Br. J. Pharmacol. (1992), 107, 566-572

'."

Macmillan Press Ltd, 1992

Thimerosal blocks stimulated but not basal release of endothelium-derived relaxing factor (EDRF) in dog isolated coronary

artery

Peter Crack & ' Thomas Cocks Baker Medical Research Institute, Commercial Road, Prahran, 3181, Victoria, Australia 1 The effect of an acetly-coA lysolecithin acyltransferase inhibitor, thimerosal, on the release of endothelium-derived relaxing factor (EDRF) was examined in the greyhound isolated coronary artery. 2 Thimerosal (1 -10 jaM) relaxed fully, ring segments of coronary artery which were contracted with the thromboxane A2-mimetic, U46619 (30nM). The response was endothelium-dependent, slow in both onset and time to reach maximum. The maximum relaxation to the highest concentration of thimerosal (10 pM) was maintained for 10-20 min before the tissue slowly regained active force (1-2 h) to the same or higher level as that prior to the addition of thimerosal. At this time the endothelium-dependent relaxation responses to acetylcholine (ACh), substance P (SP), bradykinin (BK) and the calcium ionophores, ionomycin and A23187 were abolished. The endothelium-dependent contractions to the nitric oxide synthase inhibitors, NG-nitro-L-arginine (L-NNA; 10-100 iM) and N0-monomethyl-Larginine (L-NMMA: 10-100 JIM), however, were unaffected. 3 Thimerosal (10pIM) did not affect the relaxation curve to sodium nitroprusside (SNP) nor the contraction curve to the thromboxane A2-mimetic, U46619. 4 Both the relaxation response to thimerosal and the selective block of the relaxation responses to stimulated EDRF release were unaffected by either indomethacin (10 jM) or superoxide dismutase (150 u ml-').

5 L-NNA (100JIM) significantly blocked the relaxation curves to thimerosal and A23187 but not that to SNP. 6 Abolition of stimulated EDRF-mediated responses with thimerosal was unlikely to result from maximal and maintained stimulation of EDRF release even when active U46619-induced force had returned to pre-thimerosal levels, since the relaxation curves to glyceryl trinitrate (GTN) and SNP were markedly attenuated in the presence of SNP and GTN respectively when active force was restored with endothelin-1 (ET-1). 7 Melittin (1 JIM), ionomycin (1 JIM) and A23187 (1 JM) each had selective effects on stimulated but not basal EDRF responses, similar to those of thimerosal. 8 We propose that stimulated but not 'basal' release of EDRF is dependent on the release of arachidonic acid or one of its non-cyclo-oxygenase metabolites, possibly by Ca2'-dependent activation of phospholipase A2. Keywords: EDRF; phospholipase A2; arachidonic acid; nitric oxide; L-arginine; coronary artery; thimerosal

Introduction Endothelium-derived relaxing factor (EDRF) is now thought to be nitric oxide (NO; Palmer et al., 1987; Moncada et al.,

1991) synthesized in endothelial cells from L-arginine by an enzyme termed nitric oxide synthase (NOS; Moncada et al., 1991). Release of EDRF occurs basally and in response to a variety of chemical stimuli including receptor-specific agonists like acetylcholine (ACh) as well as the calcium ionophores, A23187 and ionomycin. Although there is strong evidence to suggest that EDRF is NO, the endothelial cell signalling mechanisms underlying its release are yet to be defined. The main NOS enzyme found in endothelial cells is the constitutive type, dependent on NADPH (Moncada et al., 1991) and Ca2+ (Lopez-Jaramillo et al., 1990). The current theory behind the signal transduction of EDRF suggests that receptor-mediated EDRF agonists such as ACh and substance P (SP), upon coupling with their respective receptors, activate phospholipase C (PLC), thereby causing an increase in myo-inositol 1,4,5-triphosphate (IP3) and

I

Author for correspondence.

