European Journal of Pharmacology, 211 (1992) 235-241

235

© 1992 Elsevier Science Pubhshers B V. All rights reserved 0014-2999/92/$05 00

EJP 52282

Smooth muscle relaxation and inhibition of responses to pinacidil and cromakalim induced by phentolamine in guinea-pig isolated trachea Lone Bang and Jens Erik Nielsen-Kudsk Instttute of Pharmacology, The Barthohn Butldmg, Unwerstty of Aarhus, DK-8000Aarhus C, Denmark Recewed 30 May 1991, revised MS received 22 October 1991, accepted 19 November 1991

A concentration-dependent relaxant effect of phentolamlne was demonstated in guinea-pig isolated trachea and was probably unrelated to its a-adrenoceptor blocking action. The maximal effect of phentolamine against spontaneous tracheal tone was in the 24-100% range. However, phentolamlne produced 100% relaxation when the tone was induced by histamine, carbachol, 30 mM K ÷ or 124 mM K +. Relaxant ECs0 values ranged from 8 to 50 /zM with the highest potency found against histamineinduced contractions. Phentolamine caused no suppression of contractions elicited by prostaglandin F2, (PGF2~) or leukotriene C 4 (LTC4). At a concentration of 100/xM the a2-adrenoceptor blocker, yohlmbine, produded minor inhibition of spasmogeninduced tone, whereas the al-adrenoceptor blocker prazosin (up to 10 tzM) had no inhibitory effects in the trachealis. Propranolol (1 /xM), prazosin (1 /xM), yohimbine (100/zM), tetrodotoxin (3/zM), glibenclamlde (10/xM), tetraethylammonium (8 mM), 4-aminopyridine (5 mM), procaine (100/zM), dipyridamole (3/~M) or methylene blue (100/zM) did not influence the relaxant responses to phentolamine. In tracheal preparations contracted by PGF2a or LTC4, phentolamine (1, 10 and 100/zM) antagonized the relaxant action of the K + channel openers, pinacidil and cromakalim. The concentration-relaxation curves for pinacidil were shifted 30-fold to the right without change in the maximal effects, whereas the maximal cromakahm-lnduced relaxant responses were markedly suppressed by phentolamlne. Phentolamine, a-Adrenoceptors; Pinacidil; Cromakalim; K + channels; Trachea

1. Introduction

Pinacidil and cromakalim are prototype drugs for a new and rapidly increasing group of compounds which relax smooth muscle by opening of cell m e m b r a n e K + channels and subsequent hyperpolarisation (Cook and Quast, 1990; Edwards and Weston, 1990). Both drugs are potent relaxants of vascular and airway smooth muscle. Pinacidil is used clinically for the treatment of hypertension and cromakalim has recently been demonstrated to possess bronchodilator and antiasthmatic properties in addition to its vasodilator action (Williams et al., 1990). It has been demonstrated that among the numerous different K + channels, ATP-sensitive K + channels, which can be blocked by the sulfonylurea, glibenclamide, are the target for these drugs in arterial smooth muscle cells (Standen et al., 1989). Glibenclamide also inhibits the bronchorelaxant effects

Correspondence to: J.E. Nielsen-Kudsk, Institute of Pharmacology, The Barthohn Building, University of Aarhus, DK-8000 Aarhus C, Denmark. Tel 45 86.20.27.11 ext 3761, fax 45 86 19 12.77.

of pinacidil and cromakalim (Murray et al., 1989; Nielsen-Kudsk et al., 1990), suggesting that K ÷ channels of the same type are the site of action for these drugs in airway smooth muscle. McPherson and Angus (1989, 1990) have demonstrated that the a-adrenoceptor blocker, phentolamine, although less potent than glibenclamide, antagonizes the relaxation induced by cromakalim and pinacidil in isolated arteries. Similar observations have been made for cromakalim on guinea-pig trachealis (Murray et al., 1989). The exact nature of this interaction is unknown, but it is possible that phentolamine, unrelated to its a-adrenoceptor action, may act as another blocker of ATP-sensitive K ÷ channels. Evidence has recently been presented that phentolamine inhibits ATP-sensitive K ÷ channels in mouse pancreatic fl-cells (Plant and Henquin, 1990). In the present study, we examined the ability of phentolamine to antagonize the relaxant responses to pinacidil and cromakalim in guinea-pig isolated trachealis. During these experiments we observed that phentolamine itself relaxed the trachealis. We now also report on this previously undescribed bronchorelaxant action of phentolamine, which was studied on tracheal

236 preparations with spontaneous tone or contracted by histamine, carbachol, leukotriene C 4 (LTC4) , prostaglandin F2~ (PGF2~), 30 mM K + or 124 mM K +.

