominal cavity was cut open and approximately 2 cm of the hepatic portal vein was freed of surrounding connective tissue and isolated. This section was placed through a syringe needle, very carefully cleansed of blood and remaining connective tissue to avoid rubbing off the endothe­ lium, and slit open longitudinally. Then, a second lon­ gitudinal cut produced 2 strips from each portal vein segment. Each strip was tied at one end to a glass rod and placed in a 20-ml jacketed tissue bath containing Krebs-Henseleit (K-H) buffer, maintained at 37 °C and bubbled vigorously with 95% 02/5% C 0 2. of the following composition (in mmol/I): NaCl 118. KCI 4.7, MgSOj 1.2, KH2P 0 4 1.2. CaCl2 2.5, glucose 11.1 and NaHC'Oi 25. The other end of the strip was tied via 4-0 silk thread to a force displacement transducer (Grass model FT03) and añera 15-min equilibration period, a 1-gram preload tension was applied followed by fre­

quent washouts for the next 30-45 min. In the urinary bladder detrusor assay, the bladder was isolated, and the portion above the ureteral orifices was removed and placed in a Petri dish containing oxygenated K-H buffer. The dome section was then cut away and 4 hor­ izontal strips 2-3 mm in width, 7-10 mm in length were obtained from the middle region of each bladder. The two ends of the strips were tied to the glass rod and the transducer, respectively, and allowed to equilibrate under a 2-gram preload tension. During the following 45- to 60-min equilibration period the tissues were fre­ quently washed with fresh buffer and the preload ten­ sion adjusted to 2 g. The K+-free, rubidium K-H buffer used in some experiments was obtained by equimolar replacement of KCI and KH2P 0 4 by RbCl and NaH2P 0 4, respectively. After the equilibration period, the spontaneous or KCl-induced mechanical activity of the tissue was measured under isometric conditions for a period of 5 min. averaged and recorded as control average myo­ genic activity. A cumulative dose-response curve for each putative K+ channel opener was generated by adding the opener to the bath in half log-unit incre­ ments. At each concentration, a 5-min averaged myo­ genic activity of the tissue was taken after a 5-min con­ tact time with the opener. The mechanoinhibitory effect of an opener at each concentration was ex­ pressed as a percent of the maximum inhibition of the myogenic activity. This maximum response from the tissue was defined by the difference between the value of the control average myogenic activity and the value at which all myogenic activity was abolished. An ICj« value, defined as the concentration of the opener that produced 50% of the maximum response, was ob­ tained using Marquardt’s nonlinear iterative tech­ niques to fit the dose-response curve to the function: R = D7(DS+ Ks), where R is the normalized response, D is the concen­ tration of the opener in the bath, K is equivalent to the fitted ICjo value and s is the fitted slope. In some stud­ ies, the IC50 values were calculated simply from the linear regression of the steep portion of the probittransformed dose-response data. In the studies of the effects of K‘ channel blockers, the tissues were incubated with the blockers for at least 20 min before the generation of a cumulative doseresponse curve of the K+channel openers. The interac­ tion between the K* channel blocker and opener was characterized by applying the formalism of drug recep­ tor interaction, where the K+ channels were viewed as receptors, and the blockers and openers were antago­ nists and agonists, respectively. The antagonist affinity

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pharmacology. Furthermore, since K+ chan­ nels appear to be both ubiquitous and diverse [6], the development of agents that interfere with some aspect of their physiological func­ tion^) may have a propitious therapeutic ef­ fect. Such is the case with the development of the hypoglycemic sulfonylureas like glibenclamide and tolbutamide, which derive their therapeutic effects from their blocking actions on K+ channels in p-cells [7], It has also been suggested that K+ channel openers could be useful in the treatment of the irritable bladder syndrome [8]. provided that a new generation of drugs could be developed whose actions are both tissue specific and lacking the in vivo hypotensive effects normally associated with these agents. To that end, this work attempts to characterize and compare the in vitro ef­ fects of K+ channel openers and blockers on the mechanical activity of vascular and nonvascular smooth muscles, typified by the guinea pig portal vein and detrusor, respec­ tively. Portions of the present study have been presented earlier in abstract form [9, 10],