diacylglycerol (DAG; see Berridge & Irvine, 1989). Intracellular free Ca2" is then increased primarily by influx of extracellular Ca2" through a receptor-regulated channel as well as mobilization from intracellular pools (see Angus & Cocks, 1989; Lopez-Jaramillo et al., 1990). This increase in intracellular Ca2" then stimulates NOS from its basal level of activity, which is also dependent on Ca2" (Lopez-Jaramillo et al., 1990), resulting in an increased rate of NO formation. Calcium inophores like A23187 and ionomycin on the other hand increase intracellular Ca2" directly without interacting with PLC. Analogues of L-arginine modified in either one of the guanidino nitrogens like N0-monomethyl-L-arginine (LNMMA) and NG-nitro-L-arginine (L-NNA) have been shown to inhibit NOS, most probably as competitive substrate inhibitors (Rees et al., 1990). The effects of these inhibitors is to raise vascular tone if basal EDRF is present and inhibit vasodilator responses to EDRF releasing agents like ACh, SP and A23187. Several pieces of evidence suggest that phospholipid metabolism may be involved in EDRF production (for review, see Angus & Cocks, 1989). Putative inhibitors of phospholipase A2 (PLA2) such as mepacrine were found to

THIMEROSAL AND EDRF RELEASE

inhibit EDRF-mediated relaxation in a range of bioassays for EDRF (Furchgott, 1981; Cocks et al., 1984; Forstermann & Neufang, 1985; for review see Angus & Cocks, 1989). Also, the bee-venom peptide, melittin, a potent stimulator of PLA2 (Schier, 1979; Zeitler et al., 1991) induces endotheliumdependent relaxation resistant to cyclo-oxygenase inhibition (F6rstermann & Neufang, 1985). Similarly, thimerosal, a comparatively specific inhibitor of acyl-CoA: lysolecithin acyltransferase (LAT) in macrophages (Goppelt-Striibe et al., 1986) has been reported to cause a long-lasting release of EDRF from both endothelial cells in situ and in culture (Forstermann et al., 1986a,b). Thus, inhibition of LAT, which has been found in endothelial cells (Forstermann et al., 1986b), would be expected to induce effects similar to stimulation of PLA2 since these two enzymes catalyze arachidonic acid release from and re-incorporation into membrane glycerolipids respectively, in particular lecithin. This cycle of arachidonic acid metabolism is known as the Lands cycle (Lands, 1960; Lands & Merkl, 1963) and its activity within endothelial cells suggests that the resultant continuous release of arachidonic acid from the phospholipid membrane may be important for the production of EDRF. Interestingly, the original hypothesis on the nature of EDRF prior to the discovery of NO and NOS centred on the role of arachidonic acid or an arachidonate metabolite in EDRFmediated vasorelaxation (for reviews see Furchgott, 1981; 1984; Angus & Cocks, 1989). The present study, confirms that thimerosal induces endothelium-dependent relaxations in situ in the dog isolated coronary artery. The relaxations to thimerosal were not sustained and tone in the artery segments slowly returned to control values within 1-2 h. The surprising finding, however, was that following recovery from the thimerosal relaxation all response to EDRF-releasing agents like ACh, SP, bradykinin (BK), A23187 and ionomycin were abolished, yet the contractions to the NOS substrate inhibitors, L-NNA and L-NMMA remained normal. Melittin, as well as ionomycin and A23187 also relaxed the coronary artery and again, following return of tone, relaxations to all EDRF-releasing agents were blocked but contractions to L-NNA and LNMMA were unaffected. From these results we propose that the Lands cycle or more specifically arachidonate metabolism may play an integral role in the regulation of EDRF release.

Methods

Isolated tissues Greyhounds of either sex (20-30 kg) were deeply anaesthetized with sodium pentobarbitone (Euthatal: 60 mg kg-1, i.v.) and their hearts removed. The circumflex coronary artery was then dissected free from the surrounding myocardium and connective tissue and placed in a modified Krebs solution (Cocks et al., 1985) which was continually gassed with 95% 02, 5% CO2 and kept at room temperature (21-23°C). The artery was then cut into 3 mm long rings with a twin-bladed scalpel holder. Some rings had their endothelium removed by gently abrading the luminal surface with a Krebs-moistened filter paper taper (Cocks et al., 1985). Each ring was then mounted on two parallel stainless steel hooks in a water-jacketed, 25 ml organ bath containing Krebs solution maintained at 37°C. The lower hook was attached to a plastic (PVC) support leg and the upper hook to a Grass force-displacement transducer. Changes in isometric, circumferential force were amplified and displayed on a flat-bed chart recorder. After an equilibration period of 60 min the rings were stretched to 4 g tension and allowed 30 min recovery after which time they were again stretched to 4 g. When a steady plateau of resting force was attained (30-40 min) the rings were contracted to a steady level of active force with the thromboxane A2-mimetic, U46619 with

