Cultured endothelial cells from distinct vascular ...

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Cultured endothelial cells fro~ndistinct vascular areas show differentid responses to agonists N . WOODLEY~ A N D J.K. BARCLAY School of Numn Biology, Universify of Guelph, Guelph, ON N%G 2W%, Canada Received February 16, 1994 WQQDEEY, N., and BARCLAY, J.K. 1994. Cultured endothelial cells from distinct vascular areas show differential responses to agonists. Can. J. Physiol. Pharmacol. 72: 1007- 1012. We compared the ability of cultured endothelid cells isolated from rabbit aorta, vena cava, ventricular chamber, and pulmonary microvasculature to produce relaxing factor(s) in response to acetylcholine (ACh) and bradykinin @K). Endotheliumdenuded rabbit aortic rings were precontracted with 1 yM phenylephrine and superfused at 2 mL/rnin with Krebs -Henseleit bicarbonate buffer. Rings were exposed to 3-mL bolus control challenges of 1 yM ACh or 1 pM BK. Boluses of ACh or BK were added to dishes of cultured endothelid cells that had been incubated for 45 min in media either with or without 10 C1M[ NG-nitro-L-arginine (NNLA). The resulting solution was applied over the rings within 8 s. Only left ventricular endothelid cells stimulated with ACh and BK, and pulmonary microvascular endothelial cells stimulated with BK produced products that relaxed rings by approximately 6 f 2 % . Incubation with NNLA attenuated these relaxations. Our findings indicate there are differences in the abilities of endothelid cells of different anatomical origins to release nitric oxide derived relaxing factors in response to ACh and BK. Key words: endothelium-derived relaxing factor, aorta, in vitro bioassay, acetylcholine, bradykinin, superoxide dismutase, sodium nitropmsside. WOODLEY, N., et BARCLAY, J.K. 1994. Cultured endothelial cells from distinct vascular areas show differential responses to agonists. Can. J. Physiol. Phamacol. 72 : 1007- 1012. Nous avons compare la capacitC des cellules endothtliales isolks de l'aorte, de la veine cave, de la chambre ventriculaire et du systkme microvasculaire pulmonaire du lapin de produire un ou des facteurs de relaxation en rCponse B 1'acCtylcholine (ACh) et B la bradykinine (BK). Des anneaux aortiques de lapin dCpouillCs d'endothklium ont kt6 pr6contractCs avec 1 yM de phCnylCphrine et suprffisCs B 2 Hlm&/minavec une solution tampon bicarbonatke de Krebs -Henseleit. L a anneaux ont CtC exgodes B des Cpreuves tkmoins d'embols de 3 mL de 1 yM d'ACh ou 1 pM de BK. Les embols d'ACh et de BK ont CtC ajoutCs B des cultures de cellules endothklides qui avaient Ctk incubCes pendant 45 min dans des milieux contenant ou non 10 pM de NG-nitro-L-argirnine(NNLA). La solution ainsi obtenue a CtC rkpandue sur les anneaux en moins de 8 s. Seules les cellules endothCliales ventriculaires gauches stimulkes avec ACh et BK et les cellules endothCliales microvasculaires pulmonaires stimulkes avec BK ont donnC des produits qui ont relaxi les anneaux d'approximativement 6 f 2 R . L'incubation avec la NNLA a attCnuC ces relaxations. Nos rdsultats indiquent qu'il existe des diffkrences dans la capacitC des cellules endothkliales de divers sites anatomiques de libCrer des facteurs de relaxation dCrivCs de l'oxyde nitrique en rCpsnse B 19ACh et B la BK. Mots clds : facteur de relaxation dCrivC de l'endothklium, aorte, dosage biologique in vitro, acetylcholine, bradykinine, superowyde dismutase, nitropmssiate de sodium. [Traduit par la RCdaction]

Introduction In 1980, Furchgott and Zawadski. demonstrated that the endohelium of an intact aorta segment was capable of releasing endothelium-derived relaxing factor (EDRF). Five years later, Cwks et al. (1985) demonstrated that cultured endothelial cells released an EDW. The cultured endothelial cells used in bioassay experiments for EBRF have generally been obtained from bovine or porcine thoracic aortae (Cocks et al. 1985; D'Brlems-Suste et d.1989; Hartmm et al. 1987; Kondo et d. 1989; fivers et d . 1990). Recently Koller et al . (199 1) reported that cdturd microvascular endothelial cells from rat epididymal fat pads challenged with acetylcholine (ACh) released an EBRFlike substance that dilated the arterioles of rat cremaster muscle. Thus, cultured endothelial cells from both conduit and microvessels are capable of releasing an EBRF(s). The aim of the present study was to test the hypothesis that endoakelid cells, regardless of their vascdar origin, are capable of releasing relaxing factors in response to known EDRF agonists. To address this hypothesis, we cultured rabbit endothelial cells isolated from several anatomical locations (aorta, vena cava, chamber of the left ventricle, and pulmonary 'Author for correspondence. Rinted in Canada 1 Imprim6 au Canada

microvessels) and assessed whether they could produce vasoactive factors in response to ACh and b;adykinin (BK),which would relax endothelium-denuded rabbit aortic rings.

Methods All experimental procedures were approved by the University of Guelph institutional animal care and use committee and conducted in accordance with the American Physiological Society's Guiding Principles in the Care and Use of Animals.

Isolation and cultivation of encfethekial cells New Zealand white rabbits of both sexes weighing 1-6-2 -3 kg were euthanized with 2-mL injections of sodium pentobarbitol(65 mg/mL) administered into the right ear vein. The lungs, heart, thoracic aorta, and thoracic portion of the inferior vena cava were excised and placed in a sterile beaker containing approximately 15 mL of sterile supplemented Dulbecco's calcium- and magnesium-free phosphate-buffered saline. The composition of this solution was (in mM) KCI, 2.68; KW,P04, 1.47; NaCl, 136.89; Na,HP04 7H,O, 8.06; anhydrous L-glutamine, 200; and anhydrous D-glucose, 14; supplemented with 20% nutrient mixture F-10 (HAM) (Gibco, Grand Island, N.Y.), 0.4% bovine albumin (fraction V) (Sigma Chemical, St. Louis, Mo.), and adjusted to pH 7.36-7.40 at 37°C with 1 M sodium hydroxide (Simionescu and Simionescu 1978). The aorta and vena cava were clipped away from the heart, cleared

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of excess fat and connective tissue, cut into strips, and placed into flasks containing 20 mL of collagenase solution (CLS, 200 units/mg, Worthington Biochemical, Freehold, N .J .), (Carsen and Haudenchild 1986; Simionescu and Simionescu 1998). The bulk of the lungs was trimmed away from the esophagus, trachea, large bronchi, and major blood vessels, and the techniques described by Gerritson and Cheli (1983) for the isolation of microvessel endothelid cells were utilized. The prepared tissue was placed into a flask containing 20 mL CLS. The heart was rinsed out with supplemented Dulbecco's calciumand magnesium-free phosphate-buffered saline via a cannula placed into the left atrium. CLS was injected into the left ventricle, and the heart was suspended within the mouth of a 125-m&Erlenmeyer flask. The flask containing the heart was incubated in a water bath, and the flasks containing the other tissues were incubated in a Dubnoff metabolic shaking incubator, using moderate agitation. The aorta, vena cava, and heart were incubated with CCLS for 20 min, and the pulmonary tissue was incubated for 1 h. All solutions used for the isolation and cultivation of endothelial cells were filter sterilized and kept at 37°C. Following the incubation, CLS within the left ventricle was expelled into a beaker by inverting and gently squeezing the heart. CLS was separated from the pulmonary mince by filtration through an 80-pm nylon mesh filter and rinsing the retentate with supplemented Dulbaco's calcium- and magnesium-free phosphate-buffered saline. Ten-millaitre aliquots from each flask were pipetted into 15-EL glass centrifkage tubes and centrifuged at 120 x g for 7 min. The supernatant was discarded, the tissue pellets were resuspended in supplemented Dulbecco's calcium- and magnesium-free phosphatebuffered saline, and the tubes were centrifuged again. The same procedure was repeated twice in order to rinse away any remaining CLS, and each final pellet was resuspended in 8 mL of bicarbonatebuffered Dulbecco's modified Eagle's medium (pwder, high glucose; Gibco) containing 18% rabbit semm (Flow Lab Inc., McLean, Va.), 1% antibiotic-mtimycotic solution (Sigma Chemical), and gentamycin sulphate (50 mg/L; Sigma Chemical). Each tube was emptied into a separate 60 X 15 m Falcon Primaria culture dish, which has a modified plastic matrix that facilitates endothelial cell attachment and inhibits fibroblast attachment and proliferation. All dishes were maintained at 37°C' 5% CO, - 95% air, in a Queue incubator. The culture medium was replaced 24 h after initial cell seeding and then twice per week hereafter. Initial cell confluency of endothelial cells from all cell sources occurred within 14 days. At confluency the cells were lifted by exposure to a solution of tqpsin (0.05%) EDTA (0.02%) (Flow Labs) for 28 min (Carson and Haudenchild 1986). Each dish was subdivided into four new dishes, which in turn became confluent within 3 days. Confluent dishes of cell passages 2-7 were used in bioassay experiments. Cell ident@ccaeion Endothelid cells were identified (i) by their typical cobblestone morphology when viewed under an Olympus inverted microscope at 2 0 0 ~magnification and (ii) by the uptake of acetylated low density lipoprotein labelled with I , 1'-diochdecyl-3J.3 ' ,3 '-tetramethylindocarbcymine perchlorate (DiI-Ac-LDL) (Biomedical Tech., Stoughton, Mass.) (Carson and Haudenchild 1986; Marks et al. 1985; B o ~ c h et al. 1990). For DiH-Ac-LDL uptake, endothelial cells from all cell sources were seeded onto sterile 2 2 - m glass cover slips placed within the culture dishes. At confluency the regular medium was replaced with medium containing 10 pg/mL DiI-Ac-LBL and incubated for 4 h at 37°C. The cover slips were removed from the culture dishes; rinsed three times in phosphate-buffered saline (I 85 mL of 35.33 H-ra%%a NaHPO,, 65 mL of 66.72 mM KH2P04, and 250 mL of 145.45 mM NaCl; pH 7.4 at 23"C), with each wash lasting 5 min; and fixed in 3 % formaldehyde - phosphate-buffered saline for 20 min at 23 OC. Each cover slip was -dipped in distilled water, then mounted onto slides with Aqua-mount, allowed to dry, and examined for fluorescence under a Zeiss microscope equipped with rhodamine excitation emission filters. Controls for autofluorescence used fixed cells that

had not been exposed to DiI-Ac-EDL; and those for nonspecific fluorescence used aortic smooth muscle cells that had been exposed to DiH-Ac-LDE under the same conditions as the endothelial cells. Bioassay system for endothelieern-derivedproducts The aortae obtained fmm rabbits were cleared of excess fat and connective tissue, and cut into rings 4 -5 m wide. The rings were denuded of their endothelium by gently rubbing the luminal surfaces with the wooden end sf a cotton swab (Furchgott and Zawadsk 1980). The rings were suspended in 10-mE glass muscle chambers with one end fixed and the other attached to a force transducer by a hollow stainless-steel rod. They were then equilibrated for B R under 1 g of tension while being superfused at 2 mL/min with oxygenated Krebs Henseleit bicarbonate buffer (Krebs) at 37°C. Atropine (10 pM), a muscarinic antagonist, and indomethacin (10 pM), a cyclo-oxygenase inhibitor (Sigma Chemical) were also included in the superhsion solution of endothelium-denuded bioassay rings. The composition of Krebs was (in mM) NaCl, 118; KCl, 4.75; CaCl,, 2.54; MgSO,, 1.18; KH2P04, 1.18; NaHCO,, 24.76; D-glucose, 11.1; and I0 units insulin The buffer was aerated with 95 % 0,- 5 ETc CO,, resulting in a pH of 7.2-7.4 Immediately following the equilibration period, the rings were contracted by the addition of 1 pM L-phenylephrine to the superfusion solution. This concentration of phenylephrine produced moderate contradons of the bioassay rings, based on preliminary dose -response studies over a range of 100 nM to 1 mM. The force was allowed to plateau before further testing was conducted. Three-dlilitre bolus challenges were delivered with a hand-held syringe through a plastic fkannel with a 2-cm neck positioned directly above the bioassay ring. Experimental prsescsl Experiments were conducted to determine whether endothelial products released from various anatomical sources could relax bioassay rings and to identify the products released (n = 29). The responses of bioassay rings to direct applications of agonists (1 pM ACh and 1 pM BK) were compared with their responses to cell products released from cultured endothelial cells (aortic, n = 6; pulmonary microvascular, n = 7; left ventricular and vena caval, n = 8) stimulated by the same agonists. These challenges were then repeated with the inclusion sf 15 units/mE of superoxide dismutase (SOD), an inhibitor of the breakdown of EDRF, in the agonist sdutions. The responses of these rings to cell products were also assessed after the incubation of endothelial cells in media containing I0 pM NG-nitro-L-arginine(NNLA), an inhibitor of EDWF, for 45 min prior to the application of ACh or BK. To obtain cell products, a 3-rnL bolus of either ACh or BK was introduced into a dish of confluent endothelial cells after the medium was discarded. The solution was withdrawn and applied over the bioassay ring. In pilot experiments (n = 10) where the time for the admimistration, withdrawal, and application of cell challenges over the bioassay rings was greater than 30 s, no effects of these challenges could be detected. Therefore, in all other experiments, the transit time was reduced as much as possible, with the total time from the introduction of the challenge into the dish to the application onto the bioassay ring being approximately 8 s. All challenges were then repeated with the presence of 164 p M methylene blue in the superhsion solution to determine whether relaxations would be inhibited. The order of agonist challenges was alternated and 6 min was allowed between each challenge. Cell challenges were conducted on confluemt dishes of endothelid cells isolated from aorta (passages 4 -7), pulmonary microvasculature (passages 3 -6), chamber sf the left ventricle (passages 3 -7), and vena cava (passages 3 -7). Controls in time controls (n = 12), each ring was expsed t s 12 challenges of either the vehicle (Krebs) (n = 4) or five sequential applications of each of the agonist controls (1 pM ACh and 1 pM BK) (n = 8)

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to determine if the response of the bioassay ring to a given challenge would change over t h e . The latter commenced and e d e d with a Krebs challenge, and the order of the agonists was alter& among rings to test for possible ordering effects. In a separate set of experiments, the direct effects of endotheliumdependent challenges (1 yM ACh and 1 pM BK with and without the inclusion of 15 unitsiml SOD) (pa = 25) and an endotheliumindependent challenge (100 rA4 sodium nitropmsside (SNP)) (n = 18) were evaluated. Control bioassay experiments using the solution resulting from the application of ACh or BK on cultured aortic endothelial cells of passages 1-4 were assessed (81 = 12). This was done to determine whether the repeated passage of cultured endothelial cells affected their ability to release relaxing factors. Additional cell controls were conducted to assess whether cell products could be released in response to the application of the vehicle control. Responses of bioassay rings to direct applications of Krebs or Krebs that had been exposed to cultured endothelial cells i m e d i ately prior to its application were assessed (n = 29). Cultured endothelid cells exposed to the Krebs challenges included aortic cells (passages 4 -7), pulmonary ~movascularcells (passages 3 -6, cells from the chamber of the left ventricle (passages 3 -7), and vena caval cells (passages 3 -7). Staining of bioassay rings The absence of intact endothelium was assessed at the end of an experiment by adapting the silver nitrate staining techniques of Poole et al. (1958). Rings were pinned open and submerged into 5 % D-glucose for 3 min. This was followed by 2.5 min in 0.25% silver nitrate; 0.5 min in 5 % s-glucose; 2.5 min in 3% cobaltous bromide and 1% ammonium bromide; 0.5 rnin in D-glucose; and 1 h in 4% formaldehyde - phosphate-buffered saline. The fixed tissue could then be mounted onto slides for examimtion under a light microscope. Statistical analyses Tension was standardized as a percent of the active tension i m e d i ately preceding a challenge. The response to each treatment atwas evaluated at 10 s and 2, 4, and 6 min for all control experiments. In experiments designed to identify possible differences in cell products released from endsthelid cells of different vascular origins, the responses of bioassay rings to each treatment were evaluated at 10, 30, 60,90, and 120 s. Data were analyzed using a two-way analysis of variance (ANOVA) and contrasts for preplanned comparisons were tested for significance at a 95 % confidence level (Sndecor and CoskKm 1985). All data are presented as mean f SE, and n indicates the number of bioassay rings tested.

Results All primary cultures reached confluency within 14 days. There were no differences among cell sources in attaining confluency in subsequent passages once they had undergone their first passage. Confluent aortic, pulmonary microvascular , vena cavd, and left ventricular endothelid cells d l formed monolayers; displayed the cobblestone appearance typicd of endothelid cells; and were 98 - 100%pure as assessed by uptake of DiI-Ac-LDL. Staining of endothelium-denuded rings showed 0.9 f 0.4 % coverage (n = 9). Aortic rings developed 3 f 0.2 g tension to 1 pM phenylephrine. Controls The response of bioassay rings to repeated challenges of Krebs, ACh, or BK did not change over time or when their order of application was dtered. The results of the various control experiments are summarized in Fig. 1. There were no significant effects of direct

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FIG, 1. The effect on developed force of endothelium-denuded rabbit aorta rings of 3-rnL bolus challenges of Krebs (dB), Krebs exposed to endothelial cells (aortic, vena caval, left ventricular, and pulmonary rnicrovaxular) prior to application onto the ring (s), 1 yM ACh (A), 1 pM BK (n), and 100 nrn SNB (9). "Responses significantly different from Krebs (8).

applications of endothelium-dependent agonists (ACh, BK, ACh plus SOD, and BK plus SOD) when compared with Krebs challenges. The response of bioassay rings to Krebs that had been exposed to cultured endothelid cells from any one of the four sources prior to its application was also not different from the response to direct applications of Krebs. Therefore, the pooled response from d l four sources is presented. "Phe endothelium-independent agomist, 100 pM SNP, induced significant relaxations of the bioassay rings compared with Krebs from 30 to 120 s (Fig. 1).

Cell products In control experiments, the responses of bioassay rings to products released from cultured aortic endothelial cells stimulated by ACh or BK were independent of the number of times the cells had been passaged. It was assumed that the response to products released from endothelial cells cultured from other vascular origins would also be unaffected by serial passages of the cells. Only products released by left ventricular endothelid cells exposed to ACh produced significant relaxations of bioassay rings (Fig. 2). A relaxation of 6 f 1 % from the initial active tension occurred at 60 s, and relaxations were significantly different from the responses produced by ACh alone over the entire time frame evduated (I20 s). These responses were not altered by the inclusion of SOD in the chdlenge p i g . 2). The incubation of left ventricular endothelid cells with NNLA prior to the application of ACh greatly attenuated or eliminated relaxations of the bioassay rings to these cell products (Fig. 2). No significant changes were detected for cell p d u c t s released from pulmonary, aortic, and vena caval endothelid cells in response to ACh or ACh plus SOD. BK induced the release of products from left ventricular endothelial cells (Pig. 3) that significantly relaxed bioassay rings at BO s as compared with BK alone. The inclusion of SOD in the challenge prolonged these relaxations to 60 s, whereas the relaxations were greatly attenuated or eliminated by the incubation of the cells with NNLA prior to the application of BK. Products of pulmonary rnicrovascaalar endothelid cells induced by BK (Fig. 4) produced significant relaxations of the bioassay

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FIG. 2. The effect on developed force of endohelium-denuded rabbit aorta rings s f 3 - r d bolus challenges of 1 pM ACh (A), 1 pM ACh plus left ventricular endothelid cell products (o),1 p M ACh plus SOD (15 unitslml) and left ventricular endothelial cell products ( A ) , and 1 pM ACh plus left ventricular endothelid cell products incubated with 10 pM NNLA (0). *Responses significantly different from ACh (A).

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FIG. 4. The effect on developed force of endogkelium-denuded rabbit aorta rings of 3-mL bolus challenges of 1 pM BK (m), H pM BK plus pulmonary micmvascular endothelid cell prducts (o), 1 pM BK plus pulmonary microvascular endothelial cell products incubated with 10 yM NNLA (V), and 1 pM BK plus SOD (15 unitslml) and pulmonary microvascular endothelial cell products go). *Responses significantly different from BK (M) .

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FIG. 3. The effect on developed force of endothelium-denuded rabbit aorta rings of 3-mL bolus challenges of B pM BK (E), 1 p M BK plus left ventricular endohelial cell products (a), 1 yM BK plus left ventricular endothelid cell products incubated with 10 pM NNLA (81, and H pM BK plus SOD (25 unitslrnl) and left ventricular endothelhl cell products (n). *Responses significantly different from bradykinin (M) .