2. Materials and methods

2.1. Guinea-pig isolated tracheahs Albino-guinea pigs of either sex (body weight 260480 g) were killed by a sharp blow to the back of the neck. The trachea was removed and, under a microscope, cleaned of adhering tissue and cut into tubular segments each comprising two adjoining cartilage rings. At each experimental session, six tubular preparations were transferred to six temperature-regulated (37 o C) 5-ml organ baths containing Krebs solution (composition in mM: NaC1 118.0, KCI 4.6, CaC12 2.5, MgSO 4 1.15, NaHCO 3 24.9, KH2PO 4 1.15, glucose 5.5, pH = 7.4) gassed continuously with a mixture of 95% 0 2 and 5% CO 2. The preparations were mounted between two fine stainless steel pins submerged in each bath, and isometric tension was measured using strain-gauge transducers. The myograph has been described previously (Nielsen-Kudsk et al., 1986a). A tension of 0.6 g was initially imposed on the tissues. The preparations were allowed to equilibrate and gain spontaneous tone for 0.5-1 h before the addition of drugs.

2.2. Experiments The direct relaxant effects of phentolamine were assessed in tracheal preparations with spontaneous tone or in tissue contracted by histamine (1 tzM), carbachol (0.2/~M), LTC 4 (0.01/xM), PGF2~ (10/xM) or K+-rich (30 mM or 124 mM) Krebs solution. The concentrations of the structurally specific agonists were chosen in order to achieve about the same contraction level as with 30 mM K ÷ and 124 mM K ÷. In experiments using tracheas with spontaneous tone, the preparations were initially fully relaxed using theophylline (1 mM) in order to determine the pen position at zero tone (baseline). Tone was restored by repeated replacements of the bathing solution. Indomethacin (1 tzM) was present in the organ baths in experiments using tracheas contracted by spasmogens. Indomethacin completely suppressed the developed spontaneous tone. The tension level after addition of indomethacin was used as baseline. Indomethacin is necessary to obtain reproducible contractile responses to high concentrations of K + in guinea-pig trachealis (NielsenKudsk et al., 1986b). When a stable contraction plateau was reached, phentolamine was added cumulatively to the tissue baths in order to obtain concentration-relaxation curves. Tension was allowed to stabilize at a new plateau between concentration increments (about

20 min). Two other a-adrenoceptor blockers, prazosin and yohimbine, were tested for relaxant effects on the same type of contractions. The phentolamine responses were tested after pretreatment with different blocking agents: propranolol (1/zM), prazosin (1 izM), yohimbine (100 /zM), tetrodotoxin (3 /zM), glibenclamide (10 /~M), tetraethylammonium (8 mM), 4aminopyridine (5 mM), procaine (100 ~M), dipyridamole (3 tzM) or methylene blue (100 /xM). The drugs were added 30 min before phentolamine. Only one phentolamine concentration-relaxation curve was obtained with each tissue. Time-matched control tissues were run in parallel to assure that spasmogeninduced contractions were stable over the time of phentolamine addition. The antagonistic effects of phentolamine on responses to pinacidil and cromakalim were studied in tracheal preparations contracted by PGF2~ (10/zM) or LTC 4 (0.01 izM). Phentolamine was without relaxant action on contractions induced by these particular agonists. When a steady contraction level was reached, phentolamine was added, followed by cumulative addition of pinacidil or cromakalim after 30 min. Concentration-relaxation curves for pinacidil or cromakalim were obtained in the absence and in the presence of phentolamine at various concentrations (1, 10 and 100 /zM). The experiments were run in parallel with only one concentration-relaxation curve for pinacidil or cromakalim obtained with each tissue. Other a-adrenoceptor blockers (prazosin, 1 /zM and yohimbine, 100 /~M) were tested in the same way.

2.3. Data analysis Relaxant responses were expressed as % suppression of spontaneous or spasmogen-induced tracheal tone (means + S.E.). Reversal of the tension to its baseline value was taken as 100% relaxation. In order to calculate ECs0 and Emax values related to the concentration-relaxation curves, the mean data were computer-fitted by non-linear iterative regression analysis using the Hill function: E = EmaxfS/(EC~0 + C s) as a pharmacodynamic model. S is the Hill coefficient expressing the slope of the curve. S.E. values were determined simultaneously as described by Waud (1975). Differences between the parameters calculated were evaluated using Student's t-test for paired or unpaired data. P < 0.05 was considered significant.