567

a concentration (30 nM) which caused approximately 80% of the maximal response to U46619 (Cocks & Angus, 1984). Single concentration-responses or concentration-response curves to the compounds being tested were then obtained. At the conclusion of the experiment, the maximal relaxation to sodium nitroprusside (SNP) was obtained and all relaxation responses were then expressed as percentages of this maximal SNP response.

Statistics All relaxation curves were normalized as percentages of the maximal relaxation to SNP. Each normalized relaxation curve was then computer-fitted with a logistic equation which gave the estimates of the concentration (ECO) of the relaxing agent necessary to give 50% of the maximal response (see Nakashima et al., 1982; Angus et al., 1986). Differences between pairs of mean ECso's and mean maximal responses were tested for significance by an unpaired Students t test. A value of P< 0.05 was considered to be statistically significant.

Drugs and their source (parentheses) The following were used: U46619, (1,5,5-hydroxy-11,9(epoxymethano)prosta-5Z,13E-dienoic acid, Upjohn: Kalamazoo, U.S.A.); A23187, (Calbiochem, U.S.A.); endothelin-l (Peninsula Labs, U.S.A.); acetylcholine bromide, substance P triacetate, ionomycin, thimerosal, melittin, isoprenaline, NGnitro-L-arginine (L-NNA), indomethacin, superoxide dismutase (bovine erythrocytes) (all from Sigma, U.S.A.); N0-monomethyl-L-arginine (L-NMMA: Institute of Drug Technology, Australia); bradykinin triacetate (Fluka, Switzerland); sodium nitroprusside (Roche, Australia); glyceryl trinitrate (David Bull Laboratories, Australia).

Results

Effect of thimerosal In rings of artery contracted to a steady level of active force with U46619, thimerosal caused endothelium-dependent relaxations that were concentration-dependent for both magnitude and time to reach maximum. Low concentrations of thimerosal (0.03-1 !M) caused delayed, slowly developing relaxations over 20-40 min with maintained maxima. Higher concentrations of thimerosal (3-10I1M) caused more rapid and maximal relaxations. These relaxations, however, were not maintained. At 10 .M thimerosal, the tissue recovered its U46619-induced contraction fully over 1-2 h after maximal relaxation was maintained for 10-20 min (see Figure 1). After the tissue had relaxed maximally and recovered its U46619 active force in the presence of thimerosal (10LM), the endothelium-dependent relaxing agents, ACh, SP, BK and the divalent cation ionophore, A23187 were all unable to cause any further relaxation (Figure 1 and Table 1). The endothelium-dependent relaxation response to the calcium ionophore, ionomycin was blocked by 80 ± 7% by this concentration of thimerosal (Table 1). The contractions to the EDRF blocking agent L-NNA (10-1001iM), however, were unaffected by thimerosal (Figure 1 and Table 2). Endothelium-dependent contractions to L-NMMA (10100 gM) were also unaffected by thimerosal (data not shown). Similar effects as those found with thimerosal were obtained in further experiments with A23187 (n = 6), ionomycin (n = 6) and melittin (n = 3; see Figure 2 and Table 2). For ionomycin and A23187, the relaxation responses were endothelium-dependent but not maintained. Following return of the U46619-induced contraction, addition of other endothelium-dependent relaxing agents failed to cause further relaxations, yet the tissues were able to contract normally to L-NNA (see Figure 2 and Table 2). The same pattern of response was also seen with melittin, except the degree of

