Br. J. Pharmacol. (1992), 105, 51-58
,'-.
Macmillan Press Ltd, 1992
The resistance of some rat cerebral arteries to the vasorelaxant effect of cromakalim and other K + channel openers 'Grant A. McPherson & Andrew P. Stork Baker Medical Research Institute, Commercial Rd, Prahran, 3181, Victoria, Australia 1 Cromakalim (0.01-30gM) and sodium nitroprusside (SNP, 0.01-100guM) were tested for their ability to relax a number of pre-contracted small arteries (approximate diameter 200-700gM at 100mmHg) from the rat, rabbit and guinea-pig. 2 In the rat, SNP (0.01-100gM) caused near maximal relaxation in all vessels studied including the middle cerebral, anterior cerebellar, basilar, mesenteric and renal arteries. Cromakalim (0.01-30guM) relaxed pre-contracted mesenteric and renal arteries but was only a weak relaxant of all the rat cerebral arteries with the exception of the basilar artery. Similar experiments using mesenteric and cerebral vessels from the rabbit and guinea-pig showed cromakalim could relax pre-contracted vessels in a concentrationdependent manner. 3 Two other K+ channel openers, nicorandil and pinacidil, were also tested for their ability to relax rat cerebral arteries. Nicorandil (0.01-100guM) was ineffective in the rat anterior cerebellar artery at concentrations up to 100,UM. Pinacidil (0.01-100guM) caused significant vasorelaxation, although high concentrations were required (> 10,UM) and the response was insensitive to the effects of glibenclamide (3pM). 4 Electrophysiological experiments with the rat anterior cerebellar artery showed that cromakalim (up to 30gM) failed to influence the resting membrane potential of impaled single smooth muscle cells. 5 The results showed that some rat small cerebral arteries were resistant to the effects of K+ channel openers including cromakalim, pinacidil and nicorandil. This is peculiar to this vascular tree since the same vessels from other species do not exhibit the same behaviour. Such a phenomenon could result from a lack of K+ channels opened by cromakalim in rat cerebral arteries. Keywords: K+ channels; cromakalim; pinacidil; nicorandil; cerebral arteries; vascular specialization
Introduction Cromakalim is representative of a new class of smooth muscle relaxants (Hamilton et at., 1986; Edwards & Weston, 1990) which act by opening a type of K+ channel (termed K+ in this paper) in the cell membrane resulting in membrane hyperpolarization. There is a great deal of interest in the vascular actions of cromakalim, not only does it represent a new class of therapeutic agent, but also its haemodynamic profile appears to differ from other anti-hypertensives, in particular Ca2+ antagonists such as nifedipine (Buckingham, 1988; Shoji et al., 1990). That cromakalim and other K+ channel openers may exhibit some organ specificity is an intriguing property. A number of in vivo studies have shown cromakalim to dilate selectively some vascular beds compared with others (Buckingham et al., 1986; Hof et al., 1988). In addition cromakalim and other K+ channel openers appear to be more efficacious, in vivo at least, in ischaemic tissues (Angersbach & Nicholson, 1988; Maruyama et al., 1989). The mechanisms behind such selectivity are currently unknown. In some preliminary studies we observed that cromakalim was an unusually poor vasorelaxant in some isolated small cerebral arteries of the rat. The purpose of the present work was to extend these observations to determine the extent of such resistance in a variety of vessels of the same size. We found that such resistance was confined to the rat cerebral circulation and possibly results from a lack of K+ channels in this vascular bed. A preliminary account of this work was presented at the 24th annual meeting of the Australian Society of Clinical and Experimental Pharmacologists (Stork et al., 1990).
Methods Isolation of small resistance arteries Wistar Kyoto (WKY) or spontaneously hypertensive (SHR) rats were killed by CO2 asphyxia. In some experiments WKY 'Author for correspondence.
rats were also killed by a blow to the head. Guinea-pigs were killed by a blow to the head and rabbits were killed by an overdose of pentobarbitone (60 mg kg- l i.v.). The organ under study was rapidly removed and placed in ice cold Krebs solution (composition in mM: NaCl 119, KCl 4.7, MgSO4 * 7H20 1.17, NaHCO3 25, KH2PO4 1.18, CaCl2 2.5 and glucose 11) gassed with 5% CO2 in 2. Two mm segments were mounted in a small vessel myograph to record isometric tension development as previously described (Angus et al., 1988). Briefly two 40,um wires were threaded through the lumen of the vessel segment. One wire was attached to a stationary support driven by a micrometer while the other was attached to an isometric force transducer which measured force development. Force development was recorded on a dual flat bed recorder (W&W Scientific Instruments, model 320). Vessels were allowed to equilibrate under zero tension for 30 min at 36°C. A passive length-tension curve was constructed as previously described (Mulvany & Halpern, 1977). Since some vessels (the rat cerebral vessels) displayed spontaneous myogenic tone, sodium nitroprusside (SNP, 0.1 mM) was included in the bath to cause full relaxation while constructing the curve. The vessel was set at a diameter 0.9 times the diameter of the vessel at 100mmHg. This point on the passive length-tension curve corresponds to the point of maximum active tension development (Mulvany & Halpern, 1977). Non-linear curve fitting of the passive length-tension curve was achieved using a custom written program for the IBM PC (NORMALIZE, GA McPherson) which uses the Marquart-Levenberg modification of the Gauss-Newton technique (McPherson, 1985). In the text, vessel diameter is expressed as the diameter at an equivalent transmural pressure of 100mmHg (D100). Upon completion of the passive length-tension curve, vessels were washed and allowed to equilibrate for 30min before the addition of any drugs. The viability of the tissue was then assessed by adding a K+ depolarizing solution (in which all NaCl in the Krebs buffer was replaced by an equimolar concentration of KCI) to activate the tissue maximally. Unless otherwise stated the vasorelaxant activity of cromakalim was assessed in tissues precontracted with a sub-
52
G.A. McPHERSON & A.P. STORK
maximal concentration of 5-hydroxytryptamine (5-HT; approximately 0.3-1AuM). The rat cerebral vessels were unusual in that they displayed spontaneous myogenic tone. However, in the majority of experiments with these vessels, active tone was induced with a sub-maximal concentration of 5-HT so that a constant level of tone could be generated throughout the duration of the experiment. Vasorelaxant responses of the drugs tested were expressed as a percentage of the maximum level of tone. This was calculated as the difference between tone in the presence of the constrictor and that in the presence of SNP (0.1 mM), which was added at the end of every concentration-effect curve constructed to a vasorelaxant. Cumulative concentration-effect curves were constructed to cromakalim (0.01-30giM) and SNP (0.01-100gM) which was used as the control vasorelaxant. In additional experiments, we also determined the ability of pinacidil and nicorandil (K+ channel openers, 0.01-100gM) and three other vasorelaxants (acetylcholine 0.01-3pM, glyceryl-trinitrate 0.01-3 fM and nitric oxide 0.1-100gUM) for their ability to cause vasorelaxant responses in selected tissues. Nitric oxide (NO) solutions were prepared from acidified sodium nitrite as previously described (Cocks & Angus, 1990). The final concentration of NO generated was assumed to be equivalent to the final concentration of acidified sodium nitrite used. We were also interested in the effect of cromakalim on spontaneous myogenic tone which was displayed by the rat small cerebral arteries. It has been our experience that myogenic behaviour of small resistance arteries is better observed when vascular reactivity is assessed under isobaric/isotonic conditions. Consequently in some experiments the ability of cromakalim to alter vascular tone of the small resistance arteries was assessed isobarically using a newly developed computer based system (ISOTONIC, McPherson, unpublished data). In these experiments the manual micrometer on the small vessel myograph was replaced with a computer controlled motor (Burleigh Inchworm motor controlled by a 6000 series controller, Burleigh, U.S.A.). Wall tension in the small resistance artery was continually monitored using an analogue to digital card (DASH16, Metrabyte, U.S.A.) and transmural pressure calculated. In isobaric mode the computer program could adjust the separation distance of the wires, by the use of the inchworm motor, such that pressure was held constant at a pre-set level. Vascular reactivity was therefore assessed by monitoring changes in vessel diameter.
(Hoechst); sodium nitroprusside (Nipride, Roche); glyceryltrinitrate (David Bull Laboratories, Australia); acetylcholine bromide (Sigma); U46619 (1,5,5-hydroxy-1 la,9a (epoxymethano) prosta-E2,13-dienoic acid, Upjohn); endothelin-1 (Austpep, Australia). Cromakalim (10mM), pinacidil (10mM), nicorandil (10mM) and glibenclamide (1 mM) stock solutions were made in 100% methanol. All other drugs were made up on the day of experiment. Dilutions of all drugs were made in distilled water.
Results
Characteristics of the small arteries studied Figure 1 shows a schematic diagram of the rat cerebral circulation showing the origin of the cerebral vessels studied. These were the basilar, anterior cerebellar and the middle cerebral arteries. In selected studies anatomically similar vessels were also taken from the rabbit and guinea-pig. Several peripheral small arteries were also studied including a mesenteric and renal artery. The diameters of all vessels studied, at an estimated transmural pressure of 100 mmHg, are listed in Table 1. The vessels ranged in size from 200-700gum depending on the species and origin of the vessel. The viability of each vessel was assessed at the commencement of each experiment by fully activating the preparation using a K+ depolarizing solution. All vessels developed significant amounts of tension, which were similar when the values were standardized for differences in vessel diameter (Table 1).
Comparison of the vasorelaxant effect of cromakalim in cerebral and peripheral vesselsfrom rat, rabbit and
guinea-pig SNP was an effective vasorelaxant in all SHR and WKY rat cerebral and peripheral vessels studied although its potency did vary to some extent (Figures 2, 3 and 5; Table 2). In the mesenteric artery SNP had a pEC50 of approximately 6.6 while in the anterior cerebellar it was approximately 20 fold less potent (5.3). In all cases SNP could reverse nearly all the vasoconstrictor-induced tone (greater than 95% relaxation), except in the WKY small renal vessel where 80% of active tone was reversed at the highest concentration of SNP used.