2. 4. Drugs The drugs used were phentolamine hydrochloride, prazosin hydrochloride, yohimbine hydrochloride, histamine dihydrochloride, carbachol (carbamylcholine chloride), indomethacin, tetraethylammonium chloride, 4-aminopyridine, procaine hydrochloride, tetrodotoxin

237 (Sigma, St. Louis, U S A ) , p i n a c i d i l (Leo, C o p e n h a g e n , Denmark), cromakalim (SmithKline Beecham, UK), g l i b e n c l a m i d e ( H o e c h s t , G e r m a n y ) , p r o p r a n o l o l chloride (ICI, U K ) , d i p y r i d a m o l e h y d r o c h l o r i d e ( B o e h ringer, I n g e l h e i m ) , l e u k o t r i e n e C 4 ( U l t r a f i n e C h e m i cals, E n g l a n d ) , p r o s t a g l a n d i n F2,, ( U p j o h n , U S A ) . P h e n t o l a m i n e h y d r o c h l o r i d e was dissolved in distilled w a t e r i m m e d i a t e l y b e f o r e use a n d was p r o t e c t e d f r o m light. Pinacidil (0.1 M ) was dissolved in H C I (0.1 M). C r o m a k a l i m (0.01 M ) was p r e p a r e d in 70% ethanol. G l i b e n c l a m i d e (25 m g ) w a s s o n i c a t e d in 1 ml N a O H (0.1 M ) a n d d i l u t e d with 4 ml glucose solution (50 g / l ) to m a k e a stock s o l u t i o n o f 0.01 M. I n d o m e t h a c i n was dissolved in 5 % N a H C O 3. T h e i s o - o s m o l a r K+-rich K r e b s solutions w e r e similar to t h e s t a n d a r d K r e b s solution e x c e p t t h a t e q u i m o l a r a m o u n t s of N a C I w e r e r e p l a c e d by KCI to m a k e 30 m M K ÷ a n d 124 m M K ÷ solutions.

TABLE 1 ECs0 (/zM) and Hill coefficient (S) values with S E.s for the smooth muscle relaxation produced by phentolamlne m guinea-pig isolated trachea Tracheal tone was either spontaneous or reduced by the agents stated. The values were obtained by non-linear iteratwe regression analys~s of the mean data presented as concentrationrelaxation curves m fig. 1 Ema x w a s set equal to 100% m the fitting procedure. Phentolamme

ECs0 (~tM)

Emax (%)

S

n

Spontaneous Histamine Carbachol K + (124 mM) K + (30 mM)

35.0 _+10 0 82+0.5 40.6_+09 364+3.4 49.8+_24

100 0 100.0 1000 100.0 1000

0.6 -+0.1 1.5_+01 38_+0.3 2.9_+0.9 47_+04

8 6 5 6 5

3. Results

L T C 4 (0.01 /xM) 2.1 + 0.1, PGF2~ (10 /xM) 2.2 + 0.1. T h e i s o - o s m o l a r 124 m M K + K r e b s solution c a u s e d a b i p h a s i c r e s p o n s e . T h e s e c o n d a n d tonic p a r t of the c o n t r a c t i o n h a d a t e n s i o n o f 1.7 + 0.3 g. T h e r e s p o n s e to the i s o - o s m o l a r 30 m M K + K r e b s solution was m o n o p h a s i c with a s t e a d y t e n s i o n level of 1.6 + 0.3 g.

3.1. Effects of contractile agents

3.2. Relaxant effects of phentolamine

The mean spontaneously developed tracheal tone b e f o r e a d d i t i o n o f p h e n t o l a m i n e was 1.8 + 0.2 g (n = 8). A l l s p a s m o g e n s c a u s e d stable t o n i c c o n t r a c t i o n s with t h e following m e a n t e n s i o n s (g + S.E., n = 6): hist a m i n e (1 /zM) 2.1 + 0.1, c a r b a c h o l ( 0 . 2 / x M ) 2.2 + 0.1,

C o n c e n t r a t i o n - r e l a x a t i o n curves for p h e n t o l a m i n e a r e p r e s e n t e d in fig. 1. C o r r e s p o n d i n g EC50, Ema x a n d Hill coefficient values a r e given in t a b l e 1. P h e n t o l a m i n e ( 0 . 1 - 1 0 0 /zM) p r o d u c e d c o n c e n t r a t i o n - d e p e n d e n t s u p p r e s s i o n o f t r a c h e a l p r e p a r a t i o n s with s p o n t a -