Kb = [antagonist]/(dose ratio - 1), where [antagonist] is the molar concentration of antag­ onist and the dose ratio is the ratio of equieffective agonist concentrations obtained in the absence and presence of the antagonists. Affinity constants were expressed as -log values (p K r). Schild analysis was per­ formed in order to determine the apparent mechanism of action ofglibenclamide and pA? values were defined as the x-axis intercept of the linear regression line fit­ ting the data points from the Schild plot [ 12]. K* Channel Modulators Glibenclamide and apamin were obtained from Sigma (St. Louis, Mo., USA). Charybdotoxin (lot num­ ber 0689) was obtained from Latoxan (Rosans, France), while cromakalim, lemakalim. pinacidil and nicorandil were synthesized in-house by the Chemistry Department. Minoxidil sulfate was kindly supplied by the Upjohn Company (Kalamazoo, Mich., USA). Apart from the two peptide K+ channel blockers apamin and charybdotoxin. all other K* channel mo­ dulators were dissolved in dimethyl sulfoxide at a stock concentration of 20 mmol/1, and then further diluted serially. Apamin was dissolved in double-dis­ tilled water, while charybdotoxin was dissolved in a saline solution containing 118 mmol/1 NaCl to mini­ mize adsorption of the toxin to glass vials. Statistical Analysis Values are expressed as means with 95% confi­ dence limits. Whenever significance levels are given, they are obtained from Student's t test (paired or unpaired) and a probability of p < 0.05 is taken to indicate significantly different values.

Results Mechanoinhibitory Effects o f K+ Channel Openers The K+ channel openers cromakalim, pina­ cidil, minoxidil sulfate and nicorandil dose dependently abolished both the spontaneous myogenic activity of the portal vein and the 15 mmol/1 KCl-induced activity of the detru­ sor (fig. 1). The rank order of potency of these compounds, constituted by the relative mag­

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nitude of their respective IC50, was croma­ kalim > pinacidil > nicorandil > minoxidil sulfate in both tissues (tables 1, 2). Croma­ kalim and pinacidil yielded similar doseresponse curves in both tissues, abolishing myogenic activity with similar potencies ( < 2-fold difference in IC50 values), while mi­ noxidil sulfate was found to be the least active of all 4 openers tested. When IC50 values were compared, cromakalim and pinacidil were about 30- to 40- but minoxidil sulfate was about 86-fold less potent in the detrusor than in the portal vein. Mild membrane depolarization, induced by increasing the KC1 concentration in the bath from 5 to 10, 15 or 20 mmol/1, increased the tissue myogenic activity but decreased the effectiveness of cromakalim to abolish the myogenic activity in both portal vein and detrusor strips. Comparing the data obtained with both tissues exposed to the same extra­ cellular KC1 concentrations, we found that cromakalim was approximately 7-10 times more potent in portal vein than in detrusor strips, but in both tissues it exhibited a similar dependence on extracellular K+ (fig. 2a, b). Replacement of K+ by Rb+ in the K-H buffer, or addition of 5 mmol/1 RbCl to the bath greatly enhanced the spontaneous or KC1evoked myogenic activity in both the detrusor and portal vein preparations (fig. 3c). Both of these maneuvers decreased the effectiveness of cromakalim and produced 10- to 13-fold increases in its IC50 values in spontaneously contracting detrusor and portan vein alike. This attribute of extracellular Rb+ to func­ tionally antagonize the mechanoinhibitory activity of cromakalim decreased with in­ creasing KC1 concentrations (fig. 2a, b). In detrusor strips that were exposed to 20 mmol/1 KC1. addition of 1 mmol/1 RbCl shifted the mean IC50 value of cromakalim 2fold, while addition of 3 mmol/1 RbCl pro­ duced an apparently insurmountable inhibi-

K‘ Channel Modulators in Detrusor and Portal Vein

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constant (Kb) was determined by the dose-ratio method [II] using the following equation:

Fig. 1. Dosc-rcsponse curves of the mechanoinhibitory effects of the K* channel openers crornakalim (a), pinacidil (o). nicorandil (♦) and minoxidil sulfate (■) in spontaneously contracting guinea pig portal vein (a) and guinea pig detrusor stimulated with 15 mmoL/l KCI b). Each point repre­ sents the mean ± SEM from at least 4 experiments and is ex­ pressed as a percent of maximum inhibition of myogenic activity.