Electrophysiological experiments In some experiments the intracellular resting membrane potential was monitored in rat anterior cerebellar vessels mounted in the small vessel myograph. A conventional glass electrode (see McPherson & Angus, 1991 for details) was used to impale a single smooth muscle cell. Cumulative concentrations of cromakalim were added to the bath and membrane potential and tension changes were monitored simultaneously. Since the rat anterior cerebellar artery displayed intrinsic myogenic tone, vasorelaxant responses could be assessed without the addition of a vasoconstrictor agent.
Statistics and data analysis Statistical comparisons between two groups were made by use of Student's t test. Results in the text are the mean + s.e.mean for the specified number of experiments. The potency of the vasorelaxants are expressed as the -log concentration of the drug required to produce 50% relaxation (pEC50). Relaxation responses were calculated as a percentage of active tone with 100% implying full relaxation.
Drugs The following drugs were used: cromakalim (Beecham); pinacidil monohydrate (Leo); nicorandil (Chugai); glibenclamide
Middle cerebral
4-Anterior cerebellar
Figure 1 Dorsal view (schematic diagram) of the rat brain showing the basilar, anterior cerebellar and middle cerebral arteries which were used. Anatomically similar vessels were also obtained from the rabbit and guinea-pig.
CROMAKALIM VASORELAXATION
53
Table 1 (A) Arterial diameters (un) at 100mmHg, of the vessels studied estimated during the normalization procedure and (B) mean active tension (mN mm'- ) developed to a K + depolarizing solution which was used to assess tissue viability
Animal A WKY SHR Rabbit
Guinea-pig B WKY SHR Rabbit Guinea-pig
Mesenteric
303 (14) 298 (11) 383 (45) 398 (16) 4.7 (0.6) 5.3 (0.4) 5.5 (0.9) 6.3 (0.9)
Renal 486 (46)
5.3 (0.4)
Basilar
Anterior cerebellar
Middle cerebral
Diameter (pm) 399 (16) 251 (8) 351 (19) 226 (12) 788 (54) 443 (10) 362 (8)
266 (8) 233 (5) 397 (19)
Tension (mN mm-') 5.9 (0.7) 2.9 (0.5) 7.4 (0.6) 2.9 (0.6) 7.7 (1.0) 4.6 (0.6) 4.4 (0.05)
2.7 (0.5) 2.7 (0.3) 3.8 (0.7)
Values are the mean (s.e.mean) of 4-10 separate determinations.
In contrast, a number of peripheral rat vessels were found to be sensitive to cromakalim. Thus in the WKY mesenteric and renal artery and SHR mesenteric artery, cromakalim was an effective vasorelaxant with a pEC50 of approximately 6.4 and with an ability to cause greater than 95% relaxation (Figure 5, Table 2). For comparative purposes the ability of cromakalim to relax pre-contracted cerebral and mesenteric vessels from the rabbit and guinea-pig was also studied. In contrast to the results obtained in the rat, cromakalim was an effective vasorelaxant in rabbit and guinea-pig cerebral arteries (Figures 2, 3 and 5; Table 3). In all cases the pEC50 value for cromakalim was approximately 6.4. In addition cromakalim caused greater than 90% relaxation. As in the rat cerebral vessels, SNP was approximately 10 times less potent on the anterior cerebellar artery than on the mesenteric artery in the rabbit (Figure 3, Table 2). However, in the guinea-pig both cromakalim and SNP appeared to be relatively potent vasorelaxants (Figure 3, Table 2).
Cromakalim was particularly ineffective as a vasorelaxant on rat small cerebral arteries with the exception of the basilar artery. In the WKY and SHR basilar artery, cromakalim was able to produce significant vasorelaxant response in tissues pre-constricted with 5-HT (Figure 2, Table 2). In the WKY vessels approximately 60% and in the SHR 35% of the active tone was reversed. These were not significantly different (P > 0.05, unpaired t test). In addition cromakalim was approximately 10 fold more potent in rat mesenteric and renal small arteries (pEC50 basilar 5.58 cf 6.42 mesenteric). In the anterior cerebellar and middle cerebral artery from both strains cromakalim was a particularly poor relaxant, with only the highest concentration of cromakalim used (30pM) causing any marked relaxation (Figure 3). Approximately 20% of active tone could be reversed by cromakalim in these tissues. In the majority of studies using the rat, vessels were obtained from animals killed by CO2 asphyxia. Cerebral vessels are known to be quite sensitive to CO2. Consequently we repeated some experiments with rats killed by a blow to the head to determine whether the method of killing the animals affected the response to cromakalim. Figure 4 shows the vasorelaxant responses to cromakalim in anterior cerebral or basilar arteries from these rats in which the tissues were precontracted with either 5-HT or a low concentration of K+ (20mM). Again, particularly in the anterior cerebellar artery, cromakalim was a weak relaxant causing only 20-40% maximum relaxation at the highest concentration of cromakalim used (30 mM).
Responses of the rat anterior cerebellar artery to pinacidil and nicorandil Nicorandil (0.01-100gM), like cromakalim, was ineffective as a vasorelaxant in the WKY anterior cerebellar artery (Figure 6). This compound caused only 11% relaxation at a concentration of 100puM. However, pinacidil (0.01-100gM) was able to cause significant relaxation (approximately 75% relaxation of
Table 2 pEC50 and maximum response values for cromakalim and sodium nitroprusside (SNP) obtained in rat cerebral and peripheral small resistance arteries pre-contracted with 5-hydroxytryptamine (0.3-1 pM) Cromakalim %relax. pEC50 WKY Basilar Ant. cereb. Middle cer. Mesenteric Renal SHR Basilar Ant. cereb. Middle cer. Mesenteric Renal
5.58 (0.07)
-
61 (10) 17 (4) 23 (3)
6.42 (0.08) 6.18 (0.07)
96(1) 96 (2)
5.48 (0.1)
35 (9)
6.28 (0.1) NS
8
(4)
19 (4) 95 (2)
SNP
pEC50
%relax.
5.86 (0.06) 5.29 (0.07) 5.52 (0.07) 6.56 (0.2) 6.21 (0.3)
97 (2)
5.74 5.52 5.58 6.98
(0.1) (0.2) (0.1) (0.1)
Results are the mean (s.e.mean) of 4 to 12 separate determinations. pEC50 is the -log (M) of the concentration of drug required to cause 50% relaxation. % relax. is the percentage reduction in total active tone. * Curve could not be processed by the computer due to an absence of points, therefore, the highest concentration of cromakalim used (304uM). NS, not studied.
95 (4) 98 (1) 94 (2) 80 (5) 91 (3)
95 (3) 94 (4) 97 (1)
%relax. is the maximum relaxation observed at
54
G.A. McPHERSON & A.P. STORK a lO0i
100-
A\ \Jii I 0~\ x
0\
A
50-
I\
E
R.\
50-
0\
0
x
VA
E
LU
f%
~A
CO -
NO 0--
0)
0-o w 2lo
. C )ot ic a)
Al z::n70-
=
b *~~~~~~~~~~ 11 T
3
I
I--A
0-
;0
II\T
5 -
iOh
o
-
9
8
7
6
O
6 5 Conc. (-log M)
4
Figure 4 Mean concentration-effect curves for cromakalim in the anterior cerebellar (A, A) or the basilar artery (O. 0) from WKY rats killed by a blow to the head. Tissues were precontracted with 5hydroxytryptamine (solid symbols, 0.3-1 pM) or K+ (open symbols, 20 mM). Results are expressed as a percentage of total constrictor tone (%Em.x) defined by sodium nitroprusside (0.1 mM) and are the mean from 4 to 6 separate experiments.
T I
5
7
4
Conc. (-log M)
Figure 2 Mean concentration-effect curves for sodium nitroprusside (SNP) (a) and cromakalim (b) in the basilar artery from WKY (0), SHR (0) and rabbit (A). Tissues were precontracted with 5hydroxytryptamine (0.3-1 gM). Results are expressed as a percentage of the total amount of constrictor tone (%Emax) defined by a maximal concentration of SNP (0.1 mM) added at the end of each experiment. Values are the mean from 4 to 12 separate experiments.
active tone) with a pEC50 of approximately 4.5 (Figure 6). Additional experiments showed that the response to pinacidil (30M) in the presence of glibenclamide (3pM, 50 + 6% relaxation) was not significantly different from that in its absence (53 + 5% relaxation, n = 3). 100
E 0~
FH5 0)1 -
a
50-
7 6 Conc. (-log M)
x
wE
Figure 5 Mean concentration-effect curves for sodium nitroprusside (SNP) (a) and cromakalim (b) in the mesenteric artery from WKY (0), SHR (0), rabbit (A) and guinea-pig (A). Tissues were precontracted with 5-hydroxytryptamine (rat, 0.3-1 pM) or noradrenaline (rabbit and guinea-pig, 30-1OOpuM). Results are expressed as a percentage of the total constrictor tone (%Em.x) and are the mean from 4 to 12 separate experiments.
-0 0)
H
00b A
A
* 1 Too!~~~~~~~
4
*-. \
Electrophysiological characteristics of the rat anterior
50-
cerebellar artery A 0
A--AA OhAl , , A~A-4_
8
9
8
7
6
5
4
Conc. (-log M)
Figure 3 Mean concentration-effect curves for sodium nitroprusside (SNP) (a) and cromakalim (b) in the anterior cerebellar artery from WKY (0), SHR (0), rabbit (A) and guinea-pig (A). Tissues were precontracted with 5-hydroxytryptamine (rat and rabbit, 0.3-1 PM) or histamine (guinea-pig 1-30M). Results are expressed as a percentage of total constrictor tone (%Emax) and are the mean from 4 to 12 separate experiments.
The rat anterior cerebellar artery had a resting membrane potential (Em) of -38 + 2 mV (7 impalements from 3 arteries) in the absence of any vasoconstrictor agent. The membrane potential was unstable and oscillated with an amplitude of between 2 to 5 mV. Figure 7 shows the results from an experiment examining the effects of cromakalim. Cromakalim caused only a small hyperpolarization (-2 + 1 mV, n = 3) even at concentrations up to 30M. In addition there was no effect on the spontaneous tone displayed by these vessels. In contrast SNP (0.1 mM) caused a hyperpolarization (-14 + 5 mV, n = 3) and relaxation (Figure 7) in vessels displaying spontaneous myogenic tone.