100

100

80

80 z

60 4o

40

20

2O

,,-fi, gK

0

0 i

i

i

L

i

[PHENTOLAMINE] (M)

100

100

80

80

60

z

60

40

x ,,-H,

40

,,-4, 20 0

KI

{/{./ A

a

A

~

CO-B 10-7 lO-e 10-5 10-4 10-3 [PHENTOLAMINE] (M)

• _ . t i

~

i

i

i

10-8 10-7 10-e 10-5 10-4 [0-3 [PHENTOLAMINE] (M)

10-8 10-7 10-B 10-5 10-4 10-3

z

60

20

l

0 i i ~ i i lO-a 10-7 10-6 10-5 10-4 10-3 [PHENTOLAMINE] (M)

Flg. 1. Concentration-response curves demonstrating the relaxant action of phentolamine on guinea-pig isolated trachea. Tracheal tone was induced by 124 mM K + ( • ) , 30 mM K + (n), histamine ( • ) , spontaneously (e) or by carbachol ( • ) . Phentolamine was without relaxant activity on tracheal preparations contracted by LTC 4 (O) or PGF2,, (zx). S.E. values are shown as vertical bars (n = 5-8).

238

neous tone. There was considerable variation as reflected by the relatively high S.E. values. In five out of eight preparations phentolamine was able to cause full suppression of spontaneous tone, whereas in three preparations the maximal effect of phentolamine was 24, 31 and 63%, respectively. The ECs0 value was 35 + 10 /~M and the mean maximal inhibition of tone was 77.1 + 11.9%. Phentolamine produced complete relaxation when contraction was induced by histamine, carbachol or high concentrations of K ÷. The drug was most potent against histamine-induced contractions (table 1). The potency was about the same on contractions elicited by carbachol, 30 mM K ÷ or 124 K ÷ with ECs0 values of about 40/xM. At a concentration of 30 IzM, the drug was significantly more effective against 124 mM K ÷ than against 30 mM K÷-induced contractions. The steepness of the phentolamine relaxation curves varied considerably as expressed by the Hill coefficients which were highest in relation to the carbachol- and 30 mM K+-induced contractions (table 1). Phentolamine had no relaxant action when tracheal tone was induced by LTC 4 or PGF2~. Prazosin (0.1-10 /zM) was without relaxant effects of guinea-pig trachealis with intrinsic tone or on contractions induced by any of the contractile agents. Yohimbine (0.1-100 /xM) failed to relax preparations with spontaneous tone or those contracted with PGF2~ or LTC 4. At a concentration of 100/xM it caused small

reductions (approximately stated) in histamine- (40%), carbachol- (15%), 30 mM K ÷- (15%) and 124 mM K÷-induded (35%) contractions. Propranolol (1 IxM), prazosin (1 /~M), yohimbine (100/xM), tetrodotoxin (3 /xM), glibenclamide (10 IxM), tetraethylammonium (8000/zM), 4-aminopyridine (5000/~M), procaine (100 /~M), dipyridamole (3 /xM) or methylene blue (100 /zM) failed to influence the relaxant effect of phentolamine on histamine-contracted trachealis. Only tetraethylammonium (TEA) and 4-aminopyridine had effects themselves on tracheal tone. TEA further increased histamine-induced tone by about 30%, whereas 4-aminopyridine caused a transient relaxation of 70%.

3.3. Antagonistic effects of phentolamine on relaxant responses to pinacidil and cromakalim Pinacidil (0.1-1000 p.M) produced complete and concentration-dependent relaxation of tracheal preparations contracted by LTC 4 or PGF2~ (fig. 2). Phentolamine (1, 10 and 100 p.M) caused a rightward displacement of the pinacidil concentration-relaxation curve for LTC4-contracted preparations. The curve was shifted 3-, 10- and 30-fold to the right with the most pronounced effect at a phentolamine concentration of 10 /.~M. There was no suppression of the maximal relaxant pinacidil response. Phentolamine, at a concentration of 1 /~M, had no significant effect on the

100

100

@

CE

/,,//

8O

80 z

60 40

4o

20

2O

0 i i i ~ i ~O-a 10-7 10-6 10-5 ~0-4 10-3

~0-8

0

[PINACIDIL] (M)