P. CRACK & T. COCKS

568

recovery over the experimental time was less than that for the other three agents (Figure 2). Melittin had no significant effect on the contraction elicited by L-NNA (Table 2). Thimerosal (10 fIM) did not cause a contraction in either

a

4g[

* U46619 30nM

b

* U46619 30 nM

c

a U46619 30 nM

Figure 2 The selective effect of ionomycin (left panel), A23187 (middle panel) and melittin (right panel) on relaxations to EDRF agents (A23187, acetylcholine (ACh), substance P (SP) and ionomycin (Iono)) in rings of endothelium-intact, dog coronary artery contracted to a steady level of force with U46619. In each case, the contraction to NG-nitro-L-arginine (L-NNA) was unaffected after recovery of the U46619 tone. The time calibration for the duration of the arrows represents 6min. b

Table 2 The effect of thimerosal, ionomycin, A23187 and melittin on the contractile response to N0-nitro-L-arginine (L-NNA) in the dog coronary artery. 4g[

Drug

L-NNA

100 FiM

41:

4 g[

1 h

* U46619 30 nM

Figure 1 Representative tracings of original chart recordings from endothelium-intact rings of dog, isolated coronary artery contracted to a steady level of force with U46619. (a) Relaxations to cumulative (-log M) additions of acetylcholine (ACh), substance P (SP) and ionomycin. The time calibration bar represents 40 and 4 min before and after the vertical arrow respectively. (b) Maximal but not sustained relaxation to thimerosal followed by total block of responses to ACh, SP and ionomycin (Iono). The tissue was still able to contract normally to NG-nitro-L-arginine (L-NNA) as compared to the time control shown in (c). The time calibration for the duration of the arrow is 6 min. Note that the break in the trace in (c) corresponds to approximately 100 min.

Table 1 The effect of thimerosal (1Op1M) on the relaxation responses to maximal concentrations of acetylcholine, substance P. bradykinin, A23187 and ionomycin in the dog coronary artery

[Agonist]

Agonist

Acetylcholine Bradykinin Substance P A23187 lonomycin

JiM

n

10.0 0.003 0.01

7 4 4 4 7

1.0 0.3

= number of rings from separate animals. Values are given as mean ± s.e.mean.

n

Thimerosal (% inhibition)

97± 1.8 100 ± 0.0 100±0.0 100± 0.0

80± 6.9

[Drug] JiM

0.003

U46619 (control) Thimerosal

I10.0

Ionomycin

1.0

A23187

1.0

Melittin

1.0

[L-NNA]

A

L-NNA

n

("M)

(% U46619)

4 9 4 9 4 5 4 5 4 5

30 100 30 100 30 100 30 100 30 100

28.9 ± 5.9 31.8 ± 4.7 26.2 ± 4.3 30.5±4.4 24.7 ± 6.6 32.8 ± 7.2 32.7 ± 5.2 34.3± 7.2 14.6 ± 6.7 20.7 ± 3.2

The contractions are expressed as percentage increases in the contraction to U46619 (30 nM). n = number of rings from separate animals. Values are given as mean ± s.e.mean.

endothelium-intact or denuded rings of artery without U46619-induced active force, whereas L-NNA (100 tiM) contracted the endothelium-intact artery but had no effect on the denuded preparation (see Figure 3). Both the relaxation and the subsequent inhibition of further EDRF(NO) release by thimerosal were uneffected by pretreatment of the tissue with either indomethacin (1O iM) or superoxide dismutase (150 u ml1') (data not shown). Concentration-relaxation response curves to thimerosal, A23187 and SNP were obtained in the presence and absence of L-NNA (100 ptM) in endothelium-intact rings of coronary artery (Figure 4). L-NNA caused an approximate 10 fold significant rightward shift in the curve to thimerosal (mean EC50's ± s.e.mean (- log M) for control and L-NNA treatment were 7.0 ± 0.1 and 6.1 ± 0.2 (n = 3) respectively; P = 0.008, unpaired t test). The maximal relaxation to thimerosal was unaffected by L-NNA. L-NNA (100 pIM) also caused an approximate 10 fold significant rightwards shift in the relaxation curve to A23187 (mean EC50's (- log M) for control and L-NNA treatment were 7.8 ± 0.1 and 6.9 ± 0.1 (n = 6 and 7) respectively; P = 0.0002, unpaired t test) although here again the maximal relaxation was not