Log Cclrug] M olar

Table 1. The mechanoinhibitory activity of K+ channel openers and antagonism by glibenclamide in the spontaneously contracting (5 mmol/l KCI) and KCl-stimulated guinea pig portal vein Bath KCI mmol/l 5

K~ channel opener

Control mean IC50 pmol/1

n

Gliben­ clamide gmol/l

Treatment mean IC50 gmol/l

pKB'

cromakalim

0.020 (0.011-0.035) 0.028 (0.014-0.054) 0.179 (0.044-0.735) 2.28 (0.60-9.05)

4

1

7.34 ±0.10

4

1

4

1

7

0.3

0.465 (0.18-1.21) 0.339 (0.191-0.602) 4.17 (0.5-35.1) 254 (166-387)

pinacidil nicorandil minoxidil sulfate

7.04 ±0.16 7.35 ±0.17 8.55 ±0.29

10

cromakalim

0.027 (0.022-0.033)

6

1

0.794 (0.39-1.63)

7.452

15

cromakalim

0.045 (0.027-0.075)

7

1

1.21 (0.90-1.63)

7.402

20

cromakalim

0.054 (0.047-0.063)

8

1

0.841 (0.58-1.22)

7.I62

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Values in parentheses are 95% confidence limits. 1 IC50 values o f the K' channel openers in the absence and presence o f glibenclamide (paired data) were used to calculate the pKB value. 2 For the unpaired data. pKg values were obtained from shifts o f mean IC50 in the absence and presence o f glibenclamide.

Fig. 2. Effects of extracellular KC1 and RbCl on the mechanoinhibitory potency of cromakalim in the portal vein (a) and the detrusor (b). □ = Absence. ■ = presence of 5 mmol/1 RbCl. Each data point is from at least 4 experiments and represents the mean [C50 in nmol/i ( ± SEM) of cromakalim. (c) Doseresponse curv es of cromakalim (log molar unit) in 20 mmol/1 KCl-stimulated detrusor strips in the ab­ sence (□) and presence of 1 (o) and 3 (•) mmol/1 RbCl.

Table 2. The mechanoinhibitory activity of K* channel openers and antagonism by glibenclamide in isolated detrusor strips exposed to various concentrations of extracellular KC1

Bath KCI mmol/1

K’ channel opener

Control IC50 pmol/l

10

cromakalim

0.14 ±0.02

15

cromakalim

Treatment IC50 pinol

pA2

Slope

1.0 3.0 10.0

0.36 ±0.01 0.67 ±0.12 4.58 ±1.40

6.25

1.06

0.57 ±0.07

0.3 1.0 3.0

1.98 ±0.29 6.03 ±1.61 12.00 ± 1.74

7.01

0.91

pinacidil

1.11 ±0.07

0.3 1.0 3.0

5.00 ±0.37 9.70± 1.04 29.18 ±2.18

7.08

0.86

minoxidil sulfate

197 ±54

0.3

844 ±98

7.04'

lemakalim

0.53 ±0.09

1.0

5.55 ±0.61

6.98'

cromakalim

1.01 ±0.06

0.3 1.0 3.0

3.74 ±0.53 14.4 ±4.2 51.6 ± 11.5

7.00

pinacidil

5.01 ±0.86

nicorandil

1.10

17.08 ±3.03

Values are given as mean ± SEM from at least 4 experiments. pAi and slope values were calculated from Schild analysis with dose ratios derived from shifts in the mean ICjo values in the absence or presence o f glibenclamide. 1 pKa value for unpaired data.