FIG. 5. The effect on developed force of endothelium-denudd rabbit aorta rings of 3-mL bolus challenges of 1 pM BK (m), B pM BK plus aortic endothelial cell products (o), H pM BK plus aortic endothelid cell prducts incubated with 18 pM NNNEA (v), and 1 pM BK plus SOD (15 units1mL) and aortic egldothelial cell products (o). *Responses significantly different from BK (H).

rings as compared with BK alone. These relaxations were present over the entire 126 s and were not altered by the inclusion of SOD in the challenge. Relaxations were not detected when the cells were incubated with NNLA prior to the application of BK. No relaxations of bioassay rings were detected to products from aortic endothelid cells induced by BK (Fig. 5). However, the inclusion of SOD in the challenge revealed significant relaxations from 30 to 60 s, as compared with BK alone. Relaxations were greatly attenuated or eliminated when the cells were incubated with NNLA prior to the application of BK. No significant changes were detected for vena cavd endothelial cell products released by BK or BK plus SOD. Responses of bioassay rings to d l challenges following the addition of methylewe blue to the superfusion solution were very variable, and no clear pattern of response was evident.

Discussion The present study demonstrated that cultured rabbit endothelial cells isolated from a variety of vascular sites differed in the release of vasodilator factors in response to ACh or BK. Verification of endotheliak cells The purity of cultured endothelid cells from d l four vascular sites (aorta, vena cava, pulmonary rnicrovessels, and left ventricle) was verified by the uptake of DiI-Ac-LDL. Only endothelial cells, monocytes, and macrophages have been shown to take up and metabolize the aeetylated form of LDL, making this an effective marker for cultured endothelial cells (Potzch et d.11990; Carsen and Haudenschild 1986). The high purity of all cultured endothelial cells used in our experiments ruled out possible contributions of relaxing factors from cells other than endothelial cells.


WOBDLEY AND BARCLAY

CQ~~~O&S Bioassay rings and cultured endothelial cells were obtained from the same animal source, which eliminated the possibility of changes attributable to species differences. Controls for the direct application of challenges showed that there were no significant responses of bioassay rings to vehicle (Krebs) or agonist (ACh and BK) challenges and that these responses were reproducible over time and were not affected by the order of application. The absence of relaxations in response to direct applications of ACh or BK confirms the lack of response of endothelium-denuded rabbit aorta rings to these agonists (Furchgott and Zawadslci 1980; B'Brleans-Juste et al. 1989; Gryglewski et al. 1986; Hartmann et al. 1987; Koller et d. 1991). Bioassay rings also did not relax to the solution resulting from vehicle application to a dish of cultured endothelial cells. This removed the possibility of relaxing factors being released in response to the physical administration of a bolus challenge, as EDRF has been shown to be released in response to the constant flow of superfusion solution (Gryglewski et al. 1986; Koller et al. 1991). The absence of relaxations in the above controls was not attributable to the smooth muscle being compromised, as bioassay rings showed marked relaxations to sodium nitropmsside. Sodium nitropmsside is an endothelium-independent agonist that spontaneously liberates nitric oxide in aqueous solution (Ignarro et al. 1981). Because direct applications of agonists did not relax bioassay rings, any relaxations following the exposure of cultured endothelial cells to these agonists were assumed to be the result of endothelium-derived vasodilator products. Also, as reported elsewhere (Cocks et al. 1985), the serial passage of cultured cells had little effect on agonist-induced relaxations. Production of vasodilator factors The solution resulting from the exposure of left ventricular endothelial cells to ACh or BK and pulmonary microvascular endothelial cells to BK relaxed the bioassay rings. This indicated the release of a relaxing factor(s) from these cultured endothelid cell sources. No relaxations were detected when ACh or BK were exposed to cultured endothelial cells isolated from rabbit aorta or vena cava. Therefore, our findings indicate that there are differences in the ability of endothelid cells to respond to ACh or BK. It is unlikely that the differences we detected in the ability of cultured endothelid cells to respond to ACh or BK were attributable to culture conditions. Although it has been reprted that cultured bovine and porcine aortic endothelial ceUs may be unable to respond to AGh as a result of damage s f the muscarinic receptors from the culturing process (Cocks et al. 1985; Hartmann et al. 1987; Schmidt et al. 1989; Tracey and Peach 1992), it has also been reported that cultured rat microvascular endothelia?. cells can respond to ACh (Koller et al. 1991). The reported differences in the ability of cultured endothelid cells to respond to AGh may be due to species and (or) vessel differences. Differential abilities of endothelium-intact vessels to release EDRF in response to ACh and BK have been previously reprted m o n g different m m d i a n species, among vessels within a given mammal, and even for the endothelium of the same vessel (Christie et al. 1989; Forstermann et al. 1986; Hgnarro et al. 1986, 1987; Gmetter and Lemke 1986). In our experiments, all endothelial cells were from the same