THIMEROSAL AND EDRF RELEASE

A further series of experiments (n = 3) was carried out to assess the toxicity of thimerosal on the tissue. Cumulative concentration-contraction curves to U46619 (0.1-100 nM) were obtained 5, 30 and 60 min after the addition of thimerosal (Figure 5). The curve at 5 min was markedly depressed and then showed a time-dependent recovery which approached control responses at 60 min (Figure 5).

Plus endothelium 0

L-NNA 100 FIM

4g 10 FM

569

Nitrovasodilators 1 h

Minus endothelium

49g

0

L-NNA

10 jIM

100 jIM

1 h

Figure 3 Tracings of original chart recordings from two rings of dog coronary artery showing the effect of removal of the endothelium on the responses to thimerosal and NG-nitro-L-arginine (L-NNA). Each ring of artery was stretched twice to 4 g before the experiment was started.

b

a

Endothelium-intact rings of artery precontracted with U46619 were exposed continuously to either SNP or glyceryl trinitrate (GTN) at concentrations that caused maximal relaxations (see Figure 6). In each of three separate experiments the relaxation response recovered only poorly during maintained exposure to each of the vasodilators. A three fold higher concentration of U46619 also failed to return the active force to control levels and another constrictor, ET-1 (10 nM) was used to increase active force towards control levels. When these contractions had reached a new steady level of active force, cumulative concentrationrelaxation response to the alternative nitrovasodilators were obtained and compared to those in tissues not treated with the nitrovasodilators (Figure 6). Under these conditions of maintained stimulation with either SNP or GTN, further relaxations to the alternate nitrovasodilators were virtually abolished. The tissues, however, were able to relax sensitively to a non-nitrovasodilator, isoprenaline (Figure 6). Thimerosal (100 pLM) has no inhibitory effect upon cumulative concentration-relaxation curve to SNP (Figure 7).

100 C.)

Discussion

801

0 CU

co 0

60

\t

40

't

. .-

40

201_

I I ' *' s | . -5 '9'-11 -10 -9 -8 -7 -6 -'3 -10 -9 -8 -7 A23187 (log M) Thimerosal (log M) I

O

'1 '

The major finding from this study was that thimerosal, a compound known to inhibit lysolecithin acetylCoA-transferase (LAT), within endothelial cells (Forstermann et al., 1986a,b), first released EDRF then abolished any further response to a variety of EDRF-releasing agents like ACh and the calcium ionophores, ionomycin and A23187. The

endothelium-dependent contractions to the EDRF synthesis inhibitors L-NNA and L-NMMA, however, were unaffected. The relaxation to thimerosal was probably due to release of arginine-derived NO or a related compound, since the res-

C

100 0)

C-)

0 CU 0

80

60140 F 20 0 F-4

' - 10 -9 -8

-7 -6 -5

I4g

SNP (log M) Figure 4 The effect of NG-nitro-L-arginine (L-NNA, 0, 100 fAM) on the concentration-relaxation curve to thimerosal (a), A23187 (b) and sodium nitroprusside (SNP, c) in the dog coronary artery. Open circles (0) depict the control curve for each agent. Data points are means ± s.e.mean (vertical bars).

30 min

Figure

significantly reduced. The relaxation curve to the endothelium-independent relaxing agent, SNP was unaffected by the same concentration of L-NNA (mean EC,0's (- log M) for control and L-NNA treatment (n = 6 and 7) were 7.5 ± 0.1 and 7.4 ± 0.07 respectively; the maximal responses were not significantly different).

5 The effect of thimerosal (10 JM) on the cumulative contractions to U46619 in endothelium-intact rings of coronary artery from the dog. Contraction curves were carried out in rings not contracted previously (see Figure 3). The inset shows the time course of a typical response to thimerosal (Thim) in a ring of artery contracted with U46619 (U4) and the corresponding times at which the contraction curves to U46619 were carried out after addition of thimerosal.

570

P. CRACK & T. COCKS a

0~

~ ~ ~

-7

active 30~~

Control

~~ U4-7

cumulative

T oSPadGNrsetvl.Nt 4eaaincre iU46619 10m m

30

flm

Figure 6

Tracings of

maintained, (GTN: a)

maximal

and

original chart recordings depicting the effect of relaxing concentrations of glyceryl trinitrate

sodium

cumulative relaxation

active constrictor force

vasodilators

with

nitroprusside

curves was

higher

to

(SNP: b)

SNP and GTN

the subsequent respectively. Note

on

restored in the presence of both nitro-

concentrations

of U46619 (U4) and (ET-1). The initial U46619 contraction for the controls have been omitted for clarity. The breaks in the traces represent 20 -30 min Isoprenaline (Iso) was added to demonstrate normal endothelin-I

sensitivity of the nitrovasodilator-treated tissues to non-nitrovasodilators under the conditions of excess U46619 and endothelin.

1001

a)

801-

4-

606

.,-

401-

x

201

c:

I

-10

I

-9

I

-8

I

-7

a

-6

a

-5

SNP (log M)

IM) on the relaxation sodium nitroprusside (SNP) in rings of dog coronary artery. Open circles (0) depict the control curve for SNP. Data points are means ± s.e.mean (vertical lines). Figure 7

curves

The effect of thimerosal (0, 100

to

blocked by both L-NNA and L-NMMA. Although the block with L-NNA for thimerosal was relatively small (approximately 10 fold rightwards displacement of the EC50 at 100 fiM L-NNA with no change in range), it was nevertheless similar to that for A23187. We have previously found that EDRF-mediated relaxations in the greyhound coronary artery in vitro are relatively resistant to block by the Larginine analogues (Cocks & Angus, 1991), which probably reflects either tighter coupling or more reserve of the NOSNO/cyclic GMP transduction system in this tissue than in other tissues like the rabbit aorta (see Martin et al., 1992). ponse was

Thimerosal was unlikely to have interacted with NO directly since the relaxation curve to SNP was unaffected by thimerosal. LAT, which mediates the re-incorporation of arachidonic acid (as arachidonyl CoA) into membrane lecithin via lysolecthin constitutes one of the enzymatic arms of the Lands cycle (Lands, 1960; Lands & Merkl, 1963), the other being phospholipase A2 which catalyzes the breakdown of membrane-bound lecithin to lysolecithin and arachidonic acid. Arachidonic acid and/or its metabolites are known to interact with other transducing systems to modulate or amplify their signals. Therefore, taken together with the early evidence that EDRF was an arachidonic acid metabolite (see below), we suggest that arachidonic acid or one of its metabolites plays an important role in the regulation of stimulated EDRF release. Such an action would be not only novel, but it would also bring together much of the pre- and post-NOS/NO literature into a unified mechanism for the regulation of EDRF synthesis. Many studies on the nature of EDRF prior to the discovery of the NOS/NO signalling system (see Moncada et al., 1991) clearly suggested a role for arachidonic acid in its synthesis and/or release (see Angus & Cocks, 1989). For example, quinacrine, a supposedly specific phospholipase A2 inhibitor (Flower & Blackwell, 1976) was one of the most commonly used blockers of EDRF-mediated relaxation responses in isolated blood vessels (Furchgott, 1981; 1984; Cocks & Angus, 1984; Angus & Cocks, 1989). Also, the potent phospholipase A2 inhibitor, p-bromophenacyl bromide (p-BPB) completely and irreversibly inhibited relaxation responses of isolated vessels to acetylcholine and A23187 without affecting those to endothelium-independent relaxants like sodium nitroprusside and isoprenaline (Furchgott, 1983; Chand et al.,1987). Endothelial cell viability may be a problem with p-BPB (see Furchgott, 1983) but Chand et al. (1987) also showed that indomethacin-sensitive and endotheliumindependent relaxations of rabbit pulmonary artery were selectively blocked by p-BