The endothelium contributes to the contractile ...

contributes to the contractile responses of the human ydroxytryptamine and endothelin-1 under low but not high Po2 conditions HUHX I E ~AND CHRISTOPHER R. Department of Pharmacs!ogy and Xherqeutics, Faculty of Medicine, B e Health Sciences Centre, B e University of Calgary, Calgary, AB T2N 4NB, Canada

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Hubei university on 06/03/13 For personal use only.

Received April 2 1, 1994 C.R. 1994. The endothelium contributes to the contractile responses of the human umbilical artery XIE, H . >and T~IGGLE, to 5-hydroxytryptamine and endothelin-l under low but not high Po, conditions. Can. J. Physiol. PharmacoI. 72: 1171-1179. To determine the influences of both Po, and the presence of the endothelium on contractile responses of the human umbilical artery (HUA), the effects of a series of vasoconstrictors were compared in ring preparations with and without endothelium at low (2.5% 02,Po, < 55 mmHg (1 mmHg = 133.3 Pa)) and high Po, (95 % Q,, Po, > 600 mmHg). The results demonstrate the following. (i) 5Nydroxytsyptamine (5-HT) and endothelin-1 (ET-1) contracted the HUA at either low or high Po,. At low Po,, removal of the endothelium significantly reduced receptor-mediated responses. (ti) The nitric oxide synthase inhibitor Nu--nitro-L-arginine methyl ester @-NAME, 180 yM) did not modulate 5-HT-initiated contractions at either level of Po,. (iii) a-Methyl-5-hydroxytryptamine (a-Me-5-HT) and 5-carboxamidotryptamine (5-CT), relatively selective 5-HT,,/5-HT, and 5-HT,-like receptor agonists, respectively, elicited contractions in the HUA, and the responses were reduced at low Po2 but unaffected by removal of the endothelium. (iv) Responses of the HUA to high potassium (hK+) were unaffected by either changes in Po2 or removal of the endothelium. (v) The 5-HT, receptor antagonist ketanserin at low concentration (10 nM) inhibited contractile responses to 5-HT in an apparently competitive manner. However, with 100 nM ketanserin and at low Po,, inhibition became noncompetitive. Removal of the endothelium did not influence the action of ketanserin. (vi) Regardless of Po,, the Ca2+channel antagonist nifedipine (I yM) significantly inhibited 5-HT- and ET-1-mediated contractions. Depletion of extracellular Ca2+ also greatly reduced 5-HT-induced contractions. We conclude that, in the HUA, endothelial cells and changes in Po, differentially modulate contractions initiated by 5-HT agonists and ET-1, possibly via the release of an endothelium-derived contracting factor (EDCF). Key word%^: endothelium, contracting factors, vascular smooth muscle, oxygen tension.

H., et TRIGGLE,C.R. 1994. The endothelium contributes to the contractile responses of the human umbilical artery to 5-hydroxytryptarnine and endothelin-1 under low but not high Po, conditions. Can. J. Physiol. Pharmacol. 72 : 11'91-1B79. Pour dCteminer les influences de la Po, et de la presence de l'endothClium sur les rkponses contractiles de l'artkre ombilicale humaine (AOH), les effets d'une sCrie de vasoconstricteurs ont CtC compares dans des preparations d'anneaux avec et sans endothClium, 2 une Po, faible (2,5% d90,, Po2 < 55 mmHg (1 mmHg = 133'3 Pa)) et B une Po, ClevCe (95% d'O,, Po, > 600 rnmHg). Les risultats dCmontren\t que (i) la 5-hydroxytryptamine (5-HT) et I'endothCline-l (ET-1) ont contract6 1'AOH tant 2 bane Po, ClevCe que faible. A une Po2 faible, le retrait de l'endothtlium a significativement rCduit les rCponses mCdiCes par un rkcepteur. ( i i ) L'inhibiteur de l'oxyde nitrique synthase, L-NAME (100 pM), n'a pas module les contractions induites par la 5-HT tant B une Po, faible que ClevCe. (iii) L'a-methyl-5-hydroxytryptamine(a-Me-5-HT) et la 5-carboxarnidotryptamine (5-CT), compsCs relativement sklectifs des agsnistes des recepteurs de type 5-HT,,/5-HT, et 5-HT, respectivernent, ont induit des contractions dans l'AOH, et les rCponses ont Ctt rdduites h une Po, faible mais n'ont pas CtC affectCes par le retrait de I'endothClium. (iv) Les rCponses de 19AQHh une forte concentration de potassium (fK9) n'ont CtC affectCes ni par des variations de Po, ni par le retrait de 19endothClium.(v) L'antagoniste des rCcepteurs de 5-HT2 kChnsCrine, 2 faible concentration (10 nM), a inhibe les rCponses contractiles B la 5-HT d'une manikre apparemment compttitive. Toutefois, en presence de 100 nM de kCtansCrine et B une Po, faible, l'inhibition est devenue non compCtitive. Le retrait de l'endothClium n'a pas inflbaencC l'action de la kCtansirine. (vi) Sans Cgards 5 la Po,, l'antagoniste des canaux Ca2+, nifkdipine (1 yM), a significativement ihibC les contractions induites par l'ET-1 et la 5-HT. La dtplttion du Ca2' extracellulaire a aussi considCrablement rCduit les contractions induites par la 5-HT. Nous concluons que, dans l'AOH, les cellules endothCliales et les variations de Po, modulent diffkremment les contractions induites par les antagonistes de la 5-HT et 1'ET-1, possiblement via la libCration d'un facteur de contraction dCrivC de I'endothClium (EDCF). Mots cl& : endothClium, facteurs de contraction, muscle lisse vasculaire, pression d90xyg&ne. [Traduit par la RCdaction] %HE,

Intrsductisn The existence of both endothelium-derived relaxing factors (EDRFs) and contracting factors (EDCFs) and their importance in regulating vascular tone are now well accepted 'Author for correspondence. ,Present address: Department of Pharmacology, University of Vemont Medical College, Given Medical Building, Burlington, VT 05405-64868, U .% .A. Printed in Canada / Imprim6 au Canada

(Furchgott and Zawadzki 1980; Furchgott 1983; Ignarro 1989; Luscher et al. 1992). Recent studies with the human umbilical artery (HUA) hdicate that responses to endothelium-de~endentvasorelaxants, such as acetylcholine ( A C ~ ) , are atypical and EDRFQs)may play a minimal role in regulating thetone of the HUA (xii and Triggle 1994). others-have also shown that whereas the HUA responds in an apparently enbthelial-de~endentmanner to histamine, there was no response to ACh (Van de Vosrde et al. 1987). It is also well known that oxygen tension plays an important

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role in the regulation of vascular tone and a change in Po2 can modify the responsiveness of various blood vessels (Ckang and Detar 19883; Busse et al. 1983; Rubanyi and Vanhoutte 19851, in a partly endothelium-, species-, and tissue-dependent manner. A number s f studies indicate that hypoxia releases both vasoconstricting (Rubanyi and Vahoutte 1985) and vasodilating substances (Busse et ale 1984) from endothelial cells. Thus, the role and nature of the mediators released when Po2 changes remains controversial. Studies with the HUB have demonstrated that changes in Po2 affect vascular tone possibly by altering receptor - signal transduction interaction (McGrath et al. 1986; MacLennan et al. 1989; Templeton et al. 1991). However, interactions among P o 2 , vascular smooth muscle, and endothelial cells, particularly in human blood vessels, have not been extensively studied. This study was thus designed to determine whether changes in Po2 and endothelial cell function modulate the contractile responsiveness of human vascular smooth muscle and, if so, whether changes in Po2 affect cellular signal transduction processes. We chose the extrafetal (umbilical cord) HUA, which lacks autonomic innervation f lachenmayer 197 B ; Roach 1972), as being an appropriate tissue for studying the direct effects s f vasoactive substances as well as the influences of P o 2 , on human vascular smooth muscle. Furthermore, in vivo, the physiological oxygen tension in the HUA is low (Po2 15 mmHg (1 mmHg = 133.3 Pa)), and during parturition the Po2 of HUA blood increases, and this increase may play a role in closure of the HUA after birth (Wulf 1964; McGrath et al. 1986). In this regard the physiological regulatory mechanisms, with respect to the effects of changes in Po2, may differ in the HUA compared with, for instance, aortic tissue. The present study was undertaken to (i) study and compare the effects of 5-hydroxytryptamine (5-HT) agonists, endothelin-1 (ET- I), and high K (hK +) depolarizatation on the WUA under conditions where (a) Po2 is maintained at either < 55 or > 680 rnmHg; (b) the endotheliurn is either intact or removed; and (c)the tone of the MUA is either maintained at resting levels or elevated by agonists; (ii) determine the effect s f changes in P z on the signal transduction processes activated by 5-HT and ET-1 in the HUA; and (iii) compare the effects of changes in Po2 on 5-HT-induced contractions and the signal transduction pathway activated by 5-HT in the rat aorta (RA).

-

+

Materials and methods Prepamtion of arterial rings HUAs from normal, full-term deliveries (tissues from both vaginal delivery and elective caesarean sections were pooled) were obtained from the Calgary Foothills Hospital delivery room, and were transported to the laboratory prior to dissection in physiological salt solution (PSS; composition in mM: NaCB, 118.0; KCI. 4.7; CaCI, 2.5; MgSB, 1.2; KH,PO, 1.2; NaHCO,, 12.5; dextrose 11.1). The transportation time between delivery room and research labratory was approximately 5 - 10 min. The tissue immediately adjacent to where the umbilical cord had been "clamped" was discarded, and arteries from the placental end of the umbilical cord were carefully dissected out of the Wharton's jelly. Fresh tissues as well as the t s sues predissected then stored overnight at 4°C were used in the present study. Storing the HUB for up to 24 h at 4'C in PSS did not affect responses of the tissue to either contractile or relaxant stimuli (Xie 1994). The use of human tissue for these studies was approved by the Faculty of Medicine Conjoint Medical Ethics Committee at the University of Calgary. Male rats (Sprague - Dawley , weighing H $0 - 200 g) were used and

cared for in accordance with the Guide to the Care and Use of ExpeP-a'merrta& Animals (Canadian Council on Animal Care). The rats were sacrificed by stunning and exsanguination. The thoracic aorta was removed immediately and used the same day for the described protocols. During preparation, the tissues were kept at room temperature in PSS bubbled continuously with 95 % 0, - 5 % CO,. Care was taken not to damage the endothelium. The arteries were cleared of surrounding connective tissues. The vessels were cut into rings (5 Inm in length) and suspended between parallel stainless-steel hooks in organ baths, and aerated with different concentrations of 0,. Bath temperature was maintained at 37°C. Prior to any dmg intervention, ring preparations from the HUA were equilibrated for 3 h, and those f o m the M,for 1 h. During the equilibration the PSS was replaced at 15- to 20-min intervals. The resting tension was adjusted to the optimal preload of 2 g (Gopalakrishnan et al. 19851, and the same protocol was also used for aortic tissue. Isometric tension was measured with Grass FT-03 transducers and displayed on a model 7D Grass polygraph (Grass Instruments, Quincy, Mass.). Endothelial cells were removed from the preparations by gently rubbing the intimal surface with a Q-tip (cotton swab). The presence and absence of endothelium were verified by scanning electron microscopy (Xie and Triggle l 994). Experimental protoco&s Series P Cumulative concentration - response curves (CRCs) to 5-HT, a-Me-5-HT, 5-CT, ET- 1, and hK' , were obtained in one HUA tissue with or without endothelium. Routinely 12 preparations were used from each uanabilical cord and different agonists could thus be tested on tissues from the same cord. After obtaining at least two sets of control data the effects of a 30-min preincubation with the nitric oxide synthase inhibitor Wr"-nitro-L-argininemethyl ester (L-NAME, 100 pM) on 5-HT-initiated contractions were then examined. Effects of the 5-HT receptor agonists (5-HT, a-Me-5-HT, and 5-CT) and ET-I were also determined at both resting and agonist-elevated tone (i.e., EDso, 5-HT- or U44069-precontracted rings). The effects of 5-HT were also compared in the RA. Series 2 The effects of a I-h preincubation with 10 or 100 nM 5-HT2 antagonist, ketamserin, on 5-HT-initiated contractions in endotheliumintact and -denuded rings were determined and compared with pre-ketanserin-exposure control data. Series 3 The contribution of extracellular Ca2+ to agonist-initiated contractions was examined in two ways. (i) The effects of a 30-min preincubation with the Ca2* channel antagonist nifedipine (I pM), on responses of the MUA to 5-HT and ET-H were assessed under minimal light conditions, and the data were compared with those obtained under control conditions. The effect of nifedipine (I pM) on 5-HT-initiated contractions was also studied in the WA. (li) The effects of a 30-Enin preincubation with @a2'-free PSS plus or minus EGTA (1 w M ) on 5-HT-induced contracticdns were investigated in the HUA. AH1 studies were carried out with (i) high 8,: "standard5' gas mixture of 95% 0, - 5 % C0, (Po2 > 600 mmHg), pH 7.3-7.5 and (ii) low 0,: 2.5 % 0, - 8.5 % CO,, balanced with N, (Po, < 55 mmHg), pH 7.1 - 7.2. Po, was measured with a 1306 pH/blood gas analyzer (Instrumentation Laboratoryt Lexington, Mass.); Po, at 95% 0,: 556.8 f 7.9 mmHg (pz = 10); Po, at 2.5%: 50.9 k 5.3 (n = 8).

Dugs 5-PIT (oxalate salt), L-NAME (WCl), and nifedipine were purchased from Sigma Chemical Company (St. Louis, Mo.), ET- I was from Peptide International 'Itrc. (Louisville, Ky .) , a-Me-5-HT (maleate) and 5-carboxamido:ryptamine (5-CT, maleate) were from


XIB AND TRIGGLE

FIG. 1. Concentration-contraction curves to 5-HT in the MUA with and without endothelium (-E) and in the EL4 at low vs. high Po,. Each point represents mean f SE, n = 12. *g < 0.05. MAX, maximum. TABLE1. The effects of Po, and the endothelium on EC,, for 5-HT agonists and ET-I in the HUA

5-HT

12

a-Me-5-HT

5

5-CT

5

ET- 1

8

1 . 4 ~ los7 6~ 3.16 X (1 .O$ x -2.48 x (2.29 x 10-7-4.35 x 3.93 X 4.29 x ( 2 . 2 8 10-7-8.07 ~ x loS7) ( 1 . 8 4 10-7-8.42x ~ 1.9lXlO-'$ 2.34 X loS6? ( 9 . 6 8 ~ 1 0 - ~ - 3 . 7 5 ~ 1 0 - (~1) . 4 3 ~ 1 0 - ~ - 3 . 8 4 ~ 1 0 - ~ ) 1.91~10-" 3.21 X lo-"? ( 1 . 9 10-8-5.1 ~ x lo-') (1.28 x 10-~-2.85 x

*p < 0.05 compared with 9 E . Tp < 0.85 compared with -E at 95% 02. $p < 0.85 compared with 9 B at 95 % 0 2 .

Research Biochemicals Incorporated, (Natick, Mass.), and U44069 was a gift from Upjohn (Kalmazso, Mich.). Ketanserin (tartrate) was a gift from Jansen Phamaceutica, Belgium. The stock solutions of U4-4069, nifedipine, and ketanserin were prepared in ethanol, and that of ET-1 was prepared in 0.1 % acetic acid. All other compounds were dissolved in deionized water. The concentrations of the drugs used and depicted in the figures are the logarithm of the final molar concentration in the bathing solution. Data analysis The data points plotted in the graphs are means f SE and n values represent the number of human umbilical cords and rat a o ~ a exame ined. ECSovalues were determined from individual CRCs for each ring preparation; geometric means and 95% confidence limits were then calculated from the pooled data. Responses were expressed relative to the maximal response produced by a given tissue to 5-HT, ET-1, or KC1. Maximal responses and EC,, values were compared in rings with endothelium intact and denuded, at Iow and high Po, and analyzed using the Newman-Keuls test. Paired and unpaired data from concentration -response curves were analyzed using Student's t test. Unpaired data comparison was used for the studies of ET-1 effects in the HUA; the WUA demonstrates a significant desensitization to repeated exposure to ET-1, thus necessitating the use of different tissues, but adjacent ring preparations from the same

HUA. Unpaired data analysis was d s o used for comparing the effects of endothelium removal ( + E vs. -E) on responses to 5-HT and hKf. Paired data analysis was used for determining the effects of Po2 changes, extracellular Ca2+ removal, and nifedipine, ketanserin, and 5-WT ligands on tissue responses in the same tissue. A value of g < 0-05 was considered significant. Curves were fitted using a Sigma Plot program (Jandel Scientific, San Rafael, Calif.).

Results Respoasses of the HUA to 5-HT receptor argonksts: efiects of endothelium, Po2, and ketceasserias Storing the HUA for up to 24 h at 4'C did not affect responses of the tissue to 5-HT ( p > 8.05; data not shown). Under the experimental conditions described the 5 - m ring preparations of the HUA normally generated, in response to 5-HT, 4 g of tension, with the range being 2-8 g. Such responses were highly reproducible in some protocols for up to 18 h. Under both high and low Po2 conditions the HUA responded to 5-HIP with concentratlon-dependent contractions (Fig. 1). 5-HT-induced tension was rapid in onset and remained essentially sustained. Removal of the endothelium

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I e plus L-NAME,

100 yM

FIG.2. Effects of L-NAME (100 yM) on 5-HT-initiated contractions in the HUB at low vs. high Po2. Each point represents mean f SE, n = 4.

plus Ket., 10 nM plus Ket.. 100 nM

'I2O

1

0 At 95% 0, @ plus Ket.. 10 nM

FIG. 3. Effects of ketanserin (Ket., 10 and 100 nM) on 5-HT-initiated contractions in the HUA with (+E) and without (-E) endothelium at low vs. high Po2. Each point represents mean f SE, n = 5 -7. *p < 0.05.

significantly depressed the contractile responses to 5-HT at Bow Po2, but not at high Po2. values were increased and maxima?. responses decreased in endothelium-denuded rings when changing Po2 from high to f ow (g < 0.05) (Table 1 ; Fig. 1). Responses of the RA to 5-HT were not different under high versus low Po2 conditions (Fig. 1).

Pretreatment of the HUA with L-NAME (1W pM) for 38 min did not affect 5-NT-initiated contractions at either level of PR (Fig. 2). The relatively selective 5-HT2 receptor agonist a-Me5-HT, contracted the HUA in a concentration-dependent manner, with a decrease in sensitivity at low versus high Po2


XIB AND TRIGGLB

ET- 1, Log [MI FIG. 4. Concentration-contraction curves to ET-1 in the HUA with and without (-E) endothelium at low vs. high Po,. Each point represents mean f SE, n = 8. *p < 0.05.

KC1, Log [MI FIG. 5. Concentration-contraction curves to KC1 in the HUA with and without (-E) endothdium at Bow vs. high Po,. Each point represents mean f SE, n = 9.

( p < 0.05). The 5-HT1-like receptor agonist 5-CT behaved in a similar way, but the HUA responded with comparatively low sensitivity. Although low Po2 significantly attenuated contractions and reduced the sensitivity of the tissues to both agonists, removal of the endothelium had no effect on the responses (Table 1) . In UU069 (0.1 -0.2 pM) precontracted rings, with and without endothelium, 5-HT, a-Me-5-HT, and 5-CT either had no effect on elevated tone or produced slight contractions (data not shown) under either high or low Po2. In the presence of 10 nM ketanserin, a 5-HT2 receptor antagonist, at either high or low Po21 5-#T CRCS were

shifted to the right in an apparently parallel fashion and without a change in the maximal response. However, ketanserin, at 100 nM, shifted the 5-HT CRC to the right, and reduced the maximal response at low but not at high Po2. The presence or absence of the endothelium did not alter the nature of the antagonism produced by ketanserin (Fig. 3).

Efects of ET-1 on the HUA Under either low or high Po2, the HUA contracted to ET-1 in a concentration-dependent manner (Fig. 4). Although most HUA preparations contracted in response to ET-1 with high sensitivity (ECS0, 10-$ M, Table I), 4 of 22 tissues failed to

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[IIT

at 2.59410,

EZZ

at 9 5 % ~ ~ ~


plus Nifedipine, 1 @I

"I

j(b)

ET- 1 FIG. 6. Comparison of effects of nifedipine ( I yM) on responses to 5-HT(0.1, I , and 10 pM) and ET-1 (18, 30, and 100 nM) in the HUA at Bow vs. high Po2. n = 5 or 6 .

respond to ET-1, whereas they responded to hK9 and (or) 5-HT. In preparations with intact endothelium, changes in Po2 did not alter responses to ET- 1 (Table B ; Fig. 4). Removal of the endothelium significantly reduced ET-1initiated contractions at low Po2 but not at high Po2. At low Po2, ECs0 values for ET-1 were increased and maximal responses were decreased in endothelium-denuded rings (p < 8.05) (Table 1; Fig. 4); however, at low Po2 the maximum response to ET-l was obtained at 3 x 1W8 M, whereas at high Po2, the maximum was recorded at 1 >( 10s8 M. In 5-HT-precontracted rings, ET-1 failed to relax the HUA 8 f O % , n = 3 (data not shown). Eflects of hK+ on the HUA In contrast to the responses to 5-HT and ET-1, contractile responses to hK+ were unaffected by either rernovd of the endothelium or changes in Po2 (Fig. 5). Eficts of nifkdkptne and depletion of' extracelhlar CaD at high vs. low Po2 At either high or low P%, nifedipine (1 yM,for 30 min) significantly inhibited contractions induced by either 5-HT or ET-1 in the MUA (Fig. 6). Changes in Po2 did not alter the sensitivity of the HUA to nifedipine. Depletion of Ca2+ significantly inhibited contractions

initiated by 5-HT at both high and low Po2. 5-HT CRCs were shifted to the right and maximal responses reduced to 27 f 3 % at high Po2, and 3 1 f 3 % at low Po2 (Fig. 7). 5-HTinitiated contractions were completely abolished in Ca2+-free medium containing EGTA (I mM) (data not shown). The effects of changes in Po2 on 5-HT-initiated contractions and sensitivity to nifedipine were also compared in the RA. Nifedipine (1 pM, for 38 min) partidly antagonized 5-HT-initiated tone in the RA at both high and low Po2, and the degree of inhibition was unaffected by changes in Po2 (Pig. 8).

Discussion The major findings from this study can 1Pe summarized as (i) the endothelium contributes to the contractile responses of the HUA at low but not at high Po2 and (it) changes in Po2 may affect 5-HT -receptor interactions. We have demonstrated that changes in Po2 and removal of the endothelium from the HUA differentially affected contractile responses to 5-HT agonists and ET-1, but not hK+. Contractions initiated by either 5-HT or ET-1 were attenuated by removal of the endothelium at low but not at high Po2. Furthermore, the 5-HT- and ET- 1-mediated responses were unaffected by the presence of the nitric oxide synthase inhibitor L-NAME, at a concentration that we have previously shown should have completely eliminated any contribution from arginine-derived nitric oxide (Adeagbo and Triggle 1993). These data can be interpreted as indicating that an EDCP released under low Po2 conditions contributes to the development of active tone in the HUA. The endothelium of the HUA, however, seems to lack the ability to synthesize relaxing factors (Van de Voorde et al. 1987; Monuszko et al. 1990; Xie and Triggle 1994). That responses to hKf were not affected either by changes in Po2 or by endothelial cell removal indicates that these procedures do not directly affect the ability of HUB cells to contract and that removal of the endothelial cells did not mechanically damage the underlying smooth muscle. Hypoxia has been shown to release an EDCF(s) in a variety of vascular beds (Rubawyi and Vanhoutte 1985; Katusic and Vanhoutte 1989), but the nature of this putative factor shows tissue and species dependency. Hn the HUA contributions from endogenous eicosanoids have been implicated in mediating the effects that variations in Po2 have on responses to agonists (Bjoro et al. 1987; MacEennan et al. 1989; Templeton et al. 1991). Whether such factors have an origin from vascular endothelial or smooth muscle cell was not, however, investigated. Our data can be explained by assuming that hypoxia, in the presence of the agonist, releases a contracting factor from the endothelium that contributes to the direct effects of 5-HT and ET-1 on smooth muscle. However, the nature of this putative EBCF is unknown. Conceivably a prostanoid, such as thromboxane, is released; however, as reported by MacLennan et al. (1989), and also Xie and Triggle (1994) indomethacin has direct inhibitory effects on the HUA, thus making studies to directly test the role of cyclooxygenase products difficult to assess. Of interest is that hypoxia potentiates the contractile responses of the sheep circumflex coronary artery to 5-HT and U44069, but in the absence of the endotheliurn, responses to 5-WT are reduced (Kwan et aL 1984a, 19846). These data suggest that responses to 5-HT and hypoxia in

XIE AND TRIGGLE ,-/.-

,'

0

Ca

-free PSS

G

/

I


1

FIG. 7. Effects of Ca2+-free on responses to 5-HTin the HUA at low vs. high Po,. Each point represents mean f SE, n

the HUA and the sheep circumflex coronary artery may be similar. Our data also suggest that changes in oxygen tension modify ligmd -receptor interactions and affect agonist-induced vascular tone in the HUA. Thus (i) reduction in Po2, but not removal of the endothelium, attenuated contractile responses initiated by 5-HT agonists, a-Me-5-HT and 5-CT, but not hKf in the HUA; (ii) in the endothelium-denuded HUA, changes in Po2 from high to low level reduced contractions elicited by both 5-HT and ET-1 but not hK+; (kki) changes in Po2, but not removal of endothelium, altered the nature of antagonism produced by ketanserin. In contrast, in the RA, 5-HT-initiated contractions remained unchanged with varying Po2. 5-HT can relax vascular tissue by both endotheliumdependent and -independent processes (Cocks and Angus 1983; Eyre 1975). Our data with the HUA showed that, regardless of the presence or absence of the endothelium, neither 5-HT nor the other 5-HT agonists tested relaxed precontracted tissues. These data, together with our previous studies, demonstrate that 5-HT is a potent vasoconstrictor in the HUA and, unlike some other vascular beds, possesses no vasodilatory actions (Reiffenstein and Triggle 1974). At low Po2, contractions produced by 5-HT, but not a-Me-5-HT or 5-CT, were partially endothelium dependent. Furthermore, removal of the endothelium failed to modify the nature of the inhibition by ketanserin. These data suggest that the release of the putative EDCF is not mediated by 5-HT1 or 5-HT2 receptors. 5-CT has a comparatively high affinity for 5-HTBreceptor subtypes and a low affinity for the 5-HT2 receptor, and as-Me-5-HT has a high affinity for 5-HTlc and 5-HT2 receptor subtypes (Hoyer 1989), whereas 5-HT can also activate a variety of other 5-HT receptor subtypes, which currently include 5-HT3 and 5-HT4 (Zifa and Fillion 1992). Thus, the data presented in this study suggest that the endothelial 5-HT receptor that mediates the release of the putative contracting factor belongs to neither the 5-HT1 nor 5-HT2 subtype. The identity of the subtype(s) of 5-HT receptor that mediates endothelium-independentconstriction of the HUA is unknown.

=

5.

Fro. 8. Effects of nifedipine (I pM) on responses to 5-HTin the RA at low vs. high Po,. n = 5 .

The apparent competitive nature of the inhibition produced by 18 nM kebanserin suggests the 5-HT2 subtype. However, larger concentrations of ketanserin produced noncompetitive inhibition under low Po2 conditions. These data suggest that at least two subtypes of 5-HT receptor associated with vascular smooth muscle are involved in mediating contraction in the HUA. The conclusion that agonist-induced dependence of contractions in the HUA is dependent on extracellular Ca2+ is based on three observations. First, the Ca2 channel antagonist, nifedipine, greatly inhibited 5-HT- and ET- 1-initiated contractions. Second, the use of a Ca2+-freemedium in the presence of 1 mM EGTA abolished 5-HT-initiated contractions. Third, in the HUA depolarized by hK+ , high concentrations of 5-HT did not further enhance active tension (data not shown). Thus, L-type Ca2+ channels are very important for modulating +

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Ca2+ entry into the vascular smooth muscle of the HUB (see also Gopalakrishnan et al. 1985), although a smaller contribution sf intracellular Ca" release is suggested by the extracellular Ca2+ depletion data. Comparative studies with the WA demonstrate that, at both high and low Po2, nifedipine only partially suppressed 5-HT-induced contractions, suggesting a greater role for intracellular Ca2+ release in mediating 5-HT-induced contractions sf the RA versus the HUA. Furthermore, changes in Po2 did not affect the sensitivity of either the HUA or the WA to nifedipine, indicating that, in these tissues, the range of Po2 applied does not appear to modify Ca2 entry events via voltage-gated Ca2+ channels. Although we have shown that ET-1 is the most potent vasoconstrictor of the HUA so far identified and responses to ET-1 in the HUA resembled those observed in other blood vessels (Hughes et al. 1989; Stasch and Kazda 1989), a significant number of HUA preparations from "normaB" deliveries failed to respond to this peptide, suggesting that considerable biological variability to the effects of ET-1 may exist in the human vasculature. We also noted, in those tissues that responded to ET- 1, a marked tachyphylaxis to second, or subsequent, exposure to ET-1. Conceivably nonresponding HUA may have been desensitized to ET-1 in vivo. We have also noted the high sensitivity sf the HUA to 5-HT; plateletderived 5-HT and the effects of increases in HUA blood Po2 may thus contribute to the closure of the vessels at birth. In this study we have used HUA from both normal vaginal and elected caesarean section births. The possibility that varying periods of labor and also the use of analgesics -anesthetics m y have differentially influenced the behavior of the tissues thus has to be considered. In part, such factors may contribute to our reported requirement for a comparatively long preequilibration period 43 h) for tissue recovery. However, we also have noted that fresh versus 24-h stored tissues respond identically, suggesting that under the conditions of our experimental protocols, the HUA, regardless of the birth history, does not vary significantly in pharmacological behavior, the exception being that some tissues totally failed to respond to ET- 1. In conclusion these data indicate that in the HUA changes in Po2 and removal of the endothelium differentially modulate contractile responses to 5-HT agonists and ET-1. but not hK+. Under the normal low-oxygen conditions of the HUA, the release by either 5-HT or ET- I of an EDCF may contribute to the regulation sf tone in this vascular bed. Furthermore, changing Po2 may directly, or indirectly, affect ligand interactions with 5-HT receptors in the HUA. All of the modulations and consequent changes in vascular tone may be important in facilitating closure of the HUA at birth. +

The authors wish to thank the staff of the delivery room at the Foothills Hospital, Calgary, for their cooperation. The support of both the Alberta Heart and Stroke Foundation (C,R.T.) and the Pharmaceutical Manufacturers Association of Canada (H.X.) is gratefully acknowledged. Adeagbo, A. S.O., and Triggle, C.R. 1993. Varying extracellular [K+]:a functional approach to separating EDMF- and EBNOrelated mechanisms in perfused rat rnesenteric arterial bed. 3. Cardiovasc. Pharmacol. 21: 423 -429. Bjoro, K., Haugen, 6;., and Stray-Pedersen, S. 198'9. Altered prosta-

noid formation in human umbilical vasculatbare in response to variations in oxygen tension. Prostaglandins, 34: 34'7 - 384. Busse, R., Pohl, U., Kellner, C., and Klemm, U. 1983. Endothelial cells are involved in the vasodilatory response to hypxia. Pfluegers Arch. 397: '78 - 80. Busse, W., Forstermann, U., Matsuda, H., and Pohl, U. 1984. The role of prostaglandins in the endothelium-mediated vasodilatory response to hypoxia. Pfluegers Arch. 401: '77 -83. Chang, A.E., and Detar, W. 1980. Oxygen and vascular smooth muscle contraction revisited. Am. J. Phy siol. 238: H7 16-H428. Cocks, T.M., and Angus, J. A. 1983. Endothelibam-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature (London), 305: 627 -630. Eyre, P. 1975. Atypical tryptamine receptors in sheep pulmonary vein. Br. J. Bharmacol. 55: 329-333. Fleming, W .W., Westfall, D.P., De La Lande. I.S., and JelBett, L.B. 1972. Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. 9. Pkarmacol. Exp. Ther. 181: 339 -345. Furchgott, R.F. 1983. Role of endothelium in responses of vascular smooth muscle. Circ. Res. 53: 557 -573. Furchgott, R.F., and Zawadzki, 9.V. 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature (London), 288: 373 -376. Gopalakrishnan, V., Park, L.E., and Triggle, C.R. 1985. The effect of the calcium channel agonist, Bay K-$644 on human vascular smooth muscle. Eur. J. Pharmacol. 113: 447-451. Hoyer, D. B 989. 5-Hydroxytryptamine receptors and effector coupling mechanism in peripheral tissues. In The peripheral actions of 5-hydroxytryptamine. Edited by J.R. Fozard. Oxford Medical Publications, Oxford. pp. 72 -99. Hughes, A.D., McG. Thorn, S.A., Wodall, N., Schachter, M., Hair, W.M., Martin, G.N., and Sever, P.S. 1989. Human vascular responses to endothelin-l: observations in vivo and in vitro. J. Cardiovasc. Pharmacol. 136Suppl. 5): S225 - S228. Ignarro, L.3. 1989. Biological actions and properties of endotheliarranderived nitric oxide formed and released from artery and vein. Circ. Wes. 65: 1 -21. Katusic, Z.S., and Vanhoutte, P.M. 1989. Superoxide anion is an endothelium-derived contracting factor. Am. J . Phy siol . 257: H33 -H37. Kwan, Y.W., Wadsworth, R.M., and Kane, K.A. 1989a. Effects of hypoxia on the pharmacological responsiveness of isolated coronary artery rings from the sheep. Br. J. Pharmacol. 96: $49-856. Kwan, Y.W., Wadsworth, R.M., and Kane, #.A. 1989b7..Hypoxiaand endothelibam-mediated changes in the pharmacological responsiveness of circumflex coronary artery rings from the sheep. Br. J. Pharmacol. 96: 85'7 - 863. Lachenmayer, L. 1971. Adrenergic innervation of the umbilical vessels. Light and fluorescence microscopic studies. Z. Zellforsch. 828: 120- 136. kuscher, T.F., Boulanger, C.M., Dohi, Y., and Yang, Z.M. 1992. Endothelium-derived contracting factors. Hypertension (Dallas), 19: 117-130. MacLennan, S.J., Whittle, M.J., and McGrath, J.C. 1989. 5-HT,-like receptors requiring functional cyclo-oxygeanase and 5-HT,receptors independent of cyclo-oxygenase mediate contraction of the human umbilical artery. Br. J. Pharmacol. 97: 92 1833. McGrath, J.C., MacLennan, S.J., Mann, A.C., Stuart-Smith, K . , and Whittle, M.J. 1986. Contraction of human umbilical artery, but not vein, by oxygen. 3. Physial. (London), 386%:5 13 -519. Monuszko, E., Halevy, S., Freese, K.J., Shih, H.J., Parikh, N.S., Jelveh, Z., kiu-Barnett, M., and Cybulska, J. 1990. Umbilical vessels endothelium and vascular reactivity. Microcirc. Endsthelium Lymphatics, 6: 183-288. Reiffenstein, R. J., and Triggle, C .Ha. 1974. Cocaine-induced supersensitivity in the human umbilical artery. Can. J. Physiol. Pharmacol. 52: 687 - 488.


XHE AND TRHGGLE

Roach, M.R. 1972. A biophysical look at the relationship of structure and function in the umbilical artery. lri Proceedings of the Varcroft Symposium. Cambridge University Press, Cambridge, U .K. pp. 141-163. Rmbanyi, G.M., and Vanhoutte, P.M. 1985. PIypoxia releases a vascxonstrictor substance from the canine vascular endothelium. J. Physisl. (London), 364: 45 -56. Stasch, J .P.,and Kazda, S. 1989. Endothelin-1-induced vascular contractions: interactions with drugs affecting the calcium channel. J. Cardiovasc. Bharmacol. 134Suppl. 5) : S63 - S66. Templeton, A.G.B., McGrath, J.C., and Whittle: M.J. 1941. The role of endogenous thromboxane in contractions to U46619, oxygen, 5-HT and 5-CT in human isolated umbilical artery. Br. J . Pharmacol. 1433: 1079- 1084.

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Van de Voorde, J., Vanderstichele, M., and Leusen, I. 198'7. Release of endothelium-derived relaxing factor from human umbilical vessels. Circ. Res. 60: 51'7-522. Wulf, PI. 1964. The oxygen and carbon dioxide tension gradients in the human placenta at term. Am. J. Obstet. Gynec. $8: 38-44. Xie, PI. 1994. Human vascular reactivity. Ph.B. thesis, The University of Calgary, Calgary, Alta. Xie, W.,and Triggle, C.R. 1994. Endsthelium-independent relaxations to ACh and A23 18'7 in the human umbilical artery. J. Vasc. Wes. 31: 92-105. Zifa, E., and Fillion, 6;. 1992. 5-Hydroxytryptamine receptors. Pharmacol . Rev. $4: 40 1 -45 8.