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20

Glibenclámide gmol/l

DETRUSOR

PORTAL VEIN

3a Glibenclamide

Glibenclamide 1 umol/l t

5 mm

Cromakalim Dose-Response Glibenclamide

0.1

Pinacidil 0.1 umol/l

0.3 umol/l

i

i

5 min

0.3 100 umol/l *

Pinacidil Dose-Response

Pinacidil Dose-Response

3b

Apamin (1 pmol/l) Apam in (1pmol/l) Charybdotoxin

(10 nmol/l)

3c 5 mmol/l Rb

5 mmol/Rb

„' ,:,

*1 I ^

1 11

"I 1

5 min

5 min

KCI 10 mmol/l

• 5 mmol/l Rb

5 m in

(

I 5 mmol/l Rb)

5

1 5 'mmol/l KCI

Fig. 3. Representative tracings of the elTccts of glibenclamide (a), apamin and charybdo­ toxin (b)and RbCI (c) on the myogenic activity of spontaneously contracting portal vein (left panels) and detrusor stimulated with 15 mmol/l KCI (right panels), c The mechanocxcitatory effect of Rb* is noted also in 10 mmol/l KCl-stimulated portal vein (lower tracing after the first arrow) and in the spontaneously contracting detrusor (upper tracing).

22l

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5 mmol/l

j

L og [ C r o m a k a lim ] M o la r

Fig. 4. Cromakalim dose-response curves constructed from either spontaneously active unpaired portal vein strips (a. c) or from 15 mmol/1 KCl-stimulated unpaired detrusor strips (b. d) in the absence (a) or presence of apamin (1 pmol/l. T), charybdotoxin (1, o; 3, •; 10, 0 and 30 nmol/1. ♦). Each data point represents the mean ±SEM of the percent maximum inhibition of myogenic activity from at least 4 separate experiments at each cromakalim con­ centration.

Effects ofK * Channel Blockers on Myogenic Activity and on the Mechanoinhihitory Activity o f Cromakalim Glibenclamide (1 pmol/1) greatly stimu­ lated myogenic activity in the portal vein, but elicited no such responses in the detrusor strip (fig. 3a). In contrast, apamin and charybdo­ toxin had no significant effects on the myo­ genic activity of portal vein but greatly en­ hanced the myogenic activity of detrusor strips which had already been stimulated with 15 mmol/1 KCI (fig. 3b). However, addition of

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apamin ( 1 gmol/1) in both the spontaneously active portal vein strips (fig. 4a) and the 15 mmol/1 KCl-stimulated detrusor (fig. 4b) of­ fered no antagonism against the actions of cromakalim. Similarly, in spontaneously ac­ tive portal veins, addition of charybdotoxin ( 1. 3 and 10 nmol/1) did not antagonize the effectiveness of cromakalim in abolishing the myogenic activity (fig. 4c). In detrusor strips stimulated with 15 mmol/1 KCI, addition of 10 or 30 nmol/1 charybdotoxin produced a dose-dependent suppression of the maximum response and a shift of the mean IC50 of cro­ makalim to higher values (fig. 4d).

K- Channel Modulators in Detrusor and Portal Vein

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tion of the maximum response attainable by increasing concentrations of cromakalim (fig. 2c).

-8

-7

-6

-5

-4

Log [N ícorandil] Molar

Fig. 5. Mechanoinhibitory dose-response curves of pinacidil (a), nicorandil (b) and minoxidil sulfate (c) constructed from paired portal vein strips in the absence (o, ♦, ■) or presence (o, n, •, in a. b. and c. respectively) of 1 pmol/l glibenclamide, and of minoxidil sulfate (d) constructed from unpaired detrusor strips in the absence (■) or presence (•) of 0.3 pniol/1 glibenclamide. Each data point represents the mean ± SEM of the percent maximum inhibition of myogenic activity from at least 4 separate experiments at each concentration.

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Log [M in o x id il SxilfateJ M o lar

L o g [Crom akalim ] M olar

Glibenclamide (1 pmol/1) antagonized the actions of pinacidil. nicorandil and minoxidil sulfate in spontaneously active portal veins (fig. 5a-c). It also antagonized the actions of cromakalim in portal veins that were either spontaneously active or stimulated by elevat­ ing the KCI concentration in the bath from 5 to 10. 15 or 20 mmol/1 (fig. 6a-d: table 1). In none of these cases was a suppression of the maximum response to the K+channel openers noted. Antagonist affinity constants (pKB) calculated from shifts in IC50 values of the K4 channel openers in the absence and presence of glibenclamide were similar for cromakal­ im, pinacidil and nicorandil but significantly different for minoxidil sulfate (table 1). al­ though minoxidil sulfate also abolished 94% of the total portal vein myogenic activity in the presence of 0.3 pmol/l glibenclamide

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(fig. 5c). When tested in unpaired detrusor strips that were stimulated with 15 mmol/1 KCI. the actions of minoxidil sulfate were antagonized by glibenclamide (fig. 5d) but the pKBvalue of 7.04 was significantly differ­ ent from that of 8.55 in the portal vein (ta­ bles 1. 2). The dependence on the bath KCI concen­ tration of the antagonism of glibenclamide against the mechanoinhibitory effect of cro­ makalim was assessed in both the portal vein and detrusor strips. In the portal vein, the pKB of glibenclamide showed similar values in strips bathed in the K-H buffers with differ­ ent KCI concentrations (table 1). However, in the detrusor strips stimulated with 15 and 20 mmol/1 KCI. glibenclamide antagonized the actions of cromakalim dose dependently, yielding a pA? value of 7.0 at both KCI con-

K' Channel Modulators in Detrusor and Portal Vein

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Fig. 6. The effects of increasing KCI concentrations on the mcchanoinhibitory doseresponse curves of cromakalim constructed from paired portal vein strips in the absence (□) or presence (■) of glibcnclamidc (1 pmol/l). Each data point represents the mean ± SEM of the percent maximum inhibition of myogenic activity from at least 4 separate experiments at each cromakalim concentration.

Log [Cromakalim] Molar

Log [C rom akalim ] M olar

Log [Cromakalim] Molar

Log [Plnacidll] Molar

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Fig. 7. Mechanoinhibitory dose-response curves of cromakalim (a-c) and of pinacidil (d) constructed from detrusor strips in the absence (□, o) or presence of increasing concentrations of glibenclamide (0.3. •: I. 3, a or 10 pmol/1. ♦) Insets: Schild plots, the data points of which were derived from shifts in the mean IC50 value of cromakalim (or pinacidil) obtained in the absence and the presence of different glibenclamide concentrations. The data were fitted by linear regression to determine the x-axis intercept (pAi value) and the slope.

centrations and Schild slopes of 0.9 and 1.1, respectively (fig. 7b, c), while in detrusor strips stimulated with 10 mmol/l KC1, gliben­ clamide yielded a pA? value of 6.25 and a slope of 1.06 (fig. 7a). Glibenclamide also antagonized the me­ chanoinhibitory effects of other K+ channel openers. The dose-response curves of panicidil that were constructed from 15 mmol/l KCI-stimulated detrusor strips, in the absence or presence of increasing concentrations of glibenclamide. yielded a calculated pA? value of 7.08 and a slope close to unity (fig. 7d). Furthermore, in 20 mmol/l KCI-stimulated

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detrusor strips, lemakaiim, the (-)-enantiomcr of cromakalim, was found to be about 2-fold more potent than the racemic and > 100-fold more potent than the ^ -e n a n ­ tiomer (fig. 8a). Glibenclamide pretreatment (1 pmol/1) shifted the dose-response curve of lemakaiim 10-fold to the right of the control curve, yielding a pKB value of 6.98 (fig. 8b). These quantitative data characterizing the in­ teraction of glibenclamide with the various putative K+ channel openers are summarized in table 2. Finally, in another set of experi­ ments, dose-response curves to cromakalim that were constructed from unpaired 15-

K‘ Channel Modulators in Detrusor and Portal Vein

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Fig. 8. Mechanoinhibitory dose-response curves of the enantiomers of cromakalim (a: V and A represent the (-)- and (+)-enantiomers, respectively), of lemakaiim [(-)-cromakalim] in the absence (V) or presence (*) of 1 pmol/1 glibenclamide constructed from 20 mmol/l KCIstimulated detrusor strips (b), and of cromakalim in 15 mmol/l KCI-stimulated detrusor strips that had been pretreated with apamin (1 pmol/l) and subsequently exposed to 0 (□), 0.3 (•) and i pmol/1 (■) glibenclamide (c). Each data point represents the mean ± SEM from at least 4 separate experiments at each concentration.

Discussion In spontaneously active smooth muscle cells of vascular or nonvascular origin. K7 channel permeability is thought to deter­ mine both the resting membrane potential and. possibly, repolarization of the mem­ brane following burst activity [13]. There­ fore, interventions that either promote or in­ hibit K+ channel activity should also affect the myogenic activity of the tissue. In our study, the putative K+ channel openers cromakalim, pinacidil. nicorandil and minox­ idil sulfate all abolished myogenic activity in a dose-dependent manner. They also exhib­ ited the same rank order of potency in both the guinea pig portal vein and detrusor prep­ arations, indicating a common mechanism of action of these compounds in these two tissues. In addition, the effectiveness of both cromakalim and pinacidil in abolishing myogenic activity is about an order of mag­ nitude higher in portal vein than in detrusor. Lemakalim. the active enantiomer of croma­ kalim. was recently reported to show mechanoinhibitory activity in the rat detrusor and portal vein with potencies differing also by an order of magnitude [8]. However, in our study the potency of minoxidil sulfate to abolish the guinea pig portal vein myogenic activity was significantly lower than that in another recent report [14]. This apparent discrepancy may be due to the different sources of minoxidil sulfate. Using a small sample of the compound obtained from that source, we did observe its mechanoinhibitory activity in the guinea pig portal vein

with a potency comparable to the one re­ ported in that reference [9]. Cromakalim exhibited a similar depen­ dence on extracellular KC1 in both portal vein and bladder detrusor preparations; its po­ tency decreased with increasing KC1 concen­ trations. This observation is consistent with the proposed mechanism of action of croma­ kalim to open K+channel(s) in smooth muscle cells [15], the results of which are membrane hyperpolarization and. subsequently, reduc­ tion of Ca2+ influx and loss of myogenic con­ tractility. Increasing the extracellular KC1 concentration shifts the transmembrane po­ tential to less negative values where voltagesensitive Ca2+ channels are operational, and diminishes the mcchanoinhibitory effects of cromakalim. The sulfonylurea glibenclamide. a very po­ tent blocker of ATP-sensitive K+ channels in pancreatic [3-cells [7, 16], increased the myo­ genic activity only in the guinea pig portal vein. This mechanoexcitatory effect of gliben­ clamide has recently been noted also in rat portal vein strips [ 17]. However, glibenclam­ ide antagonized the mechanoinhibitory ef­ fects of the putative K+ channel openers in both the portal vein and the detrusor, but it exhibited a somewhat higher affinity constant (pKB) for the channels opened by cromakalim in the portal vein as compared to those in the detrusor. This difference in the action of gli­ benclamide on the vascular portal vein and the nonvascular detrusor suggests that glibenclamide-specific, ATP-sensitive K+ channels in the detrusor are probably closed under the experimental conditions and their blockade depends upon prior activation by cromakal­ im, while those in the guinea pig portal vein seem more likely to be open, permitting blockade by glibenclamide. Glibenclamide, however, did not induce the sustained eleva­ tion of tension usually noted with membrane depolarization in either the portal vein or

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mmol/1 KCl-stimulated detrusor strips, pre­ treated with apamin (1 pmol/1) followed by 0.3 or 1.0 pmol/1 glibenclamide, yielded pKB values, calculated from shifts of the mean IC50S. of 6.80 and 7.07, respectively (fig. 8c).