~ILL/

60

i

i

L

)

i

lO -7

10-6

10-5

10 -4

10-3

[PINACIDIL}

PGF2r].nduced tone

]00

100

80 @

[M)

LTC:induced tone

/~~

80

6o

z

6o

40

x

40

i--

x

d

0 lO-B



0

~0-7

~0-6

~0-5

~0 -4

[CROMAKALIM] (N] PGFaa-lnduced tone

10-3

~O-a ~0-7

~0-6

10-5

10-4

~0-3

[CROMAKALIM] (N) LfC4-;n[luced tone

Fig. 2 Concentration-response curves demonstrating the antagonistic action of phentolamlne against the relaxation induced by plnacldll and cromakahm in guinea-pig isolated trachea. The curves were obtained in the absence ( O ) and in the presence of various concentrations of phentolamme 1/zM (e), 10/zM ( n ) and 100/~M (Ill). Tracheal tone was induced by PGF2~ (10/zM) or LTC 4 (0.01 /xM). S.E. values are shown as verhcal bars (n = 5-9)

239 p i n a c i d i l c o n c e n t r a t i o n - r e l a x a t i o n curve o b t a i n e d with p r e p a r a t i o n s c o n t r a c t e d by P G F 2 , ~, w h e r e a s h i g h e r p h e n t o l a m i n e c o n c e n t r a t i o n s c a u s e d a 10-fold p a r a l l e l shift of t h e curve to t h e right. T h e a n t a g o n i s t i c effect was t h e s a m e with 10 a n d 100 # M p h e n t o l a m i n e . Cromakalim relaxed LTC4-contracted preparations by a p p r o x i m a t e l y 70% a n d P G F 2 : c o n t r a c t e d t r a c h e a s by a p p r o x i m a t e l y 90%. T h e r e l a x a n t activity o f crom a k a l i m d e c r e a s e d at c o n c e n t r a t i o n s h i g h e r t h a n 10 /zM, giving rise to b e l l - s h a p e d c o n c e n t r a t i o n - r e l a x a t i o n curves (fig. 2). P h e n t o l a m i n e (10 a n d 1 0 0 / z M ) c a u s e d r i g h t w a r d shifts of t h e c o n c e n t r a t i o n - r e l a x a t i o n curves a n d in a d d i t i o n p r o d u c e d m a r k e d s u p p r e s s i o n o f t h e m a x i m a l r e l a x a n t r e s p o n s e s (fig. 2). In P G F 2 , : c o n t r a c t e d tissues p h e n t o l a m i n e 10 / z M p r o d u c e d a s t r o n g e r a n t a g o n i s t i c effect t h a n d i d t h e 1 0 0 / z M conc e n t r a t i o n o f t h e drug. P h e n t o l a m i n e was clearly a m o r e effective a n t a g o n i s t against r e l a x a n t r e s p o n s e s to c r o m a k a l i m t h a n against pinacidil. T a b l e 2 shows the p h a r m a c o d y n a m i c p a r a m e t e r s (ECs0, Ema x a n d S) ob-

t a i n e d by n o n - l i n e a r , iterative r e g r e s s i o n analysis o f t h e mean data constituting the concentration-relaxation curves. T h e h i g h e r p h e n t o l a m i n e c o n c e n t r a t i o n s lowe r e d t h e s t e e p n e s s ( e x p r e s s e d by t h e S values) o f b o t h the cromakalim and pinacidil concentration-relaxation curves. P r a z o s i n (1 /xM) a n d y o h i m b i n e (100 /xM) h a d no a n t a g o n i s t i c actions on t h e r e l a x a n t r e s p o n s e s i n d u c e d by pinacidil or c r o m a k a l i m .

4. D i s c u s s i o n

P h e n t o l a m i n e is a classical a - a d r e n o c e p t o r b l o c k i n g drug. It has affinity for b o t h a~- a n d a 2 - r e c e p t o r s . In a d d i t i o n it has i n h i b i t o r y effects on r e s p o n s e s to serot o n i n a n d p r o d u c e s g a s t r o i n t e s t i n a l stimulation, possibly by an agonistic effect on m u s c a r i n i c a n d h i s t a m i n e H e r e c e p t o r s ( H o f f m a n a n d Lefkowitz, 1990). a A d r e n o c e p t o r s are p r e s e n t in g u i n e a - p i g a n d h u m a n

TABLE 2 ECs0,(p.M), Ema x (% relaxation of tracheal tone) and Hdl coefficient (S) values (+S.E.) (n = 5-9) for the relaxation of guinea-pig isolated

trachea reduced by plnacldil and cromakahm either alone (control) or in the presence of various concentrations of phentolamlne The values were obtained by non-hnear iterative regression analysis of the mean data presented as concentration-relaxation curves in fig. 2 Tracheal tone was reduced by PGF2~ or by LTC 4. Alone

Phentolamme 1 ~M

10 p.M

100 ~M

33 (0 2) 96.0 (1 6) 13 (0.1)

10.3 a (0.8) 100 0

99 5 a (12 1) 100.0

23.3 a (1.6) 100 0

1.2 (0 09)

1.0 (0 1)

0.8 a (0 04)

10 (0 1) 71.0 (3.7) 25 (0.8)

1.2 (0.08) 60.5 (1.9) 2.3 (0.3)

97a (2 2) 27.8 a

16.9 a (3.9) 36 4 a

08 (0.1)

1.0 (0.2)

4.3 (0 2) 97 4 (0 9) 18 (0 1)

3.7 (0.2) 98.7 (1.1) 2.7 (0.3)

53.7 a (6 7) 100 0

55.0 a (10.8) 100.0

0.8 a (0.07)

0.7 a (0.08)

08 (0.03) 92 3 (1 2) 33 (0.4)

0.9 (0.02) 89.1 (0.9) 2.8 (0.2)

18.3 a (4.4) 24.8 a

10.8 a (2.4) 56.0 a

1.0 a (0.2)

0.9 a (0.2)

Tracheal tone mduced by L TC 4

Pmacidil ECs0 Emax

S Cromakahm ECs0 Emax S

Tracheal tone mduced by PGF2~

Pinacldil ECso Emax

S Cromakalim ECso Emax

S

a Denotes a sigmficant difference from the control.

240 airway smooth muscle cells, but are probably of minor importance in regulation of muscle tone in both healthy and asthmatic airways (Goldie et al., 1985). Inhibition of exercise-induced asthma has been demonstrated with phentolamine and prazosin (Walden et al., 1984; Barnes et al., 1981) and may possibly be due to blockade of a-receptors in the bronchial vasculature (McFadden, 1990). It was demonstrated in the present study that phentolamine produces relaxation of guinea-pig airway smooth muscle. The al-adrenoceptor blocker, prazosin, failed to relax guinea-pig trachealis and the a2-adrenoceptor blocker, yohimbine, produced minor tracheal relaxation only at a very high concentration. ECs0 values for the relaxant effect of phentolamine were in the 8 - 5 0 / z M range, which is about 100 times higher than the concentrations needed for a-adrenoceptor blocking activity in isolated vascular preparations (McPherson et al., 1984). The airway smooth muscle relaxant effect of phentolamine therefore seems to be unrelated to its a-adrenoceptor blocking action. Propranolol failed to modify the phentolamine-induced relaxation of histamine-contracted trachea, indicating that release of catecholamines and subsequent /~adrenoceptor stimulation is not the mechanism of relaxation. There was no potentiation of the phentolamine response after dipyridamole, indicating that the effect is not mediated by adenosine. The phentolamine response was unaffected by methylene blue and is therefore not mediated by guanylate cyclase stimulation. The Na + channel blocker tetrodotoxin was also without effect on the relaxant response to phentolamine, suggesting that the action of phentolamine does not involve the discharge of action potentials in inhibitory neurones supplying the smooth muscle. The relaxant effect was dependent on the agent used to induce tone. The effect profile of phentolamine as tested on a panel of contractile agents (intrinsic tone, histamine, carbachol, 30 mM K +, 124 mM K +, PGF2~, LTC 4) was markedly different from that seen with xanthine derivatives, /32-adrenoceptor agonists, Ca 2+ antagonists or K* channel openers (Nielsen-Kudsk et al., 1986b, 1988). We have no explanation for the consistent finding that 30/zM phentolamine was more effective to suppress the tone induced by 124 mM K + than that induced by 30 mM K +. This is in sharp contrast to effects produced by potassium channel openers. Glibenclamide, a blocker of ATP-sensitive K + channels, failed to influence phentolamine-induced tracheal relaxant responses. Also the more unspecific K + channel blockers, TEA, 4-aminopyridine and procaine were without effect. It seems unlikely that the mechanism of relaxation involves a direct interaction with specific cell surface drug receptors, since contractions elicited by different receptor agonists (histamine, carbachol) and also by high K + were inhibited by

phentolamine. It may be speculated that phentolamine could possibly interfere with an intracellular effector or second messenger system. The contractile effects of both carbachol (via muscarinic M 3 receptors) and histamine (via H 1 receptors) seem to be mediated by inositol phospholipid breakdown and subsequent mobilisation of Ca 2+ from intracellular stores (Douglas, 1990). The complete lack of activity of phentolamine on responses to the eicosanoids PGF:~ and LTC 4 was striking and may indicate intracellular effector systems for these drugs different from those utilized by histamine and carbachol. It is of interest that multiple binding sites different from the a-adrenoceptor itself have been demonstrated for drugs like phentolamine, which possess an imidazoline structure (Michel and Insel, 1989). Further studies are obviously needed to elucidate the mechanism of airway smooth muscle relaxation produced by phentolamine. It has previously been demonstrated that phentolamine antagonizes and reverses the smooth muscle relaxation and hyperpolarisation produced by cromakalim in isolated vascular preparations and guineapig trachea (McPherson and Angus, 1989; Murray et al., 1989). The results reported by Murray et al. were obtained on tracheal preparations with spontaneous tone. Phentolamine at concentrations up to 100 ~ M was reported to be without effect on tone, which clearly contrasts with the present finding of a concentrationdependent suppression of intrinsic tracheal tone. Among several contractile agents tested in the present study, only PGF2~ and LTC 4 produced contractions that were not relaxed by phentolamine. The reasons for the disagreement between the two studies are unclear. Methodological differences may possibly be of importance. Murray et al. used open ring preparations with an imposed tension of 1.25 g and also used phentolamine mesylate instead of the chloride salt of the drug. The antagonistic effect of phentolamine on cromakalim-induced relaxation of these types of contractions was similar to the effect reported by Murray et al. who used spontaneous tone preparations. Phentolamine produced concentration-dependent displacement of the concentration-relaxation curves for cromakalim to the right and in addition caused marked suppression of the maximal relaxant responses. It was now shown that phentolamine also antagonizes the relaxation of airway smooth muscle produced by pinacidil. Concentration-relaxation curves for pinacidil on PGF2~- or LTC4-contracted tracheal preparations were shifted to the right but the maximal relaxant responses to pinacidil were not influenced by phentolamine. In terms of receptor pharmacology, phentolamine seems to act as a non-competitive antagonist against cromakalim and as a competitive antagonist against pinacidil. Exactly the same pattern was seen with phentolamine in dog isolated coronary artery (Mc-

241

Pherson and Angus, 1990). The observation that there was a more pronounced antagonism of pinacidil in LTCa-contracted tissues and of cromakalim in PGF2~contracted tissues in the presence of 10 p.M compared to 100/xM phentolamine could reflect the fact that the former concentration is at or close to the optimal inhibiting concentration. The antagonistic effect of phentolamine on cromakalim and pinacidil in guineapig trachealis seemed unrelated to the a-adrenoceptor blocking ability of the drug, since prazosin and yohimbine failed to produce similar antagonistic actions. The precise interaction between phentolamine and cromakalim/pinacidil in smooth muscle is unknown. However, strong evidence has recently been presented that phentolamine acts as a blocker of the ATP-sensitive K ÷ channel in mouse pancreatic/3-cells (Plant and Henquin, 1990). It is likely that this also applies to smooth muscle. Glibenclamide, a blocker of ATP-sensitive K ÷ channels in both smooth muscle (Standen et al., 1989) and pancreatic /3-cells, showed antagonistic effects to cromakalim and pinacidil in guinea-pig trachea (Nielsen-Kudsk et al., 1990) that were qualitatively similar to those seen with phentolamine. Glibenclamide was a more potent antagonist, but showed the same non-competitive type of antagonism against cromakalim and the same competitive type of antagonism against pinacidil as seen with phentolamine. In conclusion, phentolamine in addition to its aadrenoceptor blocking action relaxes guinea-pig airway smooth muscle by a so far unknown mechanism of action and antagonizes the tracheal relaxant responses to the K + channel openers, pinacidil and cromakalim.

References Barnes, P J , N M. Wdson and H. V,ckers, 1981, Prazosm, an alpha1-adrenoceptor antagonist, partially inhibits exercise-induced asthma, J Allergy Chn Immunol. 68, 411 Cook, N S and U Quast, 1990, Potassmm channel pharmacology, m" Potassmm Channels" Structure, Classification, Function and Therapeutic Potential, ed. N.S. Cook (Elhs Horwood Limited, England) p 181. Douglas, J S., 1990, Receptors on airway smooth muscle, Am. Rev. Resp,r Dis 141, S123. Edwards, G. and A.H. Weston, 1990, Structure-activity relationships of K + channel openers, Trends Pharmacol. Scl. 11,417. Goldle, R.G., K M Luhch and J.W. Paterson, 1985, Bronchial aadrenoceptor function m asthma, Trends Pharmacol Scl. 6, 469.

Hoffman, B B and R.J. Lefkowltz, 1990, Adrenerglc receptor antagonists, m: Goodman and Gilman's The Pharmacological Bas,s of Therapeutics, eds A.G Gdman, TW. Rail, A.S. Nies and P Naylor (Pergamon Press, U S.A.) p 221. McFadden, E.R., Jr., 1990, Hypothesis exercise-induced asthma as a vascular phenomenon, Lancet 335, 880 McPherson, G A. and J A. Angus, 1989, Phentolamme and structurally related compounds selectwely antagomze the vascular act,ons of the K + channel opener, cromakalim, Br J Pharmacol 97, 941. McPherson, G A. and J A. Angus, 1990, Characterization of responses to cromakahm and pinacldll ,n smooth and cardiac muscle by use of selective antagomsts, Br. J Pharmacol 100, 201. McPherson, G.A., I M Couper and D.A Taylor, 1984, Competitive antagonism of al-adrenoceptor mediated pressor responses in rat mesentenc artery, J Pharm. Pharmacol. 36, 338. Michel, M C. and P A Insel, 1989, Are there mult,ple imidazohne binding s,tesg, Trends Pharmacol. Scl 10, 342 Murray, M.A, J P Boyle and R C. Small, 1989, Cromakahm-,nduced relaxat,on of guinea-pig ,solated tracheahs antagomsm by ghbenclamlde and by phentolamme, Br J. Pharmacol 98, 865. N,elsen-Kudsk, F , B Poulsen, C Ryom and J E. Nlelsen-Kudsk, 1986a, A strata-gauge myograph for ,sometnc measurements of tens,on m Isolated small blood vessels and other muscle preparations, J Pharmacol. Meth. 16, 215 Nlelsen-Kudsk, J E , J.-A Karlsson and C.G A Persson, 1986b, Relaxant effects of xanthmes, a /32-receptor agomst and Ca 2+ antagomsts m gumea-p~g tracheal preparat,ons contracted by potassmm or carbachol, Eur. J Pharmacol. 128, 33 Nlelsen-Kudsk, J.E., S Mellemkj~er, C. Slggaard and C B. Nielsen, 1988, Effects of pinacldll on guinea-pig airway smooth muscle contracted by asthma medmtors, Eur J. Pharmacol 157, 221. N,elsen-Kudsk, J E., L. Bang and A.M. Brcnsgaard, 1990, Ghbenclamlde blocks the relaxant act,on of pmacldd and cromakahm m airway smooth muscle, Eur. J. Pharmacol 180, 291. Plant, T.D and J C Henqum, 1990, Phentolamlne and yohimbme inhibit ATP-sensltwe K + channels in mouse pancreatic /3-cells, Br J. Pharmacol. 101, 115 Standen, N B, J.M. Quayle, N W. Dawes, J E. Brayden, Y Huang and M.T. Nelson, 1989, Hyperpolanzmg vasoddators actwate ATP-sens,twe K ÷ channels m arterial smooth muscle, Science 245, 177 Walden, S.M, E R Bleecker, K Chahal, E J. Bntt, P Mason and S. Permutt, 1984, Effect of alpha-adrenerg,c blockade on exerciseinduced asthma and conditioned cold air, Am Rev Resplr Dis 130, 357 Waud, D R , 1975, Analysis of dose-response curves, in' Methods in Pharmacology, Vol 3, Smooth Muscle, eds E.E. Daniel and D.M. Paton (Plenum Press, New York and London) p. 471 Williams, A J., T H. Lee, G.M. Cochrane, A Hopkirk, T. Vyse, F Chew, E. Lavender, D.H. Rlchards, S Owen, P. Stone, S Church and A.A Woodcock, 1990, Attenuat,on of nocturnal asthma by cromakahm, Lancet 336, 334.

Smooth muscle relaxation and inhibition of responses to pinacidil and cromakalim induced by phentolamine in guinea-pig isolated trachea.

A concentration-dependent relaxant effect of phentolamine was demonstrated in guinea-pig isolated trachea and was probably unrelated to its alpha-adre...
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