CROMAKALIM VASORELAXATION
55
Table 3 pEC50 and maximum response values for cromakalim and sodium nitroprusside (SNP) obtained in rabbit and guinea-pig cerebral and peripheral small resistance arteries Cromakalim %relax.
Vessel
pEC50
SNP
pECGo
%relax.
100 (0) 95 (3) 98 (1) 81 (7) 95 (4) 96 (1)
Rabbit Basilar Ant. cereb. Middle cer. Mesenteric
6.39 (0.14) 6.33 (0.05) 6.28 (0.1) 6.75 (0.04)
97 (2) 99(1) 98 (1)
6.82 (0.3) 5.26 (0.2) 5.76 (0.2) 5.83 (0.2)
Guinea-pig Ant. cereb. Mesenteric
5.84 (0.06) 6.36 (0.3)
88 (6) 93 (2)
6.39 (0.1) 6.96 (0.2)
99(1)
Mesenteric arteries of both species were pre-contracted with noradrenaline (30-1OOUM). Rabbit cerebral vessels were pre-contracted with 5-hydroxytryptamine (0.3-1 pM) while the guinea-pig vessel was pre-contracted with histamine (1-30,M). Results are the mean (s.e.mean) of 4 to 7 separate determinations. pEC50 is the -log (M) of the concentration of drug required to cause 50% relaxation. %relax. is the percentage reduction in total active tone.
1cJu-
_k
,In_
h
_
"Sqf,.Ip;xllp iD'arlm"1*90,1C.(JJ o-tft-hl ine rul ur(iUor icereueiiur urtery Lgyceryt to nespow wnses nitric and acetylcholine oxide trinitrate, 2-,evf
_b
_
#^
\I ~ O~. x
E
\ o
,o-
._5
1
0
\1 U
CD
0 9
Conc. (-log M) Figure 6 Mean concentration-effect curves obtained for nicorandil
(@) and pinacidil (0) in WKY anterior cerebellar artery. Tissues were
pre-cont racted with 5-hydroxytryptamine (0.3-1 uM). Responses are the meatn from six separate experiments expressed as a percentage of total conistrictor tone.
Glyceryltrinitrate (GTN, 0.01-3 pM), nitric oxide (NO, 0.1100puM) and acetylcholine (0.01-3 pM) were tested for their ability to relax rat anterior cerebellar and mesenteric arteries precontracted with a submaximal concentration of 5-HT (0.31 pM). In the mesenteric artery all three compounds caused marked relaxant responses with approximate pEC50 values of 7.5, 6.8 and 5 for acetylcholine, GTN and NO respectively (Figure 8). Acetylcholine caused near maximal relaxation while GTN caused approximately 70% at the highest concentration used (3pM). Full concentration-effect curves to NO could not be constructed. However, at the highest concentration used (100pM) NO cause approximately 50% relaxation (Figure 8). re rhe . . . cerebellar artery were dissimilar to those in the mesenteric artery (Figure 8). In the anterior cerebellar artery acetyl-
choline was a relatively poor vasorelaxant causing only 20%
a
20-
7
6
4.5
a
SNP 4
1001
30 -
A~~,
40 E
wi
50-
Ckm
50
1\' A
60 x
A
E
70-
UJ
o
o i 0 1C
)o -
b
5-
E E z
*SNP 4
'\
0
A
I-
3-
Ckm
E c
\ .
.)
4-
5
2-
0 0)
10 300
600
9
Time (s)
Figure 7 Typical trace from experiments characterizing the electrophysiological effects of cromakalim on the rat anterior cerebellar artery. (a) Shows resting membrane potential (E.) and the bottom panel the tension trace. Tone in the vessel was present by virtue of spontaneous myogenic tone displayed by this particular vessel. Responses to cromakalim (Ckm) and sodium nitroprusside (SNP) are the -log (M). 1f
8
7
6
5
4
Conc. (-log M)
Figure 8 Mean concentration-effect curves obtained for acetylcholine (@), glyceryl trinitrate (0) and nitric oxide (NO, A) in WKY anterior cerebellar artery (a) and mesenteric artery (b) preconstricted with 5-hydroxytryptamine (0.1-3OyM). Responses are the mean of 4-8 separate experiments expressed as a percentage of the total constrictor tone.
56
G.A. McPHERSON & A.P. STORK
relaxation with a pEC50 of approximately 6. GTN displayed a similar potency as it did in the mesenteric artery (pEC50 = 7.25). However, it caused only 25% reversal of maximum active tone. Conversely, NO was a relatively powerful relaxant causing near maximal relaxation of tissue with a pEC50 of 5.25 (Figure 8).
a
300 -
4( 200._
Discussion The main finding from this work is that the effects of cromakalim on a number of rat cerebral vessels are poor compared with its actions on rat peripheral vessels (mesenteric and renal) and both cerebral and mesenteric vessels from the rabbit and guinea-pig. The resistance of the rat cerebral arteries to cro-
Ckm
cz E
7
The effect of cromakalim on spontaneously contracted rat anterior cerebellar arteries and arteries pre-contracted with U46619 We also determined whether the inability of rat cerebral vessels to relax to cromakalim depended on the nature of the vasoconstrictor used. In tissues pre-contracted with a submaximal concentration (O.1yM) of the thromboxane-mimetic U46619, cromakalim, pinacidil and SNP displayed similar vasorelaxant actions to those seen when 5-HT was used to induce tone (Figure 9). The ability of cromakalim to relax rat anterior cerebellar vessels which displayed spontaneous myogenic tone was also studied. In these studies vascular reactivity was assessed under isobaric conditions where calculated transmural pressure was held constant (100mmHg) by altering vessel diameter. Vascular reactivity was also assessed isometrically. Figure 10a and b shows the result of an experiment with a single rat anterior cerebral vessel, where cromakalim was unable to affect tone at concentrations up to 30pM under isobaric and isometric conditions. However, SNP was an effective vasorelaxant in both circumstances.
WO
56
6
SNP 10
20
b
2T
E E
Ckm
z
E
SNP
4
6
a 0
._4
Wa
n.
5
10
Time (min) Figure 10 Original trace showing the effect of cromakalim (Ckm) and sodium nitroprusside (SNP) to relax rat anterior cerebellar artery which has spontaneous myogenic activity. (a) Vascular reactivity recorded isobarically (pressure = 100mmHg) where vessel diameter is monitored. (b) Isometric recording in the same blood vessel. Concentrations are the -log M. WO washout of drug. =
makalim was not uniform. The basilar artery from both strains of rat studied (SHR and WKY) was responsive to cromakalim, although the concentrations required to cause vasorelaxation were somewhat higher (pECGO = 5.5) than those required to relax a range of vascular (this study) and nonvascular smooth muscle preparations (pEC50 = 6-6.5; McPherson & Angus, 1990). However, in the rat anterior cerebellar and middle cerebral arteries, cromakalim was a particularly weak vasorelaxant with less than 30% of 5-HT induced tone reversed by this compound at the highest concentration used. In addition, we recently tested the active isomer of cromakalim (BRL 38227) and found that it also failed to relax rat anterior cerebellar artery (data not shown), indicating that the results with cromakalim did not result from the use of the racemate.
There has been some conflicting data recently published whether K+ channel openers are, in fact, able to relax rat cerebral vessels. The studies of Wahl and co-workers (Parsons et al., 1991a; Ksoll et al., 1991), described near maximal relaxation responses to cromakalim in 5-HT pre-contracted rat basilar arteries. In their studies the pEC50 for cromakalim was approximately 6.35, while in our study the reversal of tone was only partial (40-60%) and the potency was less (pEC50 = 5.5). More recently this group (Parsons et al., 1991b) has also shown that pinacidil could relax rat pial arterioles in situ. In a separate study, the ability of cromakalim to relax isolated perfused segments of the rat superior cerebellar artery has also been described (Nagao et al., 1991). Collectively these data appear at odds with the results described in our study. However, in agreement with our results, Halpern and coworkers (McCarron et al., 1991) have recently shown in a preliminary study that perfused isolated cerebral arteries of the rat are resistant to the effects of pinacidil and cromakalim. The reasons for these differences are unclear at this time. The fact that the rat basilar (present study; Ksoll et al., 1991) and rat arterioles (Parsons et al., 1991b) respond to K' channel openers, may indicate that the resistance is only displayed by vessels of a diameter (250pum) predominantly used in our study. We also tested two other K+ channel opening compounds, nicorandil and pinacidil, and found that they were also poor vasorelaxants in the rat cerebral vasculature. There were two over
x E
wj
',
c
0
co
.0)
100
50 0
-
9
8
7
6
5
4
Conc. (-log M)
Figure 9 Mean concentration-effect curves constructed to sodium nitroprusside (SNP) (a), cromakalim (b) and pinacidil (c) in the rat anterior cerebellar artery in vessels precontracted with either 5hydroxytryptamine (0.3-1pma, 0) or U46619 (0.1 uM, 0). Responses are the mean from 4 to 10 separate experiments and are expressed as a
percentage of the total constrictor tone.
