Endothelium-dependent relaxation in rabbit aorta after cold storage

European Journal of Pharmacology - Em'tronmental Toxicology and Pharmacology Secnon, 228 (1993) 313-319

313

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Endothelium-dependent relaxation in rabbit aorta after cold storage J o z e f T 6 r 6 k , F r a n t i ~ e k K r i s t e k a n d M o n l k a Mokrfi~ovfi Institute of Normal and Pathological Physiology, Slot,ak Academy of Sciences. Brat~slaca, SloL'ak Repubhc

Received 20 October 1992, accepted 15 December 1992

The extent of preservation of endothelial, smooth muscle and neurogenlc function following cold storage was studied in rabbit thoraoc aorta Relaxation responses to acetylchollne and sodium nltroprussade were compared between fresh aortic rings and rings that had been stored in a refrigerator for 2-8 days at 4°C In fresh aortic rings, the addition of acetylchohne to precontracted vessels resulted in dose-dependent relaxation The magmtude of relaxation was gradually decreased after 4-8 days of cold storage Relaxation in response to sodium nltroprusslde did not change Following cold storage contractions of aortic rings in response to noradrenahne and phenylephrme were not reduced Contractile responses induced by transmural nerve stimulation were gradually attenuated with the length of cold storage Electron microscopy after 4 days showed partial damage of endothelial cells (shghtly vacuohzed mltochondrla) After 8 days, endothehal cells were destroyed, only membranous material was present The structure of smooth muscle cells was only partially changed even after 8 days Sympathetic nerve endings on the 4th day were partially, but on the 8th day completely destroyed These results suggest that after cold storage of rabbit aorta, the gradual reduction of endothehum-dependent relaxation is probably caused by a decrease in production of the endothehum-derived relaxing factor due to the destruction of endothelial cells Cold storage, Endothellum-dependent relaxation, Thoracic aorta (rabbit), Electron microscopy

I. Introduction It has been demonstrated that an intact endothehum is necessary for acetylchollne to induce vasodllation; when the endothehum is removed or damaged, the relaxing effect of acetylchohne is dimlnlshed or lost (Furchgott and Zawadzkl, 1980). Therefore, fresh blood vessels with an intact endothelium are the most suitable samples for experimental studies of ~ts regulatory function There are several reasons why blood vessels, includIng human blood vessels, cannot be processed on freshly obtained vascular tissue (Liascher and Vanhoutte, 1988). Therefore, several storage methods for the preservation of vascular preparations for experimental study have been introduced The most simple would seem to be to store the vessels in a physiological solution at 4°C in a refrigerator. CryopreservatIve methods which enable one to freeze and maintain vascular tissue at - 7 0 ° or - 190°C (Muller-

Correspondence to J Torok, Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Slenklewlczova 1, 813 71 Bratislava, Slovak Republic Tel 427/562 71, 427/566 18, Fax 427/565 93

Schwemltzer, 1988; Muller-Schwemltzer and Taparelh, 1986; Mtiller-Schweznltzer et al, 1986) have also been developed These techniques make It possible to preserve the contractile functions of vascular smooth muscle for several months. Recently, Thompson et al (1989) and Ku et al. (1990) showed that cryostorage is also suitable for the preservation of endothehal cells and their endothehum-dependent vasoregulatory funcUon. The freezing methods require a cryoprotectlve agent, dlmethyl sulfoxlde, but ItS effect on vascular reactivity Is still a subject of discussion (Gottlob et al, 1982, Schoeffter and Mtiller-Schwelnitzer, 1990) Storing the blood vessels at 2-6°C preserved contractlle actiwty for several days and the responsiveness to noradrenahne and adrenaline was even potentiated (Shibata, 1969) There does not to be a report on the influence of p r o l o n g e d cold storage on the endothehal-dependent relaxation of rabbit thoracic aorta in response to dilatatory agents. The present experiments were, therefore urldertaken in order to clarify vasorelaxlng responses to endothelial-dependent (acetylchohne) and endothelial-independent (sodium nitroprusside) agents in 2 - 8 day cold-stored rabbit aortic rings at 4°C, and compare them to responses in fresh tissue.