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significantly increase myogenic activity in the rat [ 18] and the guinea pig portal vein [ 14] at 0.1 pmol/1, a concentration 10-fold higher than that used presently, but it nevertheless also failed to antagonize the actions of croma­ kalim. It is therefore concluded that croma­ kalim is unlikely to act on any of the known Ca2+-activated K+ channels in the portal vein, since these channels are thought to be sensi­ tive to both apamin and charybdotoxin block­ ade [18, 19], Apamin, a relatively specific blocker of the small-conductance Ca2+-dependent K+ channels, has been reported to cause no depolarization of the guinea pig detrusor but increases both the frequency and ampli­ tude of spikes as a result of its blockade of K+ channels responsible for after-hyperpolarizing potentials [20], In our study, it induced a marked increase in myogenic activity of the detrusor but did not antagonize the actions of cromakalim or alter its maximum response. Furthermore, apamin does not affect the in­ teraction between glibenclamide and croma­ kalim in the detrusor. It appears that the small-conductance Ca2+-activated K+ chan­ nels may control the activity of the sparsely populated pacemaker cells in the detrusor. 86Rb+ is commonly used as a tracer for K+ in efflux studies from X6Rb+-loaded smooth muscle cells in the presence of K+ channel openers [1]; however, Rb+ clearly cannot sub­ stitute for K+ to maintain the same state of function in both smooth muscles examined in the present study. In our study, rubidium acts like a nonspecific K+channel blocker; replace­ ment of K+with Rb+ or addition of Rb+ to the bath increases the myogenic activity and an­ tagonizes the effectiveness of cromakalim to abolish the myogenic activity in tissues bathed in solutions of low KC1 concentra­ tions. These effects of Rb+ are. however, sig­ nificantly reduced in tissues bathed in solu­ tions of higher KCI concentrations. The par­ tial blockade of K+ channels by Rb+ through

K ’ Channel Modulators in Detrusor and Portal Vein

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detrusor. It is possible that relative to the other K+ channels in the tissues, the density and/or the conductance of glibenclamide-specific K+ channels may be low. Glibenclamide was reported to lack a significant effect on Rb+ efflux in the rat portal vein [17]; in our laboratory, we have found that glibenclamide also has no effects on Rb+ efflux in the guinea pig detrusor not activated with K+ channel openers [Trivedi. Kau and Li, unpubl. obs.]. Together, these observations raise the possi­ bility that even in the nonactivated portal veins, the glibenclamide-specific open K+ channels probably are present only in a small population of the cells, like the pacemaker cells, such that through the modulation of the K+ channels in those cells, glibenclamide stimulates the myogenic activity of the portal vein strip as a whole. Thus, the lack of glibcnclamide-induced increase in the myogenic ac­ tivity of the detrusor may underline a physio­ logical dissimilarity between the so-called pacemaker cells of the two tissues, and the higher pKBvalue of glibenclamide in the por­ tal vein may in part be attributable to the enhanced myogenic activity and the related functional antagonism. The use of the detru­ sor to estimate the affinity of glibenclamide for the channel opened by cromakalim and other K+ channel openers may more closely reflect the interaction between glibenclamide and the channel/receptor site. In contrast to the different pKB values of glibenclamide in the portal vein, we have indeed found rather similar pKB(or pAi) values for lemakalim and cromakalim in the 20 mmol/1 KCl-stimulated detrusor strips, or for cromakalim, pinacidil and minoxidil sulfate in the 15 mmol/1 KClstimulated ones. In our study of the portal vein, both apamin and charybdotoxin failed to elicit any measurable increase in myogenic activity or to antagonize the actions of cromakalim. In contrast, charybdotoxin has been reported to