CROMAKALIM VASORELAXATION
unusual observations made during these studies. First, nicorandil was particularly ineffective despite the fact that previous studies by us and others (see McPherson & Angus, 1989) have shown that nicorandil can produce vasorelaxant responses which do not involve the opening of K+ channels. Consequently our expectation was that this compound would cause a vasorelaxant response. Conversely pinacidil did cause significant vasorelaxant responses which were insensitive to glibenclamide, a compound known to antagonize the K+ opening induced by pinacidil (see McPherson & Angus, 1990; 1991). It would appear therefore that pinacidil, along with cromakalim and nicorandil, may be able to relax vascular smooth muscle using a number of mechanisms; although their predominant effect appears to result from the opening of K+ channels. We considered a number of possibilities as to why rat cerebral vessels should display such resistance in our studies. First, the inability of cromakalim to relax rat cerebral vessels may result from the type of vasoconstrictor used to induce tone. This appeared not to be the case since similar results were obtained when either 5-HT, U46619 or low concentrations of K+ (20mM) were used. In addition, the rat cerebral vessels also displayed spontaneous myogenic tone which was little affected by cromakalim but was sensitive to SNP. The fact that rat cerebral vessels display spontaneous myogenic tone may result from the absence of K+ channels in these vessels. We have previously provided evidence that K+C channels may tonically influence the resting membrane potential (by hyperpolarizing it) in selected vascular beds such as the rat small mesenteric artery (McPherson & Angus, 1991). In the case of the rat mesenteric artery it would appear that spontaneously opened K+ channels hyperpolarize the vessel from approximately -50 to -60mV (McPherson & Angus, 1991). Electrophysiological experiments revealed that the rat anterior cerebellar artery has a low resting membrane potential compared with other tissues (-38mV anterior cerebellar cf -60mV mesenteric). It is possible that, in the absence of spontaneously opened K+ channels in this tissue, voltage operated Ca2+ channels open to cause the spontaneous tone. The spontaneous myogenic activity displayed by this vessel was abolished by the Ca2 + channel antagonist felodipine (30nM), which is consistent with this hypothesis (data not shown). The finding that cromakalim did not hyperpolarize the rat anterior cerebellar artery would indicate that the K+ is absent rather than being fully closed in this vessel. SNP did cause a hyperpolarization in association with relaxation in the rat anterior cerebellar artery. We have observed that high concentrations of SNP can cause hyperpolarization in vessels obtained from a number of vascular beds. However, the mechanism behind the response is currently unknown. It is possible that the rat cerebral vessels were less responsive to the vasorelaxants studied by an, as yet, unidentified mechanism. The ability of GTN and acetylcholine to relax rat cerebral vessels was less than that observed using rat mesenteric arteries. In addition, SNP was generally more potent in rat mesenteric and rabbit and guinea-pig small arteries than in rat anterior cerebral arteries; a finding which would support this idea. However, there were exceptions, in particu-
57
lar the rabbit anterior cerebral artery where cromakalim was a more potent relaxant than SNP. Also nitric oxide (NO) was a more powerful vasorelaxant in the rat anterior cerebral artery than in the rat mesenteric artery. Consequently, there does not appear to be a clear correlation between the potency of the vasorelaxants and the ability of cromakalim to elicit a response. The possibility also exists that rat cerebral vessels were in someway damaged upon removal and mounting of the vessel in the small vessel myograph, which had a deleterious effect on responses to the vasorelaxants. We used a number of approaches to kill the animal (CO2 asphyxia or a blow to the head) and a number of vasoconstrictors to induce tone and showed that these had no effect on the poor response to cromakalim. Despite this, the relatively low and unstable resting membrane potential displayed by the anterior cerebellar artery (-40mV) is unusual and may be indicative of some form of damage. However, work by Hirst and his colleagues (Edwards et al., 1988) have also shown segments of the rat cerebral vasculature to have a similar unstable resting membrane potential associated with spontaneous myogenic activity. This does not preclude the possibility that the rat cerebral vessels were deleteriously affected in both studies. However, given that the vessels are functionally normal in terms of their response to a number of vasoconstrictors we suspect that this is not the case. This idea is also supported by a number of in vivo studies. That cromakalim may display some regional selectivity in its action is supported by a number of in vivo studies. For example, in the anaesthetized rabbit cromakalim preferentially dilates the coronary, gastrointestinal and cerebral blood vessels but to a lesser extent the renal and skeletal muscle vasculature (Hof et al., 1988). In anaesthetized cats cromakalim increases flow in carotid, mesenteric and renal beds but not to the femoral circulation (Buckingham et al., 1986). In a recent study in conscious spontaneously hypertensive rats (Shoji et al., 1990) cromakalim decreased vascular resistance in a number of beds including the heart, stomach, kidney, skeletal muscle and skin. A significant finding from that study was that cromakalim was without effect on cerebral vascular resistance. The results from this study support the idea that the rat cerebral circulation is resistant to the vasorelaxant actions of cromakalim and other potassium channel openers. In summary, the results from this study show that a number of small arteries from the rat cerebral circulation are resistant to the vasorelaxant effects of cromakalim and other K+C channel openers. The property is specific for these vessels since such resistance is not displayed in other rat vessels or in the same vessels obtained from other species. While there may be a number of reasons for this phenomenon, it is possible that these vessels may be devoid of K+ channels under the conditions in which the experiments were performed. This project was supported by an Institute Grant from the NH and MRC of Australia and Glaxo Australia. It is a pleasure to acknowledge the assistance of Simon Keily. Cromakalim, BRL 38227 (Beecham), pinacidil (Leo) and glibenclamide (Hoechst) were generous
gifts.
References ANGERSBACH, D. & NICHOLSON, C.D. (1988). Enhancement of muscle blood cell flux and P02 by cromakalim (BRL 34915) and other compounds enhancing membrane K+ conductance, but not by Ca2 + antagonists or hydralazine, in an animal model of occlusive arterial disease. Naunyn-Schmiedbergs Arch. Pharmacol., 337, 341-346. ANGUS, J.A., BROUGHTON, A. & MULVANY, M.J. (1988). Role of aadrenoceptors in constrictor responses of rat, guinea-pig and rabbit small arteries to neural activation. J. Physiol., 403, 495-510. BUCKINGHAM, R.E., CLAPHAM, J.C., HAMILTON, T.C., LONGMAN, S.D., NORTON, J. & POYSER, R.H. (1986). BRL 34915, a novel anti-
hypertensive agent: comparison of effects on blood pressure and other haemodynamic parameters with those of nifedipine in animal models. J. Cardiovasc. Pharmacol., 8, 798-804. BUCKINGHAM, R.E. (1988). Studies on the anti-vasoconstrictor activity of BRL 34915 in spontaneously hypertensive rats; a comparison with nifedipine. Br. J. Pharmacol., 93, 541-552. COCKS, T.M. & ANGUS, J.A. (1990). Comparison of relaxation responses of vascular and non-vascular smooth muscle to endothelium-derived relaxing factor (EDRF), acidified nitrite (NO) and sodium nitroprusside. Naunyn-Schmiedebergs Arch. Pharmacol., 341, 364-372.
58
G.A. McPHERSON & A.P. STORK
EDWARDS, F.R., HIRST, G.D.S. & SILVERBERG, G.D. (1988). Inward rectification in rat cerebral arterioles; involvement of potassium ions in autoregulation. J. Physiol., 404, 455-466. EDWARDS, G. & WESTON, A.H. (1990). Structure-activity relationships of K+ channel openers. Trends Pharmacol. Sci., 11, 417-422. HAMILTON, T.C., WEIR, S.W. & WESTON, A.H. (1986). Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein. Br. J. Pharmacol., 88, 103-111. HOF, R.P., QUAST, U., COOK, N.S. & BLARER, S. (1988). Mechanism of action and systemic and regional hemodynamics of the K+ channel activator BRL 34915 and its enantomers. Circ. Res., 62, 679-686. KSOLL, E., PARSONS, A.A., MACKERT, J.R.L., SCHILLING, L. & WAHL,
M. (1991). Analysis of cromakalim-, pinacidil-, and nicorandil induced relaxation of the 5-hydroxytryptamine precontracted rat isolated basilar artery. Naunyn-Schmiedebergs Arch. Pharmacol., 343, 377-383. MARUYAMA, M., FABER, N. & GROSS, G. (1989). Effect of the new potassium channel activator, END 52692, on coronary collateral blood flow in anaesthetized dogs FASEB J., 3, A897 (Abstract 3894). McCARRON, J.G., QUAYLE, J.M., HALPERN, W. & NELSON, M.T. (1991). Cromakalim and pinacidil dilate resistance-sized mesenteric arteries but not resistance sized cerebral arteries. Blood Vessel, 28, 315. McPHERSON, G.A. (1985). Analysis of radioligand binding experiments: A collection of computer programs for the IBM PC. J. Pharmacol. Methods, 14, 213-228. McPHERSON, G.A. & ANGUS, J.A. (1989). Phentolamine and structurally related compounds selectively antagonise the vascular actions
of the K+ channel open, cromakalim. Br. J. Pharmacol., 97, 941949. McPHERSON, G.A. & ANGUS, J.A. (1990). Characterization of responses to cromakalim and pinacidil in smooth and cardiac muscle by use of selective antagonists. Br. J. Pharmacol., 100, 201206. McPHERSON, G.A. & ANGUS, J.A. (1991). Evidence that acetylcholine mediated hyperpolarization of rat small mesenteric artery does not involve the K+ channel opened by cromakalim. Br. J. Pharmacol., 103,1184-1190. MULVANY, M.J. & HALPERN, W. (1977). Contractile responses of small resistance vessels in spontaneously hypertensive and normotensive rats. Circ. Res., 41, 19-26. NAGAO, T., SADOSHIMA, S., KAMOUCHI, M. & FUJISHIMA, M. (1991). Cromakalim dilates rat cerebral arteries in vitro. Stroke, 22, 221224. PARSONS, A.A., KSOLL, E., MACKERT, J.R.L., SCHILLING, L. & WAHL,
M. (1991a). Comparison of cromakalim-induced relaxation of potassium precontracted rabbit, cat and rat isolated cerebral arteries. Naunyn-Schmiedebergs Arch. Pharmacol., 343, 384-392. PARSONS, A.A., SCHILLING, L. & WAHL, M. (1991b). Effect of microapplication of pinacidil in rat pial arterioles in situ. Br. J. Pharmacol., 102, 157P. SHOJI, T., AKI, Y., FUKUI, K., TAMAKI, T., IWAO, H. & ABE, Y. (1990). Effects of cromakalim, potassium channel opener, on regional blood flow in conscious spontaneously hypertensive rats. Eur. J. Pharmacol., 186, 119-123. STORK, A.P., McPHERSON, G.A. & ANGUS, J.A. (1990). Regional variation in the vasorelaxant effect of cromakalim in small resistance arteries from the rat. Clin. Exp. Pharmacol. Physiol., Suppl. 17, 74.
(Received June 10, 1991 Revised September 3, 1991
Accepted September 4, 1991)
,'-.