314 2. Materials and methods

2 1 Rabbit aortic rmg preparation Albino rabbits of both sexes weighing 2 2 - 2 9 kg were killed by a blow on the back of the head and bled via carotid arteries. The thoracic aorta was removed and immediately placed in Krebs bicarbonate solution at room temperature The excess fat and connective tissue were removed from the vessel The aorta was then cut into 4 mm long rings The control group consisted of fresh aortas (with no cold storage) Other segments were stored in a beaker containing normal Krebs solution at 4°C for 2 - 8 days in a refrigerator In some experiments, the endothehum was removed by gently rubbing the internal surface of the vessel with a plexlglass rod covered with moistened filter paper The aortic rings were mounted between two plastic pins and suspended in 20 ml organ baths containing Krebs solution at 37°C, gassed continuously with 95% O 2 + 5% CO2; pH 7 2 - 7 4 Krebs solution consisting of (mM) NaC1 118, KCI 5, CaC12 2 H 2 0 2.5, MgSO 4 7 H 2 0 1 2, N a H C O 3 25, glucosa 11, C a N a z E D T A 0 03, ascorblc acid 055 The tension of the rings was recorded isometrically under a resting tension of about 20 mN with electromechanlcal transducers (Sanborn 10) and a potenhometrlc recorder (Labora, TZ 4200) The preparations were allowed to equlhbrate for 2 h in the bathing medium During this time the preparations were exposed to noradrenahne (10 -~ M) or phenylephrlne (10 -6 M) and after regaining the resting tone, the baseline tension of the rings was readjusted to 20 mN

the day of the experiment, the rings were slowly prewarmed to 37°C, then mounted in an organ bath and treated identically to the fresh preparations No gas was added to the medium during the refrigeration period The solution was changed every 48 h Some fresh and cold stored preparations before and after experimental treatment were taken for electron microscopy

2 3 Transmural nerce stimulation The aortic rings were placed between two platinum plate electrodes for electrical field stimulation of the intramural nerves Trains of pulses of 0 2 ms duration and supramaxlmal voltage were delivered at a frequency of 8 Hz for 20 s Preparations stored at 4°C were stimulated on the day of the experiment by the same parameters of stimulation as in fresh preparations All transmural nerve stimulation-induced contractile responses were eliminated by tetrodotoxln (10-6 M)

2 4 Electron mzcroscopy Following pharmacological examination, some fresh aortic rings and cold-stored rings were fLxed in 4% glutaraldehyde in 0 1 M fosfate buffer to assess the integrity of the endothelial cells, smooth muscle cells and autonomic nerve fibres After postfLxatlon in 2% osmium tetroxlde, the samples were dehydrated in alcohol and embedded in Durcupan ACM Thin sections were examined in a T E S L A BS 500 electron microscope

2 2 Experimental procedures After pretreatment with lndomethacm (10 -6 M), the rings were precontracted with a submaxlmal dose of phenylephrme (PE) or noradrenahne (NA) to produce a stable plateau of contraction The rings were then exposed to cumulative doses of acetylchohne chloride ( 1 0 - s - 1 0 6 M). Other rings from the same aortic segment were exposed to sodium nltroprusslde (10 - ~ 3 × 10 -6 M). Relaxations were expressed as a percentage of the steady state level of contractile force induced by N A or PE In addition, relaxation response to acetylchohne was also examined in rings without the endothehum and in rings treated with qulnacrme and methylene blue These drugs were added to the bath 20 mln before the addmon of noradrenahne. In some preparations, at the end of these concentration-response curves, papaverlne (300 /xM) was added to induce complete relaxation of the vascular ring. For cold storage treatment, the aortic rings were kept in the refrigerator at 4.0 + 0 5°C, for 2 - 8 days On

2 5 Drugs Freshly prepared (_+)-noradrenahne (SPOFA) was used Acetylchohne chloride, qulnacrlne chloride, methylene blue, tetrodotoxln, lndomethacln and sodium mtroprusslde were obtained from Sigma. Drugs were diluted in distilled water except for lndomethacm which was dissolved in 0.2 M Na2CO 3.

2 6 Data analysis Statistical analysis was performed by Students' t test Results are means +_ S E M. The dose of the drug (expressed as the negative logarithm of molar concentration) required to produce 50% of the maximum relaxation (EDs0) was determined from individual dose-response curves determined for each ring A value of P < 0 05 was considered to be significant

315

3. Results

3 1 Contracttle responses 3 1 1 Transmural nerve sttmulatton At the beginning of the expertment, the adrenerglc activity m each aortic preparation was tested by sttmulatton of intramural adrenergtc nerves. St~mulatton of thoractc aorta by 8 Hz created the contractile response averaging 9.39 + 0 96 mN m fresh preparattons. Thts value was gradually decreased m cold stored preparations (table 1) After 6 days the contractde response was wrtually abohshed 3.1 2. Phenylephrme and noradrenahne The contractile responses of aorttc rings induced by phenylephrme (10 -6 M) after 2, 6 and 8 days of cold storage at 4°C were not slgmficantly changed But after 4 days m cold storage, contractile responses were mcreased by 24 52% (table 1). Also, the contracttle responses to noradrenahne (10 -6 M) were increased only after 4 days m cold storage (table 1) 3.2 Relaxant responses 3 2 1 Acetylchohne-mduced relaxation After precontractton wxth phenylephrme, acetylchohne (10-8-10 -6 M) caused concentration-dependent relaxatton in fresh and m cold-stored aortic rings (fig. 1) The maximum relaxation obtained in fresh rings m response to acetylchohne was 82.2%. The relaxations were gradually reduced m preparahons stored tn the cold for 2-8 days; after 8 days' cold storage, the response was 21% of that in fresh controls

TABLE 1 Effect of cold storage on phenylephrme, noradrenahne and TNS-lnduced contraction of thoracic aorta Treatment

Control (fresh) (n) ~ 2 days cold storage (n) 4 days cold storage (n) 6 days cold storage (n) 8 days cold storage (n)

Contractde response to stxmulant mN (mean 55S E M ) Phenylephrme 10 -6 M

Noradrenallne 10 -6 M

TNS a 8 Hz

45 31 553 99 (14) 494155380 (14) 56 42 _+3 92 a (12) 48 62 553 05 (12) 42 39 + 2 81 (13)

49 50 + 2 46 (13) 5648_+545 (10) 60 26 553 31 a (10) 56 69 552 04 (10) 50 10_+ 3 19 (8)

9 39 + 0 96 (16) 441_+067 (16) 1 44 _+0 55 (14) 0 39 5=0 17 (14) 0 19 550 10 (15)

b b b b

a,t, Significant differences from the respective control values (a p < 0 05 and b p < 0 001) c (n) Number of &fferent aortic rings studied in each group a TNS Transmural nerve stimulation

i

0

*

20

~

s,**

*

*

*

40

•

60

8O

ACh (-log M) Fig 1 Cumulative concentration-response curves to acetylchohne (ACh) m thoracic aorta rings taken from fresh preparations (13, n = 12), and preparations cold-stored for 2 ( A, n = 10), 4 ( zx, n = 10), 6 (e, n = 11) and 8 ( o , n = 11) days Rings were precontracted with phenylephrlne (10 -6 M) prior to exposure to acetylchohne Relaxat~on was expressed as a percentage of phenylephrlne contrachon Data are shown as m e a n s + S E M Asterisks indicate significantly smaller relaxaUons in cold-stored preparations than in fresh (control) preparations * P < 0 05, * * P < 0 01, * * * P < 0 001

However, the endothehum-dependent relaxations on the 2nd day did not differ slgntficantly from those observed m fresh preparations. In neither fresh nor cold-stored preparations, did mdomethacm (10 -5 M) enhance the relaxattons induced by acetylchohne. Removal of the endothehum from fresh preparations taken from e~ght rabbits completely abohshed the acetylcholine-induced relaxations (data not shown). 3 2.2 Sodtum mtroprusstde-mduced relaxatton Sodtum nttroprusslde (10-9-3)< 10 -6 M) also ehcited dose-dependent relaxation of phenylephrmeprecontracted aortic rings from control and 2-8 day, cold-stored preparations ( n = 10-12). At maxamal doses, all rmgs relaxed completely. Nltroprusstde-induced relaxation, unlike that of acetylcholme, was not differ significantly between the control and the coldstored preparations. EDs0 for sodium mtroprusslde was 7.42 + 0.27 m fresh preparations and did not change significantly after 2-8 days of cold storage 3 3. Effect of qumacnne and methylene blue on acetylchohne-mduced relaxatton

Pretreatment with qumacrlne (10 -6 M), a phosphohpase A 2 inhibitor, significantly reduced the acetylchohne-induced relaxation tn noradrenahne-precon-

316

~ " - . ~,.. 20-

20

40

40 i.a

X f~

x

60-

-~ 60 ~ ¢¢

80

80-

100

100 ACh (-log M)

ACh (-log M) F,g 2 Cumulative concentratmn-response curves to acetylchohne (ACh) m thoracic aorta before (open symbols) and after addition of qulnacrme (10 - 6 M, filled symbols) taken from fresh (©) and 4 day, cold-stored ([]) preparatmns Relaxatmn was expressed as a percentage of the contraction ehclted by n o r a d r e n a h n e (10 6 M) Values are m e a n s + S E M for four different aorttc rings Slgmficantly different from corresponding control value * P < 0 05, ** P < 0 01

tracted aortic rings from the control (fresh) and 4-day cold-stored (at 4°C) samples (fig 2). Also, pretreatment of aortic rings with methylene blue (10 -5 M), an inhibitor of guanylate cyclase, inhibited the acetylchohne-mduced relaxations in control and 4 day, coldstored preparations (fig. 3) 3.4. Electron mtcroscoptc observattons Electron microscopic investigation of thoracic aorta in controls (after an experiment lasting approx. 6 h) showed continual hnmg of endothehal cells There were no observed meaningful morphological changes in endothelial and smooth muscle cells, nor In nerve fibres (fig 4A) After 4 days of cold storage, the cytoplasm of the endothehal cells was vacuohzed (fig. 4B). In some places, endothelial cells had disappeared and the internal elastic lamina was naked. The majority of smooth muscle cells were still relatwely unchanged The nerve

Fig 3 Cumulatwe concentrat,on-response curves to acetylchohne (ACh) m thoracic aorta before (open symbols) and after addLtlon of methylene blue (10 -5 M, filled symbols) taken from fresh (©) and 4 day. cold-stored ([]) preparations Relaxatmn was expressed as a percentage of the contraction ehclted by noradrenahne (10 6 M) Values are m e a n s + S E M for four different aortic rings S~gnlficantly different from corresponding control value * P < 0 05. ** P <001

terminals were partially destroyed and electron empty (fig. 4C) 8 days after cold storage the endothelial cells had largely been destroyed (fig 4D) Some of the smooth muscle ceils contain an increased number of vacuoles The cells did maintain intercellular contact with neighbourmg smooth muscle cells. The nerve termmals had been completely destroyed.

4. Discussion

The present study demonstrates that endothehumdependent relaxations m response to acetylchohne are decreased in rabbit thoracic aorta stored for 2 - 8 days in a cold environment On the other hand, the relaxatlons in response to the endothehum-independent

Fig 4 Transmission electron mlcrographs of transverse section of rabbit aorta A aorta after 6 h m pharmacological experiments Endothehal cells (E) create a continual layer separated from internal elastic lamina (L) by a narrow subendothehal space Smooth muscle cells (S) have a normal appearance Intercellular space (I) contains patches of elastm and collagen fibres Bar 1 / , m B aorta after 4 days m cold storage and after a 6 h pharmacological experiment Endothehal cells (E) contained vacuoles m cytoplasm (V), subendothehal space with d e b n s is pamally detached (D) Internal elastic lamina (L) Well preserved smooth muscle cells (S) Intercellular space (I) Bar 2 /zm C aorta after 4 days m cold storage and after a 6 h pharmacological experiment Nerve bundle on medlo-adventltlal border contains nerve fibres and varlcosmes (V) w,th an electron pale axoplasm Axolemma is interrupted at some places ( ~ ) Patches of elastm (P), collagen fibres (C) Flbroblast protrusions (F) Bar 2 ~ m D aorta after 8 days m cold storage and after a 6 h pharmacological e x p e n m e n t Endothehal cells (E) are parhally delammated from internal elastic lamina (L) Endothelial cells contain mainly m e m b r a n o u s material Sporadic b a c t e n a are present in subendothehal space ( - , ) and m tumca media Smooth muscle cells (S) are well preserved Intercellular space (I) Bar 2 / x m

317 vasoddator, sodmm nltropruss~de were preserved even after 8 days of cold storage Endothehum-derwed relaxing factor (EDRF) and

sodium mtroprusside cause relaxation by increasing cGMP m vascular smooth muscle cells (Rapoport and Murad, 1983). The preserved relaxation to sodium m-

318 troprusside in thoracic aorta would, therefore, be consistent w~th a reduced release of E D R F rather than an impaired vascular responsiveness Prolonged cold storage did not suppress the contractile response of aortic rmgs to N A and PE, but in fact contractions were even enhanced on the 4th day of cold storage These results are in agreement with the earher report by Shlbata (1969) The preservation or even enhancement of contraction is a result of various processes acting simultaneously in the vascular wall, for example: (a) increased calcium content in smooth muscle due to increase in calcium permeability (Carrier et a l , 1973); (b) decreased release of E D R F due to impairment of endothehal cells, (c) Increased sensmvity of smooth muscle in cold-denervated aortic rings due to destruction of nerve terminals, (d) a gradual impairment of mtergrlty of cell m e m b r a n e s and contractile aparatus One can speculate that the balance of these processes can maintain the relatively equal contraction of vascular smooth muscle to both PE and N A The enhancement of contraction after 4 days of cold storage could be explained by an imbalance of these processes The functional adrenerglc innervatlon of the thoracic aorta has been demonstrated in fresh preparations and has gradually attenuated in cold-stored preparations. D~sappearance of the transmural nerve shmulation-induced contractile response after 6 days of cold storage may be attributed to a reduction or lack of releasable endogenous catecholamlne resulting from adrenerglc nerve degeneration as is VlSlble in fig 4C Electron microscopy clearly reveals damage to nerve terminals on the 4th day of cold storage On the 8th day there was evident degeneration of sympathettc nerve v a n c o s m e s Similar degeneration of adrenerglc nerves was also observed m guinea pig taenia caecum (Hattori et al, 1972) Acetylchohne-induced relaxation, an effect supposedly strictly dependent on the e n d o t h e h u m in this preparation (Furchgott and Zawadskt, 1980), was markedly reduced in 4 - 8 day, cold-stored rings. This suggests that refrigerated preparations lose the ability to develop e n d o t h e h u m - d e p e n d e n t relaxation response to the muscarlnlC agomst, acetylcholine, because. - there is a decrease in E D R F production; cold-stored preparations produced, together with E D R F , e n d o t h e h u m - d e p e n d e n t constrictor factor m response to acetylcholine, the endothelial cells were impaired. The endothelial cells as well as neural cells are very fragile and susceptible to cold treatment. Inhibition of the relaxatory response by methylene blue (a guanylate cyclase inhibitor) and qulnacrme (a phosphahpase A 2 mhlbitor) was similar in both fresh and 4 day stored aortas, sugestmg unaltered enzymatic processes m

cold-stored vessels Since acute coohng does not reduce endothehum-dependent relaxation (Karakl and Nagase, 1987), the cold storage per se is probably not affecting the E D R F production Endothelial cells of cryopreserved autografts may release contractile substances (Elmore et a l , 1991) and in this way counteract the relaxatory effect of E D R F This posslbihty IS not excluded m these experiments The attenuation of acetylchohne-lnduced relaxations of precontracted aortas is probably caused by an impairment of endothelial cells W e a k relaxation m response to acetylchollne after 8 days can be explained by the presence of a decreased number of intact endothehal cells which are probably still able to produce E D R F The production of E D R F by uncovered smooth muscle cells, as mentioned by Bevan et al. (1988), is not excluded In this study, the endothehum-dependent relaxation response of preparations, stored for 2 days at 4°C, was not significantly altered These findings correspond to results obtained with freezing methods using cryoprotective agents Recently, in the rabbit central ear artery and its mare branch (Thompson et al., 1989) and the canine coronary artery (Ku et al., 1990), an endothehum-dependent relaxant response was observed after storage for several days at - 7 0 ° C and - 7 5 ° C , resp This was also observed by Ellis and Muller-Schweinitzer (1991) and Schoeffter and Muller-Schwemltzer (1991) in human pulmonary and pig coronary arteries, r e s p , after storage at - 1 9 0 ° C In studies using cryoprotective agents, however, the mechanical contractde responses of stored arteries were considerably attenuated compared to fresh tissue (Muller-Schwelnitzer, 1988, Muller-Schveinitzer and Tapparelh, 1986; Ebeigbe et al., 1988) As documentated in our results and in the results of Shlbata (1969), the contractile response of cold-stored aortas is not diminished, and therefore vessel preparations following cold storage at 4°C for up to 2 days are even more acceptable for functional studies than preparations following frozen storage.

Acknowledgement

We thank Prof Tom Heacox, Comenms UmversRy, Bratlslava, for scrutmlsmg the Enghsh translation

References

Bevan J A, E H Joyce and G C Wellman, 1988, Flow-dependent dilation m a resistance artery stdl occurs after endothehum removal Circ Res 63, 980 Carrier, Jr, O, J C Murphy and TE Tenner, Jr, 1973, Effect of cold storage on calcmm-related responses and electrolyte content of rabbit aomc strips, Eur J Pharmacol 24, 225

319 Ebelgbe. A B, E Muller-Schwemltzer and A Vogel, 1988, Effects of calcium channel blockade m canine saphenous veins after storage at -190°C, Br J Pharmacol 94, 381 Elhs, P and E Muller-Schwemltzer, 1991, Mamtenance o1 functional actwlty of human pulmonary arteries after cryopreservatlon, Br J Pharmacol 103, 1377 Elmore, J R , P GlovlczkJ, K G M Brockbank and V M Miller, 1991, Cryopreservatlon affects endothehal and smooth muscle function of canine autogenous veto grafts, J Vasc Surg 13, 584 Furchgott, R F and J V Zawadzkl, 1980, The obhgatory role of the endothehal cells m the relaxation of arterial smooth muscle by acetylchohne, Nature 288. 373 Gottlob, R , L Stockmger and G F Gestrmg, 1982, Conservation of veins w~th preservation of vmble endothehum, J Cardlovasc Surg 23, 109 Hatton, K , K Kurahashl, J Mort and S Shlbata, 1972, The effect of cold storage on the adrenerg~c mechamsms of intestinal smooth muscle Br J Pharmacol 46, 423 Karakl, H and H Nagase, 1987, Low temperature augments the endothehum-dependent relaxation m isolated rat aorta. Eur J Pharmacol 142, 129 Ku, D D , W L Wdhs and J B Caulfield, 1990, Retention of endothehum-dependent vasoddatory responses m canine coronary arteries following cryopreservatlon, Cryobiology 27, 511

Luscher. T E and P M Vanhoutte, 1988, Endothehum-dependent responses in human blood vessels, Trends Pharmacol Scl 9, 181 Muller-Schwemltzer, E , 1988, Cryopreservatlon a useful technique for storing t~ssues for pharmacological investigations Trends Pharmacol Scl 9, 221 Muller-Schwelmtzer, E and C Tapparelh, 1986, Pharmacological studies on frozen stored canine saphenous veins and basdar arteries, Naunyn-Schmledeberg's Arch Pharmacol 332, 74 Muller-Schwemltzer, E , C Tapparelh and M Vlctorson, 1986. Functional studies on human veins after storage at - 190°C, Br J Pharmacol 88, 685 Rapoport, R M and F Murad, 1983. Agomst-mduced endothehumdependent relaxation m rat thoracic aorta may be mediated through cGMP, Circ Res 52, 352 Schoeffter, P and E Muller-Schwelnltzer, 1990, The preservation of functional activity of smooth muscle and endothehum in pig coronary arteries after storage at - 190°C J Pharm Pharmacol 42, 646 Shlbata, S, 1969, Effect of prolonged cold storage on the contractile response of strips of rabbit aorta to various agents Clrc Res 24, 179 Thompson, L , J Duckworth and J A Bevan. 1989, Cryopreservatlon of innervat~on, endothehal and vascular smooth muscle function of a rabbit muscular and resistance artery Blood Vessels 26, 157