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animal source and were exposed to the same culture conditions. Therefore, differences in the ability of these cells to respond to ACh or BK were likely due to unique eharacteristics of the cells attributable to their different vascular origins. The relaxations we detected to products released from cultured left ventricular endothelial cells in response to ACh and BK expand previous reports of endothelium-dependent relaxations to products released from cultured ventricular endothelial cells in response to substance P (Smith et al. 1991; Schulz et al. 1991). Relaxations detected to BK-induced pulmonary microvascular endothelial cell products expands reports of relaxations from microvascular endothelial cell products in response to ACh and A23187 (Koller et al. 1991). We are not aware of any previous reports of cultured vena caval endothelial cells being evaluated. However, our findings are consistent with the variable endothelium-dependent relaxations reported for isolated preparations of systemic veins (DeMey and Vanhoutte 1982; Furchgott 1983; Furchgott and Vanhoutte 1989; Miller 1991; Seidel and LaRochelle 1987).

Nature of vasodilator factor Any relaxations detected to endothelium-derived factors were not attributable to cyclo-oxygenase products, as indomethacin was included in the superfusion solution of the bioassay rings and in the BK solution used on the cultured endothelial cells. No relaxations were detected when the time elapsed between the exposure of the cultured endothelial cells to the challenge and its application onto the bioassay ring was greater than 34) s. This indicated that the vasoactive cell product(s) had a short half-life similar to that reported for EDRF (Cocks et d. 1985; Gryglewski et al. 1986). It has been proposed that EDRF is an endogenous nitric oxide or nitrosothiol (Palmer et al. 1987; Ignarro et al. 1987). Therefore, the possibility that the products released from the cultured endothelial cells could be a nitric oxide derived product was evaluated. When cultured endothelial cells were incubated with NNEA prior to stimulation by AGh or BK, any relaxations of bioassay rings that were previously detected to resulting cell products were eliminated or greatly attenuated. NNLA has been demonstrated to be a potent inhibitor of EDRF formation-release (Ishii et al. 1990; Miller 1991;Hecker et d.1990; Schulz et al. 1991). NNEA is thought to exert its inhibition by competing with L-arginine for binding to nitric oxide synthase (NOS), thereby preventing the latter's conversion to L-citmlline and nitric oxide (Palmer et al. 1988; Palmer and Moncada 1989; Iskii et al. 1990). The removal of relaxation by incubation of the cultured cells with NNLA in the present study supprts the formation of a nitric oxide product by these cells. The possibility that a nitric oxide product was released from the cultured endothelial cells is also supported by the results of the SNP and SOD challenges. The smooth muscle of the bioassay rings showed qualitaGvely similar relaxations to products released from left ventricular and pulmonary microvascular endothelial cells as it had to the nitric oxide generated from SNP challenges. The inclusion of SOB in the BK solution prolonged the relaxations to products released from left ventricular endothelial cells and revealed relaxations to products from aortic endothelial cells that were not present in its absence. SOB, when present in solutions perfusing cultured endothelial cells, has been shown to enhance the response of bioassay rings to EBRF by inhibiting the generation of superoxide radicals, which


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inactivates EDRF and nitric oxide (Schulz et d. 1991; Hecker et d. 1992). Therefore, the results from our experiments support the release of nitric oxide derived products from cultured rabbit endothelid cells.

Conchsions We mast reject our original hypothesis and conclude that here were differences in the ability of the endsthelium to produce a relaxing factorgs) in response to h s w n EDRF agonists. Also, the endothelium-derived vasodilator f2ctor(s) was likely EDRF.

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