Macmillan Press Ltd, 1992
The resistance of some rat cerebral arteries to the vasorelaxant effect of cromakalim and other K + channel openers 'Grant A. McPherson & Andrew P. Stork Baker Medical Research Institute, Commercial Rd, Prahran, 3181, Victoria, Australia 1 Cromakalim (0.01-30gM) and sodium nitroprusside (SNP, 0.01-100guM) were tested for their ability to relax a number of pre-contracted small arteries (approximate diameter 200-700gM at 100mmHg) from the rat, rabbit and guinea-pig. 2 In the rat, SNP (0.01-100gM) caused near maximal relaxation in all vessels studied including the middle cerebral, anterior cerebellar, basilar, mesenteric and renal arteries. Cromakalim (0.01-30guM) relaxed pre-contracted mesenteric and renal arteries but was only a weak relaxant of all the rat cerebral arteries with the exception of the basilar artery. Similar experiments using mesenteric and cerebral vessels from the rabbit and guinea-pig showed cromakalim could relax pre-contracted vessels in a concentrationdependent manner. 3 Two other K+ channel openers, nicorandil and pinacidil, were also tested for their ability to relax rat cerebral arteries. Nicorandil (0.01-100guM) was ineffective in the rat anterior cerebellar artery at concentrations up to 100,UM. Pinacidil (0.01-100guM) caused significant vasorelaxation, although high concentrations were required (> 10,UM) and the response was insensitive to the effects of glibenclamide (3pM). 4 Electrophysiological experiments with the rat anterior cerebellar artery showed that cromakalim (up to 30gM) failed to influence the resting membrane potential of impaled single smooth muscle cells. 5 The results showed that some rat small cerebral arteries were resistant to the effects of K+ channel openers including cromakalim, pinacidil and nicorandil. This is peculiar to this vascular tree since the same vessels from other species do not exhibit the same behaviour. Such a phenomenon could result from a lack of K+ channels opened by cromakalim in rat cerebral arteries. Keywords: K+ channels; cromakalim; pinacidil; nicorandil; cerebral arteries; vascular specialization
Introduction Cromakalim is representative of a new class of smooth muscle relaxants (Hamilton et at., 1986; Edwards & Weston, 1990) which act by opening a type of K+ channel (termed K+ in this paper) in the cell membrane resulting in membrane hyperpolarization. There is a great deal of interest in the vascular actions of cromakalim, not only does it represent a new class of therapeutic agent, but also its haemodynamic profile appears to differ from other anti-hypertensives, in particular Ca2+ antagonists such as nifedipine (Buckingham, 1988; Shoji et al., 1990). That cromakalim and other K+ channel openers may exhibit some organ specificity is an intriguing property. A number of in vivo studies have shown cromakalim to dilate selectively some vascular beds compared with others (Buckingham et al., 1986; Hof et al., 1988). In addition cromakalim and other K+ channel openers appear to be more efficacious, in vivo at least, in ischaemic tissues (Angersbach & Nicholson, 1988; Maruyama et al., 1989). The mechanisms behind such selectivity are currently unknown. In some preliminary studies we observed that cromakalim was an unusually poor vasorelaxant in some isolated small cerebral arteries of the rat. The purpose of the present work was to extend these observations to determine the extent of such resistance in a variety of vessels of the same size. We found that such resistance was confined to the rat cerebral circulation and possibly results from a lack of K+ channels in this vascular bed. A preliminary account of this work was presented at the 24th annual meeting of the Australian Society of Clinical and Experimental Pharmacologists (Stork et al., 1990).
Methods Isolation of small resistance arteries Wistar Kyoto (WKY) or spontaneously hypertensive (SHR) rats were killed by CO2 asphyxia. In some experiments WKY 'Author for correspondence.
rats were also killed by a blow to the head. Guinea-pigs were killed by a blow to the head and rabbits were killed by an overdose of pentobarbitone (60 mg kg- l i.v.). The organ under study was rapidly removed and placed in ice cold Krebs solution (composition in mM: NaCl 119, KCl 4.7, MgSO4 * 7H20 1.17, NaHCO3 25, KH2PO4 1.18, CaCl2 2.5 and glucose 11) gassed with 5% CO2 in 2. Two mm segments were mounted in a small vessel myograph to record isometric tension development as previously described (Angus et al., 1988). Briefly two 40,um wires were threaded through the lumen of the vessel segment. One wire was attached to a stationary support driven by a micrometer while the other was attached to an isometric force transducer which measured force development. Force development was recorded on a dual flat bed recorder (W&W Scientific Instruments, model 320). Vessels were allowed to equilibrate under zero tension for 30 min at 36°C. A passive length-tension curve was constructed as previously described (Mulvany & Halpern, 1977). Since some vessels (the rat cerebral vessels) displayed spontaneous myogenic tone, sodium nitroprusside (SNP, 0.1 mM) was included in the bath to cause full relaxation while constructing the curve. The vessel was set at a diameter 0.9 times the diameter of the vessel at 100mmHg. This point on the passive length-tension curve corresponds to the point of maximum active tension development (Mulvany & Halpern, 1977). Non-linear curve fitting of the passive length-tension curve was achieved using a custom written program for the IBM PC (NORMALIZE, GA McPherson) which uses the Marquart-Levenberg modification of the Gauss-Newton technique (McPherson, 1985). In the text, vessel diameter is expressed as the diameter at an equivalent transmural pressure of 100mmHg (D100). Upon completion of the passive length-tension curve, vessels were washed and allowed to equilibrate for 30min before the addition of any drugs. The viability of the tissue was then assessed by adding a K+ depolarizing solution (in which all NaCl in the Krebs buffer was replaced by an equimolar concentration of KCI) to activate the tissue maximally. Unless otherwise stated the vasorelaxant activity of cromakalim was assessed in tissues precontracted with a sub-
52
G.A. McPHERSON & A.P. STORK
maximal concentration of 5-hydroxytryptamine (5-HT; approximately 0.3-1AuM). The rat cerebral vessels were unusual in that they displayed spontaneous myogenic tone. However, in the majority of experiments with these vessels, active tone was induced with a sub-maximal concentration of 5-HT so that a constant level of tone could be generated throughout the duration of the experiment. Vasorelaxant responses of the drugs tested were expressed as a percentage of the maximum level of tone. This was calculated as the difference between tone in the presence of the constrictor and that in the presence of SNP (0.1 mM), which was added at the end of every concentration-effect curve constructed to a vasorelaxant. Cumulative concentration-effect curves were constructed to cromakalim (0.01-30giM) and SNP (0.01-100gM) which was used as the control vasorelaxant. In additional experiments, we also determined the ability of pinacidil and nicorandil (K+ channel openers, 0.01-100gM) and three other vasorelaxants (acetylcholine 0.01-3pM, glyceryl-trinitrate 0.01-3 fM and nitric oxide 0.1-100gUM) for their ability to cause vasorelaxant responses in selected tissues. Nitric oxide (NO) solutions were prepared from acidified sodium nitrite as previously described (Cocks & Angus, 1990). The final concentration of NO generated was assumed to be equivalent to the final concentration of acidified sodium nitrite used. We were also interested in the effect of cromakalim on spontaneous myogenic tone which was displayed by the rat small cerebral arteries. It has been our experience that myogenic behaviour of small resistance arteries is better observed when vascular reactivity is assessed under isobaric/isotonic conditions. Consequently in some experiments the ability of cromakalim to alter vascular tone of the small resistance arteries was assessed isobarically using a newly developed computer based system (ISOTONIC, McPherson, unpublished data). In these experiments the manual micrometer on the small vessel myograph was replaced with a computer controlled motor (Burleigh Inchworm motor controlled by a 6000 series controller, Burleigh, U.S.A.). Wall tension in the small resistance artery was continually monitored using an analogue to digital card (DASH16, Metrabyte, U.S.A.) and transmural pressure calculated. In isobaric mode the computer program could adjust the separation distance of the wires, by the use of the inchworm motor, such that pressure was held constant at a pre-set level. Vascular reactivity was therefore assessed by monitoring changes in vessel diameter.
(Hoechst); sodium nitroprusside (Nipride, Roche); glyceryltrinitrate (David Bull Laboratories, Australia); acetylcholine bromide (Sigma); U46619 (1,5,5-hydroxy-1 la,9a (epoxymethano) prosta-E2,13-dienoic acid, Upjohn); endothelin-1 (Austpep, Australia). Cromakalim (10mM), pinacidil (10mM), nicorandil (10mM) and glibenclamide (1 mM) stock solutions were made in 100% methanol. All other drugs were made up on the day of experiment. Dilutions of all drugs were made in distilled water.
Results
Characteristics of the small arteries studied Figure 1 shows a schematic diagram of the rat cerebral circulation showing the origin of the cerebral vessels studied. These were the basilar, anterior cerebellar and the middle cerebral arteries. In selected studies anatomically similar vessels were also taken from the rabbit and guinea-pig. Several peripheral small arteries were also studied including a mesenteric and renal artery. The diameters of all vessels studied, at an estimated transmural pressure of 100 mmHg, are listed in Table 1. The vessels ranged in size from 200-700gum depending on the species and origin of the vessel. The viability of each vessel was assessed at the commencement of each experiment by fully activating the preparation using a K+ depolarizing solution. All vessels developed significant amounts of tension, which were similar when the values were standardized for differences in vessel diameter (Table 1).
Comparison of the vasorelaxant effect of cromakalim in cerebral and peripheral vesselsfrom rat, rabbit and
guinea-pig SNP was an effective vasorelaxant in all SHR and WKY rat cerebral and peripheral vessels studied although its potency did vary to some extent (Figures 2, 3 and 5; Table 2). In the mesenteric artery SNP had a pEC50 of approximately 6.6 while in the anterior cerebellar it was approximately 20 fold less potent (5.3). In all cases SNP could reverse nearly all the vasoconstrictor-induced tone (greater than 95% relaxation), except in the WKY small renal vessel where 80% of active tone was reversed at the highest concentration of SNP used.
