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Pharmacological evidence for the role of mediators in hypoxia-induced vasoconstriction in sheep isolated intrapulmonary artery rings
European Journal of Pharmacology, 203 (1991) 1-8 0 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50 ADONIS 0014299S91006328
EJP 52052
A. Tuncay Demiryurek, Roger M. Wadsworth and Kathleen A. Kane Department of Physiology and Pharmacology, University of Strathclyde, Glasgow Gl lxW, U.K.
Received 5 April 1991, revised MS received 25 June 1991, accepted 16 July 1991
The aim of this study was to determine the likely mediator(s) involved in the hypoxic-induced contraction in sheep pulmonary artery rings in vitro by studying the effects of selective receptor antagonists and enzyme inhibitors. Hypoxia caused a contraction in arteries under resting force and when precontracted with 5hydroxytryptamine (5HT). Flurbiprofen, a cyclooxygenasc inhibitor, reduced the hypoxic contraction in MIT-precontracted rings but augmented the first part of the hypoxic contraction under baseline force. Inhibition of nitric oxide by haemolysate increased the hypoxic contraction under resting force. Superoxide dismutase and N-t-butyl_cY-pbenylnitrone (PBN), free radical scavenging agents, and trypsin, a proteolytic enzyme, did not produce any significant effect on hypoxia-induced constriction. Propranolol plus phentolamine, p- and a-adrcnoceptor antagonists respectively, did not produce any effect on hypoxic contraction under resting force, whereas these antagonists augmented hypoxic contraction in the presence of 5-HT. This combination of antagonists also caused a reduction of 5-HT contraction which was the result of qadrenoceptor blockade. Verapamil, a calcium channel blocking drug, significantly reduced the 5-HT contraction, but did not reduce that caused by hypoxia either under resting force or in precontracted rings. These results suggest that hypoxic constriction in sheep isolated intrapulmonary artery is in part caused by reduced release of vasodilator prostanoids. This contraction does not involve voltage-operated calcium channels and L limited by release of endothelium-derived nitric oxide. Hypoxia; Intrapulmonary
artery rings (sheep); Flurbiprofen;
1. Introduction Pulmonary vasoconstriction in response to hypoxia is a component of the physiological mechanism that matches alveolar ventilation to perfusion (Voelkel, 1986). Hypoxia-induced vasoconstriction is present in lungs perfused with physiological salt solution (McDonnell et al., 19901, and in pulmonary artery strips (e.g. Madden et al., 1985; Rodman et al., 1989; Ohe et al., 1989). We have recently confirmed that such a hypoxiil-induced contraction also occurs in the intrapulmonary arteries isolated from sheep and that it is endothelium-dependent CDemiryurek et al.. 1991). Furthermore, in this preparation, hypoxia abolished acetylcholine-induced relaxation which is an enciotheliumdependent process (Demiryurek et al., 1991). These findings suggest that the endothelium plays an important role during hypoxic-induced vasoconstriction, at least, in the sheep pulmonary artery.
Correspondence to: K.A. Kane, Department of Physiology and Pharmacology, [Jniversity of Strathclyde, 204 George Street. Glasgow Gl IXW, U.K. Tel. 44.41.552.4400, ext. 2620, fax 44.41.552.2562.
Verapamil
The vascular endothelium releases both vasodilator and vasoconstrictor substances, including endorhelitimderived nitric oxide, prostacyclin and superoxide radical. Changes in PO, are known to affect the releaw of endothelium-derived vasoactive substances (Furchgott and Zawadski, 1980; Madden et al., 1986; De Mey and Vanhoutte, 1983). Moreover, the blood vessel wall contains other vasoactive substances, such as noradrenaline, and it is possible that the release of these media.tors could also be affected by hypoxia (Rorie and Tyce, 1983). The aim of this study was to determine the likely mediator(s) involved in the hypoxic-induced contraction in sheep pulmonary artery rings. Use was made of inhibitors of cyclooxygenase and nitric oxide, free radical scavengers, trypsin, a proteolytic enzyme, and adrenoceptor antagonists to identify the role if any of prostanoids, nitric oxide, free radicals, peptides and noradrenaline respectively in the response to hypoxia. The role of membrane calcium channels in mediating the hypoxia-induced contraction was investigated using the calcium channel blocking drug, verapamil. An abstract, based on some of these results, has been published (Demiryurek et al., 1990).
Sheep hmgs were obtained from a local abbatoir and delivered in cooled oxygenated physiological salt solution to the laboratory within 30 min of excision. Small (0.32-0.64 mm diameter at their optima] resting force) intrapulmonary arteries were isolated from the lungs mainly from the second branch of the main pulmonary artery. Arteries were cleared of fat and adhering connective tissue. Care was taken to avoid stretching and damage to the luminal surface. The arteries were cut mto rings of 3-4 mm long. Segments were suspended in a water-jacketed organ bath (10 ml) filled with Krebs-Henseleit solution (37°C) of the following composition (in mM): NaCl 119; NaHCO, 25; KC] 4.6; MgClz 1.2; KH2 PO, 1.2; CaCl z 2.5; glucose 11. The solution was aerated with gas mixtures containing 95% O,-5% CO, (oxygenated) or 95% Nz-5% CO, (hypoxic). The rings were suspended on a pair of stainless-steel hooks, one of which was fiied to an L-shaped rod inside the chamber and the other to a Grass isometric transducer (FTO3). Isometric contractions were recorded continuously on a Grass polygraph (Model 79 D). Arterial rings were equilibrated in Krebs-Henseleit solution gassed with 95% Oz-5% CO, for 1 h at their optimum resting force. The optimum resting force of the small pulmonary rings was determined as 0.5 g in nine preparations by comparing the tension developed by 40 mM KCI (EC,,) under different resting forces. The isometric force was calculated as force developed per cross sectional area. The cross sectional area (A) of the artery was calculated by using the equation: A = blotted weight of the artery/(h xj3) where h = the distance (cm) between the two stainless steel hooks with the artery ring under optimum resting force, and P = the density of the artery ring which has been shown to be 1.05 g crnm3 in sheep carotid artery (Keatinge, 19683. The tissues were exposed to repeated iusually three) applications of 0.3 FM 5-hydroxytryptamine (5-HT) (EC,,) with a conLact time 8-10 min until two consecutive identical responses were observed before the start of the experimental protocol. The oxygen tension of the bathing medium was measured using an oxygen electode (Strathkelvin Instruments). This electrode was calibrated to zero every day using sodium sulphite 100 mM in disodium tetraborate 10 mM. 2.1. Experimental protocol 2.1-l. Effects of hypoxia on artery rings precontracted with 5-HT or under resting force In artery rings, under resting force or precontracted
with 0.3 PM 5-HT, hypoxia was induced by changing to a 95% N,-5% CO, gas mixture. PO, values (n = 112)
(mm Hg) of 219 k 8,59 + 4, 15 + 1,8 k 1,6 + 1,5 f 0.5 and 4 + 0.5 were obtained at 0.5, 1, 2, 5, 10, 15 and 20 min respectively. After 15 (precontracted) or 20 (under resting force) min, oxygenated conditions were reestablished by washing with the Krebs-Henseleit solution aerated with 95% O,-5% CO, to yield a PO, of 652 + 4 mm Hg, n = 93. In control experiments, two consecutive responses to 5-HT under oxygenated conditions and two consecutive responses to hypoxia were obtained. 2.1.2. Effects of antagonists or enzyme inhibitors on the hypoxic contraction of artery rings precontracted with 5-HT, or lmder resting force
Following the measurement of a control hypoxic contraction, either under resting force or in a 5-HTprecontracted artery, the antagonist or enzyme inhibitor was added to the bath for 30 min, and in the continued presence of the drug a second hypoxic contraction was induced. Prior to the second hypoxic challenge, resting force was readjusted, if necessary, to the optimum resting force by reducing the stretch applied. The following drugs and concentrations were used: flurbiprofen (1 PM), a cyclooxygenase inhibitor; haemolysate (1 ~1 ml-‘) an inhibitor of nitric oxide; N-t-butyl-a-phenylnitrone (PBN, 300 PM) and superoxide dismutase (SOD, 150 U ml-‘) free radical scavenging agents; trypsin (1 U ml- ‘1 a proteolytic enzyme; propranolol (1 PM) plus phentolamine (1 PM), nonselective /3- and a-adrenoceptor blocking drugs respectively; verapamil (1 PM) a Ca2+ channel blocking drug. 2.1.3. Effects of a-adrenoceptor
antagonists and ketanserin on the S-HT-induced contraction
The effects of phentolamine (1 PM), yohimbine (1 PM), prazosin (0.1 PM) and ketanserin (0.1 FM) on the contractions induced by 5-HT at its EC,, i.e. 0.3 PM were determined. A control response to S-HT was recorded and following washout the preparations were equilibrated with one of the above antagonists for 30 min. In the continued presence of the antagonist a 5-Hl’ contraction was reobtained and c pared with the control. 2.2. Preparation of haemolysa te solution Cat arterial blood was heparinized (60 U ml-‘), centrifuged (1000 X g for 20 min at 4” 0, and the plasma and buffy coat were removed by aspiration. The erythrocytes were washed twice and resuspended in phosphate-buffered isotonic saline to restore the original volume of blood. Haemolysis was effected by pipetting 1 ml of washed erythrocyte suspension into 19 ml of hypotonic phosphate buffer (20 mOsmo1 I-‘, pH 7.41, mixing and centrifuging at 20000 x g for 30-40
3
min at 4°C with stirring against distilled water to remove low molecular weight components. The haemolysate was used at a concentration of 1 ~1 (equivalent to 0.05 ml of whole blood) per ml.
at 6 and 21 min post administration, it was 115.5 f 7.8 and 98.8 + 8 g cmW2 (n = 11) respectively. A second challenge with 5-HT resulted in a similar contraction, measurements at 6 and 21 min were 112.7 + 10.5 and 87.9 f 10.9 g cmm2 respectively. Lowering the PO1 of the bathing solution from 652 to 5 mm Hg at the peak of the 5-HT contraction (fig. 1A) caused a further contraction. As this contraction was not sustained in all of the artery rings studied, measurements were made at two time points as illustrated in fig. 1A. The hypoxic contraction was measured as the differences between the contraction obtained just prior to hypoxia (i.e. at 6 min of the 5-HT contraction) and that obtained at the peak of and again at the end of the hypoxic response (i.e. at 21 min of the 5-HT contraction). Table 1 shows the mean data obtained following two hypoxic challenges in 5-HT-precontracted arteries. It can be seen that there were no significant differences between two consecutive 5-HT plus hypoxia-induced contractions. In consecutive 5-HT contractions under oxygenated conditions, the difference in the contraction between 6 and 21 min was - 17.9 + 5 and -24.8 + 9.3 g cmp2 (n = 11) respectively. Comparison of this data with that given in table 1 indicates that hypoxia did cause a contraction even at the end of the hypoxic challenge. Under optimum resting force, introduction of hypoxia for 20 min also caused a contraction of the artery rings. This contraction was more variable than than seen in the precontracted rings. In some preparations (10 out of 124) no hypoxic induced contraction was observed under optimum resting force and in others (8 out of 124) an atypically large contraction was noted. Moreover, there was a decrease in this hypoxic response with the second hypoxic challenge (6.7 k 1.12.1 + 0.6 g cmm2, n = 13).
2.3. Drugs 5Hydroxytryptamine creatine sulphate complex, flurbiprofen, superoxide dismutase, d,l-propranolol hydro&!oride, trypsin (type XIII), verapamil, yohimbine hydrochloride (all dissolved in distiiled water and obtained from Sigma), N-tert-butyl-cu-phenylnitrone (PBN) (dissolved in saline, Sigma), ketanserin tartrate (dissolved in distilled water, Janssen), phentolamine maleate (dissolved in distilled water, Ciba) were used. Prazosin hydrochloride (Pfizer) was made up at a 100 PM stock concentation in 1% lactic acid. 2.4. Data analysis All results are expressed as means f S.E.M.; n refers to the number of lungs used. Statistical significance of differences between mean values was analyzed using Student’s paired or unpaired t-test as appropriate. P values of less than 0.05 were considered to denote statistical significance of differences.
3. Results 3.1. Effects of hypoxia on sheep intrapulmonary artery rings precmtracted with 5-HT or under resting force
Under oxygenated conditions, 5-HT at its EC,,, caused a contraction which was not sustained such that
TABLE
1
Effects of hypoxia on 5-HT (0.3 PM)-precontracted
intrapulmonaty arteries before and during treatment with antagonists/inhibitors.
n = number of lungs. Data are given as means + S.E.M. The hypoxic contraction was measured at two times. Firstly as the difference between the contraction obtained just prior to the introduction of hypoxia (i.e. at 6 min of the 5-HT contraction) and that obtained at the peak of the hypoxic response (6 min-Peak) and secondly as the difference between the contraction obtained just prior to the introduction of hypoxia and the contraction at the end of the exposure to hypoxia (i.e. at 21 min of the S-HT contraction). Treatment
n
Hypoxic contraction (g cm-‘) During treatment
Before treatment
Control Flurbiprofen Haemolysate
PBN SOD Trypsin Prop. + phentol. Verapamil
I4 I5
16.256.7 16.: k4.2
11 8 7 I1
26.5 f 5.5 10.5 f 5.4 27.Ok88.1 15.3 * 5.9
5 9
12.1 rt6.2 H.Xrt3.1
6 min - Peak
6 min - 21 min
Is+ 0.4+
8.2 8.6
13 * 3.4+
7.4 13;’
-0.9& 8.7 - 22.2 + 9.5 i’
21.?+ 6.0? 26.3k 2.0*
8.7 6.9 9.4 8.6
39.2+ l2.7* 28.1* 18.4+
5.3 6.0 10.4 7.6
6 min - 21 min
6 min -Peak -
-4.5 j 6.5 -5.7rt 16.0
26.5 + 6.2 ”
“ P < 0.05 significantly different from the appropriate control value in the absence of the antagonist/inhibitor.
34.3 + 8.2 5.5* 7.5 28.2+ II.5 -h.l+ll.h 24.1* 24.2 f
7.1 ” 7.8
TABLE
Al
6 min
Effects of antagonists/inhibitors on 5-HT-induced contraction prior to the introduction of hypoxia.
21 min
peak
I
n = number of lungs. Data are given as means+S.E.M.
G-
k
160
B 5
160
g
140
2 5
120
Treatment
0 5-HT . 5-MT
8 100
Flurbiprofen Haemolysate PBN SOD Trypsin Prop. + phentol. Verapamil
IO.3 @Jl + HYP 111 + HYP 1111 In-141
s
IAO.5 0
: 2
5
160
5 .=
160
I: E
140
c I
0 5-HT (0.3 ,,Ml + HVP In=151 m FLLIR~IPROFEN (1 I dl - 5+T+wP
120
0
5
10
15
n
15 11 8 7 11 5 9
S-HT contraction before hypoxia (g cm-*) Before treatment
During treatment
154.Ort 20.4 116.9+ 10.3 119.0 f 20.6 117.5 f 30.7 155.7 f 16.2 153.1+ 29.8 139.5 k 25.4
171.1 k20.9 117J3*11 105.2k21.2 90.1 f 27.3 125.8* 12.2 96.Ok25.4 27.5* 3.6
a a a a a
a P < 0.05 significantly different from the appropriate control value in the absence of the antagonist/inhibitor.
TIME (mid
z
2
20
25
TIME lmml Fig. 1. The effect of hypoxia on sheep intrapulmonary artery rings orecontracted with 5-HT at the EC,,. The contraction was measured at various time points up to 21 min after the administration of WIT. Hypoxia was introduced at the time indicated by the arrow. Panel (A) shows two consecutive control 5-HT plus hypoxia-induced contractions and panel \B) the effect of pretreatment with flurbiprofen on the contraction induced by 5-HT and hypoxia,
Under optimum resting force, during oxygenated conditions, flurbiprofen caused a contraction as shown in fig. 2. This increase in resting force observed in eight preparations was 8.0 f 2.2 g cm-‘. This figure also illustrates that, after the re-establishment of the optimum resting force, hypoxia in the continued presence of flurbiprofen caused a contraction which developed more rapidly than in the control and ia which the peak but not the later phase (measured at 20 min after hypoxia) was potentiated. Table 3 summarises the data obtained for two consecutive hypoxic challenges under resting force in control and drug-treated rings. As the second hypoxic contraction was less than the first in control preparations, the % change between the first and second contraction was calculated and compared for statisticai significance. The peak of the second hypoxic contraction was reduced by 64 + 9% in control experiments whereas in the presence of flurbiprofen it was increased by 89 it 58%, these values being significantly different from each other (P < 0.05).
3.2. Effect of flurbiprofen
3.3. Effect of haemolvsate As can be seen in fig 1B and table 2, pretreatment with flurbiprofen, (1 PM) slightly enhanced the contraction induced by 5-HT prior to the introduction of hypoxia. This figure also shows that flurbiprofen significantly reduced the hypoxic contraction measured both at the peak and at the end of the contraction.
1
I~lurhiprofcn
Haemosylate (1 ~1 ml- ‘1 did not affect the S-HT-induced contraction (table 2) and although it tended to increase the size of the hypoxic contraction this difference did not reach statistical significance (fig. 3 and table 1).
(I pM)
Beset
2;” Fig. 2. A typical trace showing a control response to hypoxia under resting force, the effect of flurbiprofen on resting force under oxygenated conditions, and the effect of the second hypoxic challenge in the continued presence of tlurbiprofen.
T
0
10
5
T
T
T
15
T
HYP
__
20
25
TIME [minl
Fig. 3. The effects of hypoxia on control and haemolysate-pretreated intrapulmonary artery rings precontracted with 5-I-U at its EC,,.
Similarly to flurbiprofen, haemolysate appeared to augment the hypoxic contraction obtained under resting force (table 3). The second hypoxic contraction was 23 f 15% larger than the first in the presence of haemolysate whereas in control preparations it was 64 f 9% less (P < 0.05).
0
5
10
15
20
25
TIME IminI Fig. 4. The effect of propranolol (1 PM) plus phentolamine (1 wMI on the contractions induced by 5-HT and hypoxia in intrapulmonary artery rings. o 5-I-R (0.3 PM)+ hypoxia fn = 5); * propranolol (1 kM)+phentoIamine (1 PM) - 5HT+hypoxia.
or under resting force (tables 1 and 3). It did, however, slightly reduce the 5-HT contraction (table 2).
3.4. Effect of PBN, superoxide dismutase and trypsin
3.5. Effect of propranolol plus phentolamine
Neither of the free radical scavening agents used, PBN (300 PM) or SOD (150 U ml-‘) modified the contractions induced by hypoxia in the presence of 5-HT or under resting force (tables 1 and 3). Although in the case of SOD a significant difference was noted between the hypoxic response obtained under resting force in control and drug-treated rings this was also the case prior to the introduction of drug. The % decrease between first and second hypoxic contractions was similar to that obtained in the control experiments (45.8 k 17 cf. 64 f 9%). Both drugs caused a very slight reduction in the 5-HT-induced contraction (table 2). Trypsin (1 U ml- ‘1 had no significant effect on the response to hypoxia in rings precontracted with 5-HT
As shown in fig. 4 and table 1, propranolol (1 PM) plus phentolamine (1 PM) appeared to augment the contraction caused by hypoxia in 5-HT-precontracted rings. These antagonists, however, had no effect on the resting force and did not significantly alter the hypoxic contraction under this condition (table 3). Figure 4 and table 2 also illustrate that the 5-HT contraction itself was markedly reduced by pretreatment with this CGIIIbinatiGn of antagonists. Additional experiments identified that this reduction in the 5-HT contraction was associated with a,-blockade. Phentolamine (1 PM) and yohimbine (1 PM), a selective al-antagonist, markedly reduced the size of the 5-HT contraction by 93.3 f 1.8 (n = 41 and 57 f 7% (n = 71
TABLE 3 The hypoxic contraction induced in sheep intrapulmonary antagonist or enzyme inhibitor (Hypoxia III.
artery rings under resting force in the absence (Hypoxia I) in the presence of
n = number of lungs. Data are given as means f S.E.M. n
Control Flurbiprofeu Haemolysate PBN SOD Trypsin Prop. f phentol. Verapamil
13 8 7 8 8 8 5 8
Hypoxic contraction tg cm--2) Hypoxia II
Hypoxia I -Peak
20 min
Peak
20 min
6.6* 1.5 92k2.7 13.3 f 6.6 6.4& 1.2 13.5f 1.9 il 10.7 f 4.0 3.1 *OS ” 5.6* 1.1
6.7+ 1.1 8.6* 1.9 8.4k5.2 7.3+ 1.5 14.2k3.0 a 11.6k3.5 4.3* 1.4 4.0* 1.0
2.1 kO.6 11.5f2.7 a 13.Ok4.6 ” 1.4*0.5 6.1 f 1.2 ’ 2.5 + 0.5 2.5 f 0.8 2.6+0.4
2.150.6 1.5kO.3 B 7.6k3.6 1.650.4 7.7* 1.7 n 2.7*0.6 2.4rtO.9 1.4*0.5
‘I P < 0.05 significantly different from the appropriate hypoxic response in control rings.
CI
a 5-HT 10.3 . VERAPAMIL
Al
contraction comparable to that obtained with a EC,, dose of the agonist in the absence of the Ca2+ blocking drug. However, it was shown that comparable contractions could be obtained by using 0.07 /.LM 5-HT which was an EC,, in control rings and 1 PM 5-HT in the presence of verapamil. Figure 5B shows that when hypoxia was induced under these conditions, it caused a contraction in both control and verapamil-pretreated rings and that the hypoxic contraction (measured as contraction 6 min after 5-HT - contraction 21 min after 5-HT) was slightly increased in the verapamil treated (78.3 f 16.3 g cm-*) compared with that in the control rings (39.5 * 16.4 g cme2). Under resting force, verapamil had no significant effect either alone or in the presence of hypoxia (table 3).
@4l + HYP In=91 [email protected] - 5-HT+HYP
HYP
TIME Imid 0 5-HT (0.07 e VEAAPAMIL
flMl + HYP (1 ph.+5-HT
In=71 11 IrMl+i’YP
4. Discussion
3,
OL, 0
I
5
10
15
20
2s
TIME lmml Fig. 5. The effects of hypoxia on control intrapulmonary
artery
rings precontracted
(panel A) and in rings in which the 5-HT
alld verapamil-pretreated wi!h
5-HT
at its EC,,
response is matched
absence and presence of verapamil
in the
(panel B).
respectively. whereas prazosin (0.1 FM), a selective a,-adrenoceptor antagonist, did not significantly modify the 5-HT contraction (8.3 k 15.4% inhibition, n = 5). The 5-HT contraction was also completely abolished by the 5-HT, receptor antagonist, ketanserin (0.1 PM) from 134.3 k 22.8 g cm-* to 0 g cm-* (n = ‘3). 3.4. Effect of verapamil Verapamil (1 PM) markedly reduced the contraction induced by 5-HT at its EC,; concentration (fig. 5 and table 2). In contrast to the control hypoxic contraction. in the presence of verapamil the contraction continued to rise steadily such that at the end of the hypoxic challenge the hypoxic contraction induced was greater than that in the control rings (table 1). Reintroduction of oxygenated conditions in the continued presence of 5-HT and verapamil did partially reduce this contraction (fig. 5A). Further experiments established that the blockade of the 5-HT contraction by verapamil was non-competitive so that it was not possible, in the presence of verapamil, to obtain a 5-HT
In sheep intrapulmonary artery rings in a blood free system, the contraction induced by hypoxia is abolished by removal of the endothelium (Demiryurek et al., 1991). In the present experiments, inhibition of cyclooxygenase with flurbiprofen caused contraction itself and potentiated the first phase of the hypoxic contraction recorded under baseline force. It is likely, therefore, that there is a basal release of a vasodilator prostanoid, e.g. prostacyclin, from the endothelium. Inhibition, by flurbiprofen, of the formation of this vasodilator could account both for the contraction produced Gy flurbiprofen and for augmentation of the hypoxic contraction. It may also be the case that the hypoxic contraction observed under resting force in the presence of flurbiprofen developed more rapidly than ‘In control preparations because of removal of the effect of a basally released vasodilator. The rather marked difference in the shape of the hypoxic contraction in the presence and absence of flurbiprofen may suggest that there are two phases of this response and it is possible that difference mediators are responsible for the different phases. Flurbiprofen was also shown to potentiate the 5HT-induced contraction under oxygenated conditions. Since it has been reported that 5-HT infusion can increase prostacyclin formation in the isolated blood perfused dog lung lobe (El-Kashef et al., 1990) it is likely that, in isolated pulmonary artery rings, 5-H’T also released prostacyclin. Hypoxia may reduce 5.-HTinduced prostacyclin release, accounting for at least part of the contraction caused by hypoxia in 5-HT-precontracted sheep intrapulmonary artery rings. In bovine pulmonary artery cultured endothelial cells, reduction of POZ below lo-15 mm Hg suppresses cyclooxygenase activity and hence prostacyclin production (Madden et al., 1986). It is possible that the action of flurbiprofen
7
tc reduce the hypoxic contraction in SHT-precontracted rings could be explained by its ability to prevent the hypoxia-induced reduction in prostacyclin release. Haemolysate, an inhibitor of nitric oxide, increased the hypoxic contraction recorded under optimal baseline force. This may indicate that there is a basal release of nitric oxide and inhibition of its effect led to potentiation of the response to hypoxia. This result is consistent with the finding that inhibition of the synthesis of endothelium-derived nitric oxide by NGmonomethyl-L-arginine enhanced hypoxic pulmonary vasoconstriction in rat pulmonary artery rings and in isolated lungs (Archer et al., 1989). Haemolysate did not affect the much larger hypoxic contraction produced in SHT-precontracted artery rings. Therefore, nitric oxide release appears to be low in comparison with release of prostacyclin and other mediator(s) which may play a role in the response to hypoxia in precontracted arteries. In our experiments part of the hypoxic-induced contraction remained in the presence of flurbiprofen. Thus hypoxia may also cause the release of a vasoconstrictor from the endothelium as has been demonstrated for certain other arteries (Rubanyi and Vanhoutte, 1985). We cannot, however, exclude the possibility that hypoxia may also reduce the release of another vasodilator, not studied in these experiments. In these experiments, hypoxic vasoconstriction was not affected by the free radical scavenging agents PBN or SOD, nor by trypsin. These results suggest that if a vasoconstrictor mediator is released in sheep intrapulmonary artery rings it is not a free radical nor a peptide. Hypoxic vasoconstriction was not reduced by verapamil, suggesting that it is not dependent upon activation of membrane ca!cium channels. In some studies in other species, hypoxic pulmonary vasoconstriction has been found to be abolished by calcium antagonists (Ohe et al., 1989; Rodman et al., 1989). However, in other studies a remnant of the contraction remained in the presence of calcium antagonists (Hottenstein et al., 1984). It is likely that the profile of mediators released during hypoxia depends on the species and to some extent on the experimental conditions, such as the level of precontraction. The combination of phentolamine and propranolol did not reduce the hypoxic contraction and hence these experiments did not provide any evidence for the release of noradrenaline by hypoxia, as has been observed in dog pulmonary artery (Rorie and Tyce, 1983). The contractile effect of 5-HT appeared to involve 5-HT, receptors since it was abolished by the selective 5-HT, antagonist ketanserin. It did not involve aIadrenoceptors since prazosin had no effect. However, since 5-HT comractions were reduced by phentolamine and yohimbine, there may be some linkage between
5-HT, receptors and a,-adrenoceptors
in sheep intra-
pulmonary artery as has been reported for bovine coronary arteries (Kaumann, 1983). In conclusion, the present results suggest that in sheep intrapulmonary artery rings at their optimal resting force, hypoxic constriction may be due to the reduced release of a vasodilator or may be caused by the release of a vasoconstrictor from the endothelium, whose action does not involve membrane calcium channels. This contraction is limited in size by a basal output of nitric oxide. In sheep intrapulmonary artery rings 5-HT may enhance the release of prostacyclin. The large hypoxic contraction produced in 5-HT precontracted rings, may be partly mediated by a reduction in prostacyclin release from the endothelium.
Acknowledgements A.T. Dernityurek is supported by Gazi University, Turkey. and the Turkish Higher Education Council.
References Archer, S.L., J.P. Tolins, L. Raij and E.K. Weir, 1989, pulmonaryvasoconstriction is enhanced by inhibition of
Hypoxic the synthesis of an endothelium derived relaxing factor, B&hem. Biophys. Res. Commun. 164, 1198. De Mey, J.G. and P.M. Vanhoutte. 1983. Anoxia and endotheliumdependent reactivity of the canine femoral artery, J. Physiol. 335, 65. Demiryurek. A.T., R.M. Wadsworth and K.A. Kane, 1990. Mediators involved in hypoxia-induced contraction of sheep pulmonary artery, Blood Vessels 27, 33. Demiryurek, A.T., R.M. Wildsworth and K.A. Kane, 1991, Effects of hypoxia on isolated intrapulmonary arteries from the sheep, Pulmon. Pharm. 4. 158. El-Kashef, H.A., W.F. Hofman and I.C. Ehrhart, 1990, Prostacyclin production with serotonin, increased flow, or elevated venous pressure in dog lung. J. Appl. Physiol. 69, 1283. Furchgott, R.F. and J.V. Zawadzki, 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288. 373. Hottenstein, 0.. W. Mitzner and J.T. Sylvester, 1984. Calcium dependence of isometric contractile forces during acute hypoxia in isolated rat main puimonary arteries, Fed. Proc. 43. 923. Kaumann, A.J., 1983. Yohimbine and rauwolscine inhibit S-hydroxytrypatmine-induced contraction of large cornary arteries of calf through blockade of 5-HT, receptors, Naunyn-Schmiedeb. *-rch. Fharmacol. 323. 149. Keatigne. W.R., 196X, Na flux and electrical activity of arterial smooth muscle, J. Physiol. 194. 183. Madden, J.A.. C.A. Dawson arJ D.R. Harder, 1985, Hypoxia-induced activation in small isolated pulmonary arteries from the cat, I. Appl. Physiol. 59, 113. Madden, M. C., R.L. Vender and M. Friedman, 1986, Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium, Prostaglandins 31, 1049. McDonnell, T.J., J.Y. Wescott. J. Czartolomna and N.F. Voelkel. 1990, Role of peptidoleukotrienes in hypoxic pulmonary vasoconstriction in rats, Am. J. Physiol. 259, H-751.
s he, hi.. M. Ggata. K. Shirato and T. Takishima, 1989, Effects of verapamil and BAY 8644 on the hypoxic contraction of the isolated human pulmonary artery. Tohoku 1. Exp. Med. 157. 81. Rodmsn, D.M.. T. Yamaguchi, F.R. O’Brien and I.F. McMurtry, 19S9. Hypoxic contraction of isolated rat pulmonary artery, J. Pharmacol. Exp. Ther. 248, 952. Rorie, D.K. and G.M. Tyce, 1983, Effects of hypoxia on nore-
pinephrine release and metabolism in dog pulmonary artery, J. Appl. Physiol. 55, 750. Rubanyi, GM. and P.M. Vanhoutte, 1985, Hypoxia releases a vasoconstrictor substance from the canine vascular endothelium, J. Physiol. 364, 45. Voelkel, N.F., 1986, Mechanisms of hypoxic pulmonary vasoconstriction, Am. Rev. Respir. Dis. 133, 11865.
EJP 52052
A. Tuncay Demiryurek, Roger M. Wadsworth and Kathleen A. Kane Department of Physiology and Pharmacology, University of Strathclyde, Glasgow Gl lxW, U.K.
Received 5 April 1991, revised MS received 25 June 1991, accepted 16 July 1991
The aim of this study was to determine the likely mediator(s) involved in the hypoxic-induced contraction in sheep pulmonary artery rings in vitro by studying the effects of selective receptor antagonists and enzyme inhibitors. Hypoxia caused a contraction in arteries under resting force and when precontracted with 5hydroxytryptamine (5HT). Flurbiprofen, a cyclooxygenasc inhibitor, reduced the hypoxic contraction in MIT-precontracted rings but augmented the first part of the hypoxic contraction under baseline force. Inhibition of nitric oxide by haemolysate increased the hypoxic contraction under resting force. Superoxide dismutase and N-t-butyl_cY-pbenylnitrone (PBN), free radical scavenging agents, and trypsin, a proteolytic enzyme, did not produce any significant effect on hypoxia-induced constriction. Propranolol plus phentolamine, p- and a-adrcnoceptor antagonists respectively, did not produce any effect on hypoxic contraction under resting force, whereas these antagonists augmented hypoxic contraction in the presence of 5-HT. This combination of antagonists also caused a reduction of 5-HT contraction which was the result of qadrenoceptor blockade. Verapamil, a calcium channel blocking drug, significantly reduced the 5-HT contraction, but did not reduce that caused by hypoxia either under resting force or in precontracted rings. These results suggest that hypoxic constriction in sheep isolated intrapulmonary artery is in part caused by reduced release of vasodilator prostanoids. This contraction does not involve voltage-operated calcium channels and L limited by release of endothelium-derived nitric oxide. Hypoxia; Intrapulmonary
artery rings (sheep); Flurbiprofen;
1. Introduction Pulmonary vasoconstriction in response to hypoxia is a component of the physiological mechanism that matches alveolar ventilation to perfusion (Voelkel, 1986). Hypoxia-induced vasoconstriction is present in lungs perfused with physiological salt solution (McDonnell et al., 19901, and in pulmonary artery strips (e.g. Madden et al., 1985; Rodman et al., 1989; Ohe et al., 1989). We have recently confirmed that such a hypoxiil-induced contraction also occurs in the intrapulmonary arteries isolated from sheep and that it is endothelium-dependent CDemiryurek et al.. 1991). Furthermore, in this preparation, hypoxia abolished acetylcholine-induced relaxation which is an enciotheliumdependent process (Demiryurek et al., 1991). These findings suggest that the endothelium plays an important role during hypoxic-induced vasoconstriction, at least, in the sheep pulmonary artery.
Correspondence to: K.A. Kane, Department of Physiology and Pharmacology, [Jniversity of Strathclyde, 204 George Street. Glasgow Gl IXW, U.K. Tel. 44.41.552.4400, ext. 2620, fax 44.41.552.2562.
Verapamil
The vascular endothelium releases both vasodilator and vasoconstrictor substances, including endorhelitimderived nitric oxide, prostacyclin and superoxide radical. Changes in PO, are known to affect the releaw of endothelium-derived vasoactive substances (Furchgott and Zawadski, 1980; Madden et al., 1986; De Mey and Vanhoutte, 1983). Moreover, the blood vessel wall contains other vasoactive substances, such as noradrenaline, and it is possible that the release of these media.tors could also be affected by hypoxia (Rorie and Tyce, 1983). The aim of this study was to determine the likely mediator(s) involved in the hypoxic-induced contraction in sheep pulmonary artery rings. Use was made of inhibitors of cyclooxygenase and nitric oxide, free radical scavengers, trypsin, a proteolytic enzyme, and adrenoceptor antagonists to identify the role if any of prostanoids, nitric oxide, free radicals, peptides and noradrenaline respectively in the response to hypoxia. The role of membrane calcium channels in mediating the hypoxia-induced contraction was investigated using the calcium channel blocking drug, verapamil. An abstract, based on some of these results, has been published (Demiryurek et al., 1990).
Sheep hmgs were obtained from a local abbatoir and delivered in cooled oxygenated physiological salt solution to the laboratory within 30 min of excision. Small (0.32-0.64 mm diameter at their optima] resting force) intrapulmonary arteries were isolated from the lungs mainly from the second branch of the main pulmonary artery. Arteries were cleared of fat and adhering connective tissue. Care was taken to avoid stretching and damage to the luminal surface. The arteries were cut mto rings of 3-4 mm long. Segments were suspended in a water-jacketed organ bath (10 ml) filled with Krebs-Henseleit solution (37°C) of the following composition (in mM): NaCl 119; NaHCO, 25; KC] 4.6; MgClz 1.2; KH2 PO, 1.2; CaCl z 2.5; glucose 11. The solution was aerated with gas mixtures containing 95% O,-5% CO, (oxygenated) or 95% Nz-5% CO, (hypoxic). The rings were suspended on a pair of stainless-steel hooks, one of which was fiied to an L-shaped rod inside the chamber and the other to a Grass isometric transducer (FTO3). Isometric contractions were recorded continuously on a Grass polygraph (Model 79 D). Arterial rings were equilibrated in Krebs-Henseleit solution gassed with 95% Oz-5% CO, for 1 h at their optimum resting force. The optimum resting force of the small pulmonary rings was determined as 0.5 g in nine preparations by comparing the tension developed by 40 mM KCI (EC,,) under different resting forces. The isometric force was calculated as force developed per cross sectional area. The cross sectional area (A) of the artery was calculated by using the equation: A = blotted weight of the artery/(h xj3) where h = the distance (cm) between the two stainless steel hooks with the artery ring under optimum resting force, and P = the density of the artery ring which has been shown to be 1.05 g crnm3 in sheep carotid artery (Keatinge, 19683. The tissues were exposed to repeated iusually three) applications of 0.3 FM 5-hydroxytryptamine (5-HT) (EC,,) with a conLact time 8-10 min until two consecutive identical responses were observed before the start of the experimental protocol. The oxygen tension of the bathing medium was measured using an oxygen electode (Strathkelvin Instruments). This electrode was calibrated to zero every day using sodium sulphite 100 mM in disodium tetraborate 10 mM. 2.1. Experimental protocol 2.1-l. Effects of hypoxia on artery rings precontracted with 5-HT or under resting force In artery rings, under resting force or precontracted
with 0.3 PM 5-HT, hypoxia was induced by changing to a 95% N,-5% CO, gas mixture. PO, values (n = 112)
(mm Hg) of 219 k 8,59 + 4, 15 + 1,8 k 1,6 + 1,5 f 0.5 and 4 + 0.5 were obtained at 0.5, 1, 2, 5, 10, 15 and 20 min respectively. After 15 (precontracted) or 20 (under resting force) min, oxygenated conditions were reestablished by washing with the Krebs-Henseleit solution aerated with 95% O,-5% CO, to yield a PO, of 652 + 4 mm Hg, n = 93. In control experiments, two consecutive responses to 5-HT under oxygenated conditions and two consecutive responses to hypoxia were obtained. 2.1.2. Effects of antagonists or enzyme inhibitors on the hypoxic contraction of artery rings precontracted with 5-HT, or lmder resting force
Following the measurement of a control hypoxic contraction, either under resting force or in a 5-HTprecontracted artery, the antagonist or enzyme inhibitor was added to the bath for 30 min, and in the continued presence of the drug a second hypoxic contraction was induced. Prior to the second hypoxic challenge, resting force was readjusted, if necessary, to the optimum resting force by reducing the stretch applied. The following drugs and concentrations were used: flurbiprofen (1 PM), a cyclooxygenase inhibitor; haemolysate (1 ~1 ml-‘) an inhibitor of nitric oxide; N-t-butyl-a-phenylnitrone (PBN, 300 PM) and superoxide dismutase (SOD, 150 U ml-‘) free radical scavenging agents; trypsin (1 U ml- ‘1 a proteolytic enzyme; propranolol (1 PM) plus phentolamine (1 PM), nonselective /3- and a-adrenoceptor blocking drugs respectively; verapamil (1 PM) a Ca2+ channel blocking drug. 2.1.3. Effects of a-adrenoceptor
antagonists and ketanserin on the S-HT-induced contraction
The effects of phentolamine (1 PM), yohimbine (1 PM), prazosin (0.1 PM) and ketanserin (0.1 FM) on the contractions induced by 5-HT at its EC,, i.e. 0.3 PM were determined. A control response to S-HT was recorded and following washout the preparations were equilibrated with one of the above antagonists for 30 min. In the continued presence of the antagonist a 5-Hl’ contraction was reobtained and c pared with the control. 2.2. Preparation of haemolysa te solution Cat arterial blood was heparinized (60 U ml-‘), centrifuged (1000 X g for 20 min at 4” 0, and the plasma and buffy coat were removed by aspiration. The erythrocytes were washed twice and resuspended in phosphate-buffered isotonic saline to restore the original volume of blood. Haemolysis was effected by pipetting 1 ml of washed erythrocyte suspension into 19 ml of hypotonic phosphate buffer (20 mOsmo1 I-‘, pH 7.41, mixing and centrifuging at 20000 x g for 30-40
3
min at 4°C with stirring against distilled water to remove low molecular weight components. The haemolysate was used at a concentration of 1 ~1 (equivalent to 0.05 ml of whole blood) per ml.
at 6 and 21 min post administration, it was 115.5 f 7.8 and 98.8 + 8 g cmW2 (n = 11) respectively. A second challenge with 5-HT resulted in a similar contraction, measurements at 6 and 21 min were 112.7 + 10.5 and 87.9 f 10.9 g cmm2 respectively. Lowering the PO1 of the bathing solution from 652 to 5 mm Hg at the peak of the 5-HT contraction (fig. 1A) caused a further contraction. As this contraction was not sustained in all of the artery rings studied, measurements were made at two time points as illustrated in fig. 1A. The hypoxic contraction was measured as the differences between the contraction obtained just prior to hypoxia (i.e. at 6 min of the 5-HT contraction) and that obtained at the peak of and again at the end of the hypoxic response (i.e. at 21 min of the 5-HT contraction). Table 1 shows the mean data obtained following two hypoxic challenges in 5-HT-precontracted arteries. It can be seen that there were no significant differences between two consecutive 5-HT plus hypoxia-induced contractions. In consecutive 5-HT contractions under oxygenated conditions, the difference in the contraction between 6 and 21 min was - 17.9 + 5 and -24.8 + 9.3 g cmp2 (n = 11) respectively. Comparison of this data with that given in table 1 indicates that hypoxia did cause a contraction even at the end of the hypoxic challenge. Under optimum resting force, introduction of hypoxia for 20 min also caused a contraction of the artery rings. This contraction was more variable than than seen in the precontracted rings. In some preparations (10 out of 124) no hypoxic induced contraction was observed under optimum resting force and in others (8 out of 124) an atypically large contraction was noted. Moreover, there was a decrease in this hypoxic response with the second hypoxic challenge (6.7 k 1.12.1 + 0.6 g cmm2, n = 13).
2.3. Drugs 5Hydroxytryptamine creatine sulphate complex, flurbiprofen, superoxide dismutase, d,l-propranolol hydro&!oride, trypsin (type XIII), verapamil, yohimbine hydrochloride (all dissolved in distiiled water and obtained from Sigma), N-tert-butyl-cu-phenylnitrone (PBN) (dissolved in saline, Sigma), ketanserin tartrate (dissolved in distilled water, Janssen), phentolamine maleate (dissolved in distilled water, Ciba) were used. Prazosin hydrochloride (Pfizer) was made up at a 100 PM stock concentation in 1% lactic acid. 2.4. Data analysis All results are expressed as means f S.E.M.; n refers to the number of lungs used. Statistical significance of differences between mean values was analyzed using Student’s paired or unpaired t-test as appropriate. P values of less than 0.05 were considered to denote statistical significance of differences.
3. Results 3.1. Effects of hypoxia on sheep intrapulmonary artery rings precmtracted with 5-HT or under resting force
Under oxygenated conditions, 5-HT at its EC,,, caused a contraction which was not sustained such that
TABLE
1
Effects of hypoxia on 5-HT (0.3 PM)-precontracted
intrapulmonaty arteries before and during treatment with antagonists/inhibitors.
n = number of lungs. Data are given as means + S.E.M. The hypoxic contraction was measured at two times. Firstly as the difference between the contraction obtained just prior to the introduction of hypoxia (i.e. at 6 min of the 5-HT contraction) and that obtained at the peak of the hypoxic response (6 min-Peak) and secondly as the difference between the contraction obtained just prior to the introduction of hypoxia and the contraction at the end of the exposure to hypoxia (i.e. at 21 min of the S-HT contraction). Treatment
n
Hypoxic contraction (g cm-‘) During treatment
Before treatment
Control Flurbiprofen Haemolysate
PBN SOD Trypsin Prop. + phentol. Verapamil
I4 I5
16.256.7 16.: k4.2
11 8 7 I1
26.5 f 5.5 10.5 f 5.4 27.Ok88.1 15.3 * 5.9
5 9
12.1 rt6.2 H.Xrt3.1
6 min - Peak
6 min - 21 min
Is+ 0.4+
8.2 8.6
13 * 3.4+
7.4 13;’
-0.9& 8.7 - 22.2 + 9.5 i’
21.?+ 6.0? 26.3k 2.0*
8.7 6.9 9.4 8.6
39.2+ l2.7* 28.1* 18.4+
5.3 6.0 10.4 7.6
6 min - 21 min
6 min -Peak -
-4.5 j 6.5 -5.7rt 16.0
26.5 + 6.2 ”
“ P < 0.05 significantly different from the appropriate control value in the absence of the antagonist/inhibitor.
34.3 + 8.2 5.5* 7.5 28.2+ II.5 -h.l+ll.h 24.1* 24.2 f
7.1 ” 7.8
TABLE
Al
6 min
Effects of antagonists/inhibitors on 5-HT-induced contraction prior to the introduction of hypoxia.
21 min
peak
I
n = number of lungs. Data are given as means+S.E.M.
G-
k
160
B 5
160
g
140
2 5
120
Treatment
0 5-HT . 5-MT
8 100
Flurbiprofen Haemolysate PBN SOD Trypsin Prop. + phentol. Verapamil
IO.3 @Jl + HYP 111 + HYP 1111 In-141
s
IAO.5 0
: 2
5
160
5 .=
160
I: E
140
c I
0 5-HT (0.3 ,,Ml + HVP In=151 m FLLIR~IPROFEN (1 I dl - 5+T+wP
120
0
5
10
15
n
15 11 8 7 11 5 9
S-HT contraction before hypoxia (g cm-*) Before treatment
During treatment
154.Ort 20.4 116.9+ 10.3 119.0 f 20.6 117.5 f 30.7 155.7 f 16.2 153.1+ 29.8 139.5 k 25.4
171.1 k20.9 117J3*11 105.2k21.2 90.1 f 27.3 125.8* 12.2 96.Ok25.4 27.5* 3.6
a a a a a
a P < 0.05 significantly different from the appropriate control value in the absence of the antagonist/inhibitor.
TIME (mid
z
2
20
25
TIME lmml Fig. 1. The effect of hypoxia on sheep intrapulmonary artery rings orecontracted with 5-HT at the EC,,. The contraction was measured at various time points up to 21 min after the administration of WIT. Hypoxia was introduced at the time indicated by the arrow. Panel (A) shows two consecutive control 5-HT plus hypoxia-induced contractions and panel \B) the effect of pretreatment with flurbiprofen on the contraction induced by 5-HT and hypoxia,
Under optimum resting force, during oxygenated conditions, flurbiprofen caused a contraction as shown in fig. 2. This increase in resting force observed in eight preparations was 8.0 f 2.2 g cm-‘. This figure also illustrates that, after the re-establishment of the optimum resting force, hypoxia in the continued presence of flurbiprofen caused a contraction which developed more rapidly than in the control and ia which the peak but not the later phase (measured at 20 min after hypoxia) was potentiated. Table 3 summarises the data obtained for two consecutive hypoxic challenges under resting force in control and drug-treated rings. As the second hypoxic contraction was less than the first in control preparations, the % change between the first and second contraction was calculated and compared for statisticai significance. The peak of the second hypoxic contraction was reduced by 64 + 9% in control experiments whereas in the presence of flurbiprofen it was increased by 89 it 58%, these values being significantly different from each other (P < 0.05).
3.2. Effect of flurbiprofen
3.3. Effect of haemolvsate As can be seen in fig 1B and table 2, pretreatment with flurbiprofen, (1 PM) slightly enhanced the contraction induced by 5-HT prior to the introduction of hypoxia. This figure also shows that flurbiprofen significantly reduced the hypoxic contraction measured both at the peak and at the end of the contraction.
1
I~lurhiprofcn
Haemosylate (1 ~1 ml- ‘1 did not affect the S-HT-induced contraction (table 2) and although it tended to increase the size of the hypoxic contraction this difference did not reach statistical significance (fig. 3 and table 1).
(I pM)
Beset
2;” Fig. 2. A typical trace showing a control response to hypoxia under resting force, the effect of flurbiprofen on resting force under oxygenated conditions, and the effect of the second hypoxic challenge in the continued presence of tlurbiprofen.
T
0
10
5
T
T
T
15
T
HYP
__
20
25
TIME [minl
Fig. 3. The effects of hypoxia on control and haemolysate-pretreated intrapulmonary artery rings precontracted with 5-I-U at its EC,,.
Similarly to flurbiprofen, haemolysate appeared to augment the hypoxic contraction obtained under resting force (table 3). The second hypoxic contraction was 23 f 15% larger than the first in the presence of haemolysate whereas in control preparations it was 64 f 9% less (P < 0.05).
0
5
10
15
20
25
TIME IminI Fig. 4. The effect of propranolol (1 PM) plus phentolamine (1 wMI on the contractions induced by 5-HT and hypoxia in intrapulmonary artery rings. o 5-I-R (0.3 PM)+ hypoxia fn = 5); * propranolol (1 kM)+phentoIamine (1 PM) - 5HT+hypoxia.
or under resting force (tables 1 and 3). It did, however, slightly reduce the 5-HT contraction (table 2).
3.4. Effect of PBN, superoxide dismutase and trypsin
3.5. Effect of propranolol plus phentolamine
Neither of the free radical scavening agents used, PBN (300 PM) or SOD (150 U ml-‘) modified the contractions induced by hypoxia in the presence of 5-HT or under resting force (tables 1 and 3). Although in the case of SOD a significant difference was noted between the hypoxic response obtained under resting force in control and drug-treated rings this was also the case prior to the introduction of drug. The % decrease between first and second hypoxic contractions was similar to that obtained in the control experiments (45.8 k 17 cf. 64 f 9%). Both drugs caused a very slight reduction in the 5-HT-induced contraction (table 2). Trypsin (1 U ml- ‘1 had no significant effect on the response to hypoxia in rings precontracted with 5-HT
As shown in fig. 4 and table 1, propranolol (1 PM) plus phentolamine (1 PM) appeared to augment the contraction caused by hypoxia in 5-HT-precontracted rings. These antagonists, however, had no effect on the resting force and did not significantly alter the hypoxic contraction under this condition (table 3). Figure 4 and table 2 also illustrate that the 5-HT contraction itself was markedly reduced by pretreatment with this CGIIIbinatiGn of antagonists. Additional experiments identified that this reduction in the 5-HT contraction was associated with a,-blockade. Phentolamine (1 PM) and yohimbine (1 PM), a selective al-antagonist, markedly reduced the size of the 5-HT contraction by 93.3 f 1.8 (n = 41 and 57 f 7% (n = 71
TABLE 3 The hypoxic contraction induced in sheep intrapulmonary antagonist or enzyme inhibitor (Hypoxia III.
artery rings under resting force in the absence (Hypoxia I) in the presence of
n = number of lungs. Data are given as means f S.E.M. n
Control Flurbiprofeu Haemolysate PBN SOD Trypsin Prop. f phentol. Verapamil
13 8 7 8 8 8 5 8
Hypoxic contraction tg cm--2) Hypoxia II
Hypoxia I -Peak
20 min
Peak
20 min
6.6* 1.5 92k2.7 13.3 f 6.6 6.4& 1.2 13.5f 1.9 il 10.7 f 4.0 3.1 *OS ” 5.6* 1.1
6.7+ 1.1 8.6* 1.9 8.4k5.2 7.3+ 1.5 14.2k3.0 a 11.6k3.5 4.3* 1.4 4.0* 1.0
2.1 kO.6 11.5f2.7 a 13.Ok4.6 ” 1.4*0.5 6.1 f 1.2 ’ 2.5 + 0.5 2.5 f 0.8 2.6+0.4
2.150.6 1.5kO.3 B 7.6k3.6 1.650.4 7.7* 1.7 n 2.7*0.6 2.4rtO.9 1.4*0.5
‘I P < 0.05 significantly different from the appropriate hypoxic response in control rings.
CI
a 5-HT 10.3 . VERAPAMIL
Al
contraction comparable to that obtained with a EC,, dose of the agonist in the absence of the Ca2+ blocking drug. However, it was shown that comparable contractions could be obtained by using 0.07 /.LM 5-HT which was an EC,, in control rings and 1 PM 5-HT in the presence of verapamil. Figure 5B shows that when hypoxia was induced under these conditions, it caused a contraction in both control and verapamil-pretreated rings and that the hypoxic contraction (measured as contraction 6 min after 5-HT - contraction 21 min after 5-HT) was slightly increased in the verapamil treated (78.3 f 16.3 g cm-*) compared with that in the control rings (39.5 * 16.4 g cme2). Under resting force, verapamil had no significant effect either alone or in the presence of hypoxia (table 3).
@4l + HYP In=91 [email protected] - 5-HT+HYP
HYP
TIME Imid 0 5-HT (0.07 e VEAAPAMIL
flMl + HYP (1 ph.+5-HT
In=71 11 IrMl+i’YP
4. Discussion
3,
OL, 0
I
5
10
15
20
2s
TIME lmml Fig. 5. The effects of hypoxia on control intrapulmonary
artery
rings precontracted
(panel A) and in rings in which the 5-HT
alld verapamil-pretreated wi!h
5-HT
at its EC,,
response is matched
absence and presence of verapamil
in the
(panel B).
respectively. whereas prazosin (0.1 FM), a selective a,-adrenoceptor antagonist, did not significantly modify the 5-HT contraction (8.3 k 15.4% inhibition, n = 5). The 5-HT contraction was also completely abolished by the 5-HT, receptor antagonist, ketanserin (0.1 PM) from 134.3 k 22.8 g cm-* to 0 g cm-* (n = ‘3). 3.4. Effect of verapamil Verapamil (1 PM) markedly reduced the contraction induced by 5-HT at its EC,; concentration (fig. 5 and table 2). In contrast to the control hypoxic contraction. in the presence of verapamil the contraction continued to rise steadily such that at the end of the hypoxic challenge the hypoxic contraction induced was greater than that in the control rings (table 1). Reintroduction of oxygenated conditions in the continued presence of 5-HT and verapamil did partially reduce this contraction (fig. 5A). Further experiments established that the blockade of the 5-HT contraction by verapamil was non-competitive so that it was not possible, in the presence of verapamil, to obtain a 5-HT
In sheep intrapulmonary artery rings in a blood free system, the contraction induced by hypoxia is abolished by removal of the endothelium (Demiryurek et al., 1991). In the present experiments, inhibition of cyclooxygenase with flurbiprofen caused contraction itself and potentiated the first phase of the hypoxic contraction recorded under baseline force. It is likely, therefore, that there is a basal release of a vasodilator prostanoid, e.g. prostacyclin, from the endothelium. Inhibition, by flurbiprofen, of the formation of this vasodilator could account both for the contraction produced Gy flurbiprofen and for augmentation of the hypoxic contraction. It may also be the case that the hypoxic contraction observed under resting force in the presence of flurbiprofen developed more rapidly than ‘In control preparations because of removal of the effect of a basally released vasodilator. The rather marked difference in the shape of the hypoxic contraction in the presence and absence of flurbiprofen may suggest that there are two phases of this response and it is possible that difference mediators are responsible for the different phases. Flurbiprofen was also shown to potentiate the 5HT-induced contraction under oxygenated conditions. Since it has been reported that 5-HT infusion can increase prostacyclin formation in the isolated blood perfused dog lung lobe (El-Kashef et al., 1990) it is likely that, in isolated pulmonary artery rings, 5-H’T also released prostacyclin. Hypoxia may reduce 5.-HTinduced prostacyclin release, accounting for at least part of the contraction caused by hypoxia in 5-HT-precontracted sheep intrapulmonary artery rings. In bovine pulmonary artery cultured endothelial cells, reduction of POZ below lo-15 mm Hg suppresses cyclooxygenase activity and hence prostacyclin production (Madden et al., 1986). It is possible that the action of flurbiprofen
7
tc reduce the hypoxic contraction in SHT-precontracted rings could be explained by its ability to prevent the hypoxia-induced reduction in prostacyclin release. Haemolysate, an inhibitor of nitric oxide, increased the hypoxic contraction recorded under optimal baseline force. This may indicate that there is a basal release of nitric oxide and inhibition of its effect led to potentiation of the response to hypoxia. This result is consistent with the finding that inhibition of the synthesis of endothelium-derived nitric oxide by NGmonomethyl-L-arginine enhanced hypoxic pulmonary vasoconstriction in rat pulmonary artery rings and in isolated lungs (Archer et al., 1989). Haemolysate did not affect the much larger hypoxic contraction produced in SHT-precontracted artery rings. Therefore, nitric oxide release appears to be low in comparison with release of prostacyclin and other mediator(s) which may play a role in the response to hypoxia in precontracted arteries. In our experiments part of the hypoxic-induced contraction remained in the presence of flurbiprofen. Thus hypoxia may also cause the release of a vasoconstrictor from the endothelium as has been demonstrated for certain other arteries (Rubanyi and Vanhoutte, 1985). We cannot, however, exclude the possibility that hypoxia may also reduce the release of another vasodilator, not studied in these experiments. In these experiments, hypoxic vasoconstriction was not affected by the free radical scavenging agents PBN or SOD, nor by trypsin. These results suggest that if a vasoconstrictor mediator is released in sheep intrapulmonary artery rings it is not a free radical nor a peptide. Hypoxic vasoconstriction was not reduced by verapamil, suggesting that it is not dependent upon activation of membrane ca!cium channels. In some studies in other species, hypoxic pulmonary vasoconstriction has been found to be abolished by calcium antagonists (Ohe et al., 1989; Rodman et al., 1989). However, in other studies a remnant of the contraction remained in the presence of calcium antagonists (Hottenstein et al., 1984). It is likely that the profile of mediators released during hypoxia depends on the species and to some extent on the experimental conditions, such as the level of precontraction. The combination of phentolamine and propranolol did not reduce the hypoxic contraction and hence these experiments did not provide any evidence for the release of noradrenaline by hypoxia, as has been observed in dog pulmonary artery (Rorie and Tyce, 1983). The contractile effect of 5-HT appeared to involve 5-HT, receptors since it was abolished by the selective 5-HT, antagonist ketanserin. It did not involve aIadrenoceptors since prazosin had no effect. However, since 5-HT comractions were reduced by phentolamine and yohimbine, there may be some linkage between
5-HT, receptors and a,-adrenoceptors
in sheep intra-
pulmonary artery as has been reported for bovine coronary arteries (Kaumann, 1983). In conclusion, the present results suggest that in sheep intrapulmonary artery rings at their optimal resting force, hypoxic constriction may be due to the reduced release of a vasodilator or may be caused by the release of a vasoconstrictor from the endothelium, whose action does not involve membrane calcium channels. This contraction is limited in size by a basal output of nitric oxide. In sheep intrapulmonary artery rings 5-HT may enhance the release of prostacyclin. The large hypoxic contraction produced in 5-HT precontracted rings, may be partly mediated by a reduction in prostacyclin release from the endothelium.
Acknowledgements A.T. Dernityurek is supported by Gazi University, Turkey. and the Turkish Higher Education Council.
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