tight binding to the channels has been ob­ served in excretory glands [21 ]; it may also be the direct consequence of the lower perme­ ability of Rb+ through the K+ channels. The diminished action of Rb+ in depolarized tis­ sues could reflect the characteristics of the K+ channels opened by cromakalim. It has been reported that the current-voltage relationship of K+ channels in the red blood cell arc highly nonlinear for the permeation of K+, but this nonlinearity is hardly noticeable with Rb+ [22], Thus, it is also possible that while the difference between the magnitudes of K+ and Rb+ current through the K+ channels in a highly depolarized state is small, this differ­ ence may be greatly augmented in a hyperpolarized state upon the action of cromakalim. Nevertheless, it cannot be excluded that in addition to functional antagonism, both K+ and Rb+ may complete for a site to interact

directly with cromakalim. and this interaction could depend on the membrane potential. In conclusion, we have presented the simi­ lar as well as the dissimilar myogenic re­ sponses to different K" channel modulators in a vascular and a nonvascular smooth muscle, as exemplified by the portal vein and bladder detrusor. We have further inferred that there could be physiological and perhaps morpho­ logical dissimilarities between the K+ chan­ nels in the two tissues. These dissimilarities should provide an opportunity to develop K+ channel modulators with tissue selectivity and channel specificity.

Acknowledgements The authors would like to thank Ms. Carolyn Starr for her secretarial assistance during the preparation of the manuscript.

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5 Inoue R. Brading AF: The proper­ ties o f the ATP-induced depolariza­ tion and current in single cells iso­ lated from the guinea-pig urinary bladder. Br J Pharmacol 1990:100: 619-625. 6 Rudy B: Diversity and ubiquity of K‘ channels. Neuroscience 1988;25: 729-749. 7 Sturgess NC. Ashford M U , Cook DL, Hales CN: The sulfonylurea re­ ceptor may be an ATP-sensitivc po­ tassium channel. Lancet 1985:ii: 474-475. 8 Edwards G, Henshaw M. Miller M, Weston AH: Comparison of the ef­ fects o f several potassium-channel openers on the rat bladder and rat portal vein in vitro. Br J Pharmacol 1991;102:679-686. 9 Li JH. Zografos P. Kau ST: Mechan­ ical responses to modulation o f po­ tassium channels in isolated guinea pig portal vein. FASEB J 1990;4: A333.

10 Li J, Zografos P, Pritchard W, Yasay G. Kau S: Comparison o f the effects o f potassium channel modulators on the myogenic contractility o f blad­ der detrusor and portal vein strip from guinea pig. J Urol 1991:145: 308A. 11 Furchgott RF: The classification o f adrenoceptors (adrenergic recep­ tors). An evaluation from the stand­ point o f receptor theory; in Blaschko H, Muscholl E (eds): Catechol­ amines. Berlin. Springer. 1972. pp 283-335. 12 Arunlakshana O. Schild HO: Some quantitative uses o f drug antago­ nists. Br J Pharmacol 1959:14:4858. 13 Longmore J, Weston AH: The role o f K* channels in the modulation o f vascular smooth muscle tone; in Cook NS (ed): Potassium Channels: Structure. Classification. Function and Therapeutic Potential. Chiches­ ter, Horwood, 1990, pp 259-278.

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14 Wickcnden AD, Grimwood S. Grant TL, Todd MH: Comparison o f the effects o f the K’ channel openers cromakalim and minoxidil sulfate on vascular smooth muscle. Br J Pharmacol 1991:103:» 1481152. 15 Standen NB. Quayle JM. Davies NW. Brayden JE. Huang Y. Nelson MT: Hyperpolarizing vasodilators activate ATP-sensitive K* channels in arterial smooth muscle. Science 1989;245:177-180. 16 Sturgess NC, Kozlowski RZ. Car­ rington CA, Hales CN, Ashford M U : Effects o f sulfonylureas and diazoxide on insulin secretion and nucleotide-sensitive channels in an insulin-secreting cell line. BrJ Phar­ macol 1988:95:83-94.

Comparison of the in vitro effects of K+ channel modulators on detrusor and portal vein strips from guinea pigs.

The effects of K+ channel openers and blockers on smooth muscles of vascular and nonvascular origin from guinea pigs have been investigated. Cromakali...
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