Electrophysiological experiments In some experiments the intracellular resting membrane potential was monitored in rat anterior cerebellar vessels mounted in the small vessel myograph. A conventional glass electrode (see McPherson & Angus, 1991 for details) was used to impale a single smooth muscle cell. Cumulative concentrations of cromakalim were added to the bath and membrane potential and tension changes were monitored simultaneously. Since the rat anterior cerebellar artery displayed intrinsic myogenic tone, vasorelaxant responses could be assessed without the addition of a vasoconstrictor agent.
Statistics and data analysis Statistical comparisons between two groups were made by use of Student's t test. Results in the text are the mean + s.e.mean for the specified number of experiments. The potency of the vasorelaxants are expressed as the -log concentration of the drug required to produce 50% relaxation (pEC50). Relaxation responses were calculated as a percentage of active tone with 100% implying full relaxation.
Drugs The following drugs were used: cromakalim (Beecham); pinacidil monohydrate (Leo); nicorandil (Chugai); glibenclamide
Middle cerebral
4-Anterior cerebellar
Figure 1 Dorsal view (schematic diagram) of the rat brain showing the basilar, anterior cerebellar and middle cerebral arteries which were used. Anatomically similar vessels were also obtained from the rabbit and guinea-pig.
CROMAKALIM VASORELAXATION
53
Table 1 (A) Arterial diameters (un) at 100mmHg, of the vessels studied estimated during the normalization procedure and (B) mean active tension (mN mm'- ) developed to a K + depolarizing solution which was used to assess tissue viability
Animal A WKY SHR Rabbit
Guinea-pig B WKY SHR Rabbit Guinea-pig
Mesenteric
303 (14) 298 (11) 383 (45) 398 (16) 4.7 (0.6) 5.3 (0.4) 5.5 (0.9) 6.3 (0.9)
Renal 486 (46)
5.3 (0.4)
Basilar
Anterior cerebellar
Middle cerebral
Diameter (pm) 399 (16) 251 (8) 351 (19) 226 (12) 788 (54) 443 (10) 362 (8)
266 (8) 233 (5) 397 (19)
Tension (mN mm-') 5.9 (0.7) 2.9 (0.5) 7.4 (0.6) 2.9 (0.6) 7.7 (1.0) 4.6 (0.6) 4.4 (0.05)
2.7 (0.5) 2.7 (0.3) 3.8 (0.7)
Values are the mean (s.e.mean) of 4-10 separate determinations.
In contrast, a number of peripheral rat vessels were found to be sensitive to cromakalim. Thus in the WKY mesenteric and renal artery and SHR mesenteric artery, cromakalim was an effective vasorelaxant with a pEC50 of approximately 6.4 and with an ability to cause greater than 95% relaxation (Figure 5, Table 2). For comparative purposes the ability of cromakalim to relax pre-contracted cerebral and mesenteric vessels from the rabbit and guinea-pig was also studied. In contrast to the results obtained in the rat, cromakalim was an effective vasorelaxant in rabbit and guinea-pig cerebral arteries (Figures 2, 3 and 5; Table 3). In all cases the pEC50 value for cromakalim was approximately 6.4. In addition cromakalim caused greater than 90% relaxation. As in the rat cerebral vessels, SNP was approximately 10 times less potent on the anterior cerebellar artery than on the mesenteric artery in the rabbit (Figure 3, Table 2). However, in the guinea-pig both cromakalim and SNP appeared to be relatively potent vasorelaxants (Figure 3, Table 2).
Cromakalim was particularly ineffective as a vasorelaxant on rat small cerebral arteries with the exception of the basilar artery. In the WKY and SHR basilar artery, cromakalim was able to produce significant vasorelaxant response in tissues pre-constricted with 5-HT (Figure 2, Table 2). In the WKY vessels approximately 60% and in the SHR 35% of the active tone was reversed. These were not significantly different (P > 0.05, unpaired t test). In addition cromakalim was approximately 10 fold more potent in rat mesenteric and renal small arteries (pEC50 basilar 5.58 cf 6.42 mesenteric). In the anterior cerebellar and middle cerebral artery from both strains cromakalim was a particularly poor relaxant, with only the highest concentration of cromakalim used (30pM) causing any marked relaxation (Figure 3). Approximately 20% of active tone could be reversed by cromakalim in these tissues. In the majority of studies using the rat, vessels were obtained from animals killed by CO2 asphyxia. Cerebral vessels are known to be quite sensitive to CO2. Consequently we repeated some experiments with rats killed by a blow to the head to determine whether the method of killing the animals affected the response to cromakalim. Figure 4 shows the vasorelaxant responses to cromakalim in anterior cerebral or basilar arteries from these rats in which the tissues were precontracted with either 5-HT or a low concentration of K+ (20mM). Again, particularly in the anterior cerebellar artery, cromakalim was a weak relaxant causing only 20-40% maximum relaxation at the highest concentration of cromakalim used (30 mM).
Responses of the rat anterior cerebellar artery to pinacidil and nicorandil Nicorandil (0.01-100gM), like cromakalim, was ineffective as a vasorelaxant in the WKY anterior cerebellar artery (Figure 6). This compound caused only 11% relaxation at a concentration of 100puM. However, pinacidil (0.01-100gM) was able to cause significant relaxation (approximately 75% relaxation of
Table 2 pEC50 and maximum response values for cromakalim and sodium nitroprusside (SNP) obtained in rat cerebral and peripheral small resistance arteries pre-contracted with 5-hydroxytryptamine (0.3-1 pM) Cromakalim %relax. pEC50 WKY Basilar Ant. cereb. Middle cer. Mesenteric Renal SHR Basilar Ant. cereb. Middle cer. Mesenteric Renal
5.58 (0.07)
-
61 (10) 17 (4) 23 (3)
6.42 (0.08) 6.18 (0.07)
96(1) 96 (2)
5.48 (0.1)
35 (9)
6.28 (0.1) NS
8
(4)
19 (4) 95 (2)
SNP
pEC50
%relax.
5.86 (0.06) 5.29 (0.07) 5.52 (0.07) 6.56 (0.2) 6.21 (0.3)
97 (2)
5.74 5.52 5.58 6.98
(0.1) (0.2) (0.1) (0.1)
Results are the mean (s.e.mean) of 4 to 12 separate determinations. pEC50 is the -log (M) of the concentration of drug required to cause 50% relaxation. % relax. is the percentage reduction in total active tone. * Curve could not be processed by the computer due to an absence of points, therefore, the highest concentration of cromakalim used (304uM). NS, not studied.
95 (4) 98 (1) 94 (2) 80 (5) 91 (3)
95 (3) 94 (4) 97 (1)
%relax. is the maximum relaxation observed at
54
G.A. McPHERSON & A.P. STORK a lO0i
100-
A\ \Jii I 0~\ x
0\
A
50-
I\
E
R.\
50-
0\
0
x
VA
E
LU
f%
~A
CO -
NO 0--
0)
0-o w 2lo
. C )ot ic a)
Al z::n70-
=
b *~~~~~~~~~~ 11 T
3
I
I--A
0-
;0
II\T
5 -
iOh
o
-
9
8
7
6
O
6 5 Conc. (-log M)
4
Figure 4 Mean concentration-effect curves for cromakalim in the anterior cerebellar (A, A) or the basilar artery (O. 0) from WKY rats killed by a blow to the head. Tissues were precontracted with 5hydroxytryptamine (solid symbols, 0.3-1 pM) or K+ (open symbols, 20 mM). Results are expressed as a percentage of total constrictor tone (%Em.x) defined by sodium nitroprusside (0.1 mM) and are the mean from 4 to 6 separate experiments.
T I
5
7
4
Conc. (-log M)
Figure 2 Mean concentration-effect curves for sodium nitroprusside (SNP) (a) and cromakalim (b) in the basilar artery from WKY (0), SHR (0) and rabbit (A). Tissues were precontracted with 5hydroxytryptamine (0.3-1 gM). Results are expressed as a percentage of the total amount of constrictor tone (%Emax) defined by a maximal concentration of SNP (0.1 mM) added at the end of each experiment. Values are the mean from 4 to 12 separate experiments.
active tone) with a pEC50 of approximately 4.5 (Figure 6). Additional experiments showed that the response to pinacidil (30M) in the presence of glibenclamide (3pM, 50 + 6% relaxation) was not significantly different from that in its absence (53 + 5% relaxation, n = 3). 100
E 0~
FH5 0)1 -
a
50-
7 6 Conc. (-log M)
x
wE
Figure 5 Mean concentration-effect curves for sodium nitroprusside (SNP) (a) and cromakalim (b) in the mesenteric artery from WKY (0), SHR (0), rabbit (A) and guinea-pig (A). Tissues were precontracted with 5-hydroxytryptamine (rat, 0.3-1 pM) or noradrenaline (rabbit and guinea-pig, 30-1OOpuM). Results are expressed as a percentage of the total constrictor tone (%Em.x) and are the mean from 4 to 12 separate experiments.
-0 0)
H
00b A
A
* 1 Too!~~~~~~~
4
*-. \
Electrophysiological characteristics of the rat anterior
50-
cerebellar artery A 0
A--AA OhAl , , A~A-4_
8
9
8
7
6
5
4
Conc. (-log M)
Figure 3 Mean concentration-effect curves for sodium nitroprusside (SNP) (a) and cromakalim (b) in the anterior cerebellar artery from WKY (0), SHR (0), rabbit (A) and guinea-pig (A). Tissues were precontracted with 5-hydroxytryptamine (rat and rabbit, 0.3-1 PM) or histamine (guinea-pig 1-30M). Results are expressed as a percentage of total constrictor tone (%Emax) and are the mean from 4 to 12 separate experiments.
The rat anterior cerebellar artery had a resting membrane potential (Em) of -38 + 2 mV (7 impalements from 3 arteries) in the absence of any vasoconstrictor agent. The membrane potential was unstable and oscillated with an amplitude of between 2 to 5 mV. Figure 7 shows the results from an experiment examining the effects of cromakalim. Cromakalim caused only a small hyperpolarization (-2 + 1 mV, n = 3) even at concentrations up to 30M. In addition there was no effect on the spontaneous tone displayed by these vessels. In contrast SNP (0.1 mM) caused a hyperpolarization (-14 + 5 mV, n = 3) and relaxation (Figure 7) in vessels displaying spontaneous myogenic tone.
CROMAKALIM VASORELAXATION
55
Table 3 pEC50 and maximum response values for cromakalim and sodium nitroprusside (SNP) obtained in rabbit and guinea-pig cerebral and peripheral small resistance arteries Cromakalim %relax.
Vessel
pEC50
SNP
pECGo
%relax.
100 (0) 95 (3) 98 (1) 81 (7) 95 (4) 96 (1)
Rabbit Basilar Ant. cereb. Middle cer. Mesenteric
6.39 (0.14) 6.33 (0.05) 6.28 (0.1) 6.75 (0.04)
97 (2) 99(1) 98 (1)
6.82 (0.3) 5.26 (0.2) 5.76 (0.2) 5.83 (0.2)
Guinea-pig Ant. cereb. Mesenteric
5.84 (0.06) 6.36 (0.3)
88 (6) 93 (2)
6.39 (0.1) 6.96 (0.2)
99(1)
Mesenteric arteries of both species were pre-contracted with noradrenaline (30-1OOUM). Rabbit cerebral vessels were pre-contracted with 5-hydroxytryptamine (0.3-1 pM) while the guinea-pig vessel was pre-contracted with histamine (1-30,M). Results are the mean (s.e.mean) of 4 to 7 separate determinations. pEC50 is the -log (M) of the concentration of drug required to cause 50% relaxation. %relax. is the percentage reduction in total active tone.
1cJu-
_k
,In_
h
_
"Sqf,.Ip;xllp iD'arlm"1*90,1C.(JJ o-tft-hl ine rul ur(iUor icereueiiur urtery Lgyceryt to nespow wnses nitric and acetylcholine oxide trinitrate, 2-,evf
_b
_
#^
\I ~ O~. x
E
\ o
,o-
._5
1
0
\1 U
CD
0 9
Conc. (-log M) Figure 6 Mean concentration-effect curves obtained for nicorandil
(@) and pinacidil (0) in WKY anterior cerebellar artery. Tissues were
pre-cont racted with 5-hydroxytryptamine (0.3-1 uM). Responses are the meatn from six separate experiments expressed as a percentage of total conistrictor tone.
Glyceryltrinitrate (GTN, 0.01-3 pM), nitric oxide (NO, 0.1100puM) and acetylcholine (0.01-3 pM) were tested for their ability to relax rat anterior cerebellar and mesenteric arteries precontracted with a submaximal concentration of 5-HT (0.31 pM). In the mesenteric artery all three compounds caused marked relaxant responses with approximate pEC50 values of 7.5, 6.8 and 5 for acetylcholine, GTN and NO respectively (Figure 8). Acetylcholine caused near maximal relaxation while GTN caused approximately 70% at the highest concentration used (3pM). Full concentration-effect curves to NO could not be constructed. However, at the highest concentration used (100pM) NO cause approximately 50% relaxation (Figure 8). re rhe . . . cerebellar artery were dissimilar to those in the mesenteric artery (Figure 8). In the anterior cerebellar artery acetyl-
choline was a relatively poor vasorelaxant causing only 20%
a
20-
7
6
4.5
a
SNP 4
1001
30 -
A~~,
40 E
wi
50-
Ckm
50
1\' A
60 x
A
E
70-
UJ
o
o i 0 1C
)o -
b
5-
E E z
*SNP 4
'\
0
A
I-
3-
Ckm
E c
\ .
.)
4-
5
2-
0 0)
10 300
600
9
Time (s)
Figure 7 Typical trace from experiments characterizing the electrophysiological effects of cromakalim on the rat anterior cerebellar artery. (a) Shows resting membrane potential (E.) and the bottom panel the tension trace. Tone in the vessel was present by virtue of spontaneous myogenic tone displayed by this particular vessel. Responses to cromakalim (Ckm) and sodium nitroprusside (SNP) are the -log (M). 1f
8
7
6
5
4
Conc. (-log M)
Figure 8 Mean concentration-effect curves obtained for acetylcholine (@), glyceryl trinitrate (0) and nitric oxide (NO, A) in WKY anterior cerebellar artery (a) and mesenteric artery (b) preconstricted with 5-hydroxytryptamine (0.1-3OyM). Responses are the mean of 4-8 separate experiments expressed as a percentage of the total constrictor tone.
56
G.A. McPHERSON & A.P. STORK
relaxation with a pEC50 of approximately 6. GTN displayed a similar potency as it did in the mesenteric artery (pEC50 = 7.25). However, it caused only 25% reversal of maximum active tone. Conversely, NO was a relatively powerful relaxant causing near maximal relaxation of tissue with a pEC50 of 5.25 (Figure 8).
a
300 -
4( 200._
Discussion The main finding from this work is that the effects of cromakalim on a number of rat cerebral vessels are poor compared with its actions on rat peripheral vessels (mesenteric and renal) and both cerebral and mesenteric vessels from the rabbit and guinea-pig. The resistance of the rat cerebral arteries to cro-
Ckm
cz E
7
The effect of cromakalim on spontaneously contracted rat anterior cerebellar arteries and arteries pre-contracted with U46619 We also determined whether the inability of rat cerebral vessels to relax to cromakalim depended on the nature of the vasoconstrictor used. In tissues pre-contracted with a submaximal concentration (O.1yM) of the thromboxane-mimetic U46619, cromakalim, pinacidil and SNP displayed similar vasorelaxant actions to those seen when 5-HT was used to induce tone (Figure 9). The ability of cromakalim to relax rat anterior cerebellar vessels which displayed spontaneous myogenic tone was also studied. In these studies vascular reactivity was assessed under isobaric conditions where calculated transmural pressure was held constant (100mmHg) by altering vessel diameter. Vascular reactivity was also assessed isometrically. Figure 10a and b shows the result of an experiment with a single rat anterior cerebral vessel, where cromakalim was unable to affect tone at concentrations up to 30pM under isobaric and isometric conditions. However, SNP was an effective vasorelaxant in both circumstances.
WO
56
6
SNP 10
20
b
2T
E E
Ckm
z
E
SNP
4
6
a 0
._4
Wa
n.
5
10
Time (min) Figure 10 Original trace showing the effect of cromakalim (Ckm) and sodium nitroprusside (SNP) to relax rat anterior cerebellar artery which has spontaneous myogenic activity. (a) Vascular reactivity recorded isobarically (pressure = 100mmHg) where vessel diameter is monitored. (b) Isometric recording in the same blood vessel. Concentrations are the -log M. WO washout of drug. =
makalim was not uniform. The basilar artery from both strains of rat studied (SHR and WKY) was responsive to cromakalim, although the concentrations required to cause vasorelaxation were somewhat higher (pECGO = 5.5) than those required to relax a range of vascular (this study) and nonvascular smooth muscle preparations (pEC50 = 6-6.5; McPherson & Angus, 1990). However, in the rat anterior cerebellar and middle cerebral arteries, cromakalim was a particularly weak vasorelaxant with less than 30% of 5-HT induced tone reversed by this compound at the highest concentration used. In addition, we recently tested the active isomer of cromakalim (BRL 38227) and found that it also failed to relax rat anterior cerebellar artery (data not shown), indicating that the results with cromakalim did not result from the use of the racemate.
There has been some conflicting data recently published whether K+ channel openers are, in fact, able to relax rat cerebral vessels. The studies of Wahl and co-workers (Parsons et al., 1991a; Ksoll et al., 1991), described near maximal relaxation responses to cromakalim in 5-HT pre-contracted rat basilar arteries. In their studies the pEC50 for cromakalim was approximately 6.35, while in our study the reversal of tone was only partial (40-60%) and the potency was less (pEC50 = 5.5). More recently this group (Parsons et al., 1991b) has also shown that pinacidil could relax rat pial arterioles in situ. In a separate study, the ability of cromakalim to relax isolated perfused segments of the rat superior cerebellar artery has also been described (Nagao et al., 1991). Collectively these data appear at odds with the results described in our study. However, in agreement with our results, Halpern and coworkers (McCarron et al., 1991) have recently shown in a preliminary study that perfused isolated cerebral arteries of the rat are resistant to the effects of pinacidil and cromakalim. The reasons for these differences are unclear at this time. The fact that the rat basilar (present study; Ksoll et al., 1991) and rat arterioles (Parsons et al., 1991b) respond to K' channel openers, may indicate that the resistance is only displayed by vessels of a diameter (250pum) predominantly used in our study. We also tested two other K+ channel opening compounds, nicorandil and pinacidil, and found that they were also poor vasorelaxants in the rat cerebral vasculature. There were two over
x E
wj
',
c
0
co
.0)
100
50 0
-
9
8
7
6
5
4
Conc. (-log M)
Figure 9 Mean concentration-effect curves constructed to sodium nitroprusside (SNP) (a), cromakalim (b) and pinacidil (c) in the rat anterior cerebellar artery in vessels precontracted with either 5hydroxytryptamine (0.3-1pma, 0) or U46619 (0.1 uM, 0). Responses are the mean from 4 to 10 separate experiments and are expressed as a
percentage of the total constrictor tone.
CROMAKALIM VASORELAXATION
unusual observations made during these studies. First, nicorandil was particularly ineffective despite the fact that previous studies by us and others (see McPherson & Angus, 1989) have shown that nicorandil can produce vasorelaxant responses which do not involve the opening of K+ channels. Consequently our expectation was that this compound would cause a vasorelaxant response. Conversely pinacidil did cause significant vasorelaxant responses which were insensitive to glibenclamide, a compound known to antagonize the K+ opening induced by pinacidil (see McPherson & Angus, 1990; 1991). It would appear therefore that pinacidil, along with cromakalim and nicorandil, may be able to relax vascular smooth muscle using a number of mechanisms; although their predominant effect appears to result from the opening of K+ channels. We considered a number of possibilities as to why rat cerebral vessels should display such resistance in our studies. First, the inability of cromakalim to relax rat cerebral vessels may result from the type of vasoconstrictor used to induce tone. This appeared not to be the case since similar results were obtained when either 5-HT, U46619 or low concentrations of K+ (20mM) were used. In addition, the rat cerebral vessels also displayed spontaneous myogenic tone which was little affected by cromakalim but was sensitive to SNP. The fact that rat cerebral vessels display spontaneous myogenic tone may result from the absence of K+ channels in these vessels. We have previously provided evidence that K+C channels may tonically influence the resting membrane potential (by hyperpolarizing it) in selected vascular beds such as the rat small mesenteric artery (McPherson & Angus, 1991). In the case of the rat mesenteric artery it would appear that spontaneously opened K+ channels hyperpolarize the vessel from approximately -50 to -60mV (McPherson & Angus, 1991). Electrophysiological experiments revealed that the rat anterior cerebellar artery has a low resting membrane potential compared with other tissues (-38mV anterior cerebellar cf -60mV mesenteric). It is possible that, in the absence of spontaneously opened K+ channels in this tissue, voltage operated Ca2+ channels open to cause the spontaneous tone. The spontaneous myogenic activity displayed by this vessel was abolished by the Ca2 + channel antagonist felodipine (30nM), which is consistent with this hypothesis (data not shown). The finding that cromakalim did not hyperpolarize the rat anterior cerebellar artery would indicate that the K+ is absent rather than being fully closed in this vessel. SNP did cause a hyperpolarization in association with relaxation in the rat anterior cerebellar artery. We have observed that high concentrations of SNP can cause hyperpolarization in vessels obtained from a number of vascular beds. However, the mechanism behind the response is currently unknown. It is possible that the rat cerebral vessels were less responsive to the vasorelaxants studied by an, as yet, unidentified mechanism. The ability of GTN and acetylcholine to relax rat cerebral vessels was less than that observed using rat mesenteric arteries. In addition, SNP was generally more potent in rat mesenteric and rabbit and guinea-pig small arteries than in rat anterior cerebral arteries; a finding which would support this idea. However, there were exceptions, in particu-
57
lar the rabbit anterior cerebral artery where cromakalim was a more potent relaxant than SNP. Also nitric oxide (NO) was a more powerful vasorelaxant in the rat anterior cerebral artery than in the rat mesenteric artery. Consequently, there does not appear to be a clear correlation between the potency of the vasorelaxants and the ability of cromakalim to elicit a response. The possibility also exists that rat cerebral vessels were in someway damaged upon removal and mounting of the vessel in the small vessel myograph, which had a deleterious effect on responses to the vasorelaxants. We used a number of approaches to kill the animal (CO2 asphyxia or a blow to the head) and a number of vasoconstrictors to induce tone and showed that these had no effect on the poor response to cromakalim. Despite this, the relatively low and unstable resting membrane potential displayed by the anterior cerebellar artery (-40mV) is unusual and may be indicative of some form of damage. However, work by Hirst and his colleagues (Edwards et al., 1988) have also shown segments of the rat cerebral vasculature to have a similar unstable resting membrane potential associated with spontaneous myogenic activity. This does not preclude the possibility that the rat cerebral vessels were deleteriously affected in both studies. However, given that the vessels are functionally normal in terms of their response to a number of vasoconstrictors we suspect that this is not the case. This idea is also supported by a number of in vivo studies. That cromakalim may display some regional selectivity in its action is supported by a number of in vivo studies. For example, in the anaesthetized rabbit cromakalim preferentially dilates the coronary, gastrointestinal and cerebral blood vessels but to a lesser extent the renal and skeletal muscle vasculature (Hof et al., 1988). In anaesthetized cats cromakalim increases flow in carotid, mesenteric and renal beds but not to the femoral circulation (Buckingham et al., 1986). In a recent study in conscious spontaneously hypertensive rats (Shoji et al., 1990) cromakalim decreased vascular resistance in a number of beds including the heart, stomach, kidney, skeletal muscle and skin. A significant finding from that study was that cromakalim was without effect on cerebral vascular resistance. The results from this study support the idea that the rat cerebral circulation is resistant to the vasorelaxant actions of cromakalim and other potassium channel openers. In summary, the results from this study show that a number of small arteries from the rat cerebral circulation are resistant to the vasorelaxant effects of cromakalim and other K+C channel openers. The property is specific for these vessels since such resistance is not displayed in other rat vessels or in the same vessels obtained from other species. While there may be a number of reasons for this phenomenon, it is possible that these vessels may be devoid of K+ channels under the conditions in which the experiments were performed. This project was supported by an Institute Grant from the NH and MRC of Australia and Glaxo Australia. It is a pleasure to acknowledge the assistance of Simon Keily. Cromakalim, BRL 38227 (Beecham), pinacidil (Leo) and glibenclamide (Hoechst) were generous
gifts.
References ANGERSBACH, D. & NICHOLSON, C.D. (1988). Enhancement of muscle blood cell flux and P02 by cromakalim (BRL 34915) and other compounds enhancing membrane K+ conductance, but not by Ca2 + antagonists or hydralazine, in an animal model of occlusive arterial disease. Naunyn-Schmiedbergs Arch. Pharmacol., 337, 341-346. ANGUS, J.A., BROUGHTON, A. & MULVANY, M.J. (1988). Role of aadrenoceptors in constrictor responses of rat, guinea-pig and rabbit small arteries to neural activation. J. Physiol., 403, 495-510. BUCKINGHAM, R.E., CLAPHAM, J.C., HAMILTON, T.C., LONGMAN, S.D., NORTON, J. & POYSER, R.H. (1986). BRL 34915, a novel anti-
hypertensive agent: comparison of effects on blood pressure and other haemodynamic parameters with those of nifedipine in animal models. J. Cardiovasc. Pharmacol., 8, 798-804. BUCKINGHAM, R.E. (1988). Studies on the anti-vasoconstrictor activity of BRL 34915 in spontaneously hypertensive rats; a comparison with nifedipine. Br. J. Pharmacol., 93, 541-552. COCKS, T.M. & ANGUS, J.A. (1990). Comparison of relaxation responses of vascular and non-vascular smooth muscle to endothelium-derived relaxing factor (EDRF), acidified nitrite (NO) and sodium nitroprusside. Naunyn-Schmiedebergs Arch. Pharmacol., 341, 364-372.
58
G.A. McPHERSON & A.P. STORK
EDWARDS, F.R., HIRST, G.D.S. & SILVERBERG, G.D. (1988). Inward rectification in rat cerebral arterioles; involvement of potassium ions in autoregulation. J. Physiol., 404, 455-466. EDWARDS, G. & WESTON, A.H. (1990). Structure-activity relationships of K+ channel openers. Trends Pharmacol. Sci., 11, 417-422. HAMILTON, T.C., WEIR, S.W. & WESTON, A.H. (1986). Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein. Br. J. Pharmacol., 88, 103-111. HOF, R.P., QUAST, U., COOK, N.S. & BLARER, S. (1988). Mechanism of action and systemic and regional hemodynamics of the K+ channel activator BRL 34915 and its enantomers. Circ. Res., 62, 679-686. KSOLL, E., PARSONS, A.A., MACKERT, J.R.L., SCHILLING, L. & WAHL,
M. (1991). Analysis of cromakalim-, pinacidil-, and nicorandil induced relaxation of the 5-hydroxytryptamine precontracted rat isolated basilar artery. Naunyn-Schmiedebergs Arch. Pharmacol., 343, 377-383. MARUYAMA, M., FABER, N. & GROSS, G. (1989). Effect of the new potassium channel activator, END 52692, on coronary collateral blood flow in anaesthetized dogs FASEB J., 3, A897 (Abstract 3894). McCARRON, J.G., QUAYLE, J.M., HALPERN, W. & NELSON, M.T. (1991). Cromakalim and pinacidil dilate resistance-sized mesenteric arteries but not resistance sized cerebral arteries. Blood Vessel, 28, 315. McPHERSON, G.A. (1985). Analysis of radioligand binding experiments: A collection of computer programs for the IBM PC. J. Pharmacol. Methods, 14, 213-228. McPHERSON, G.A. & ANGUS, J.A. (1989). Phentolamine and structurally related compounds selectively antagonise the vascular actions
of the K+ channel open, cromakalim. Br. J. Pharmacol., 97, 941949. McPHERSON, G.A. & ANGUS, J.A. (1990). Characterization of responses to cromakalim and pinacidil in smooth and cardiac muscle by use of selective antagonists. Br. J. Pharmacol., 100, 201206. McPHERSON, G.A. & ANGUS, J.A. (1991). Evidence that acetylcholine mediated hyperpolarization of rat small mesenteric artery does not involve the K+ channel opened by cromakalim. Br. J. Pharmacol., 103,1184-1190. MULVANY, M.J. & HALPERN, W. (1977). Contractile responses of small resistance vessels in spontaneously hypertensive and normotensive rats. Circ. Res., 41, 19-26. NAGAO, T., SADOSHIMA, S., KAMOUCHI, M. & FUJISHIMA, M. (1991). Cromakalim dilates rat cerebral arteries in vitro. Stroke, 22, 221224. PARSONS, A.A., KSOLL, E., MACKERT, J.R.L., SCHILLING, L. & WAHL,
M. (1991a). Comparison of cromakalim-induced relaxation of potassium precontracted rabbit, cat and rat isolated cerebral arteries. Naunyn-Schmiedebergs Arch. Pharmacol., 343, 384-392. PARSONS, A.A., SCHILLING, L. & WAHL, M. (1991b). Effect of microapplication of pinacidil in rat pial arterioles in situ. Br. J. Pharmacol., 102, 157P. SHOJI, T., AKI, Y., FUKUI, K., TAMAKI, T., IWAO, H. & ABE, Y. (1990). Effects of cromakalim, potassium channel opener, on regional blood flow in conscious spontaneously hypertensive rats. Eur. J. Pharmacol., 186, 119-123. STORK, A.P., McPHERSON, G.A. & ANGUS, J.A. (1990). Regional variation in the vasorelaxant effect of cromakalim in small resistance arteries from the rat. Clin. Exp. Pharmacol. Physiol., Suppl. 17, 74.
(Received June 10, 1991 Revised September 3, 1991
Accepted September 4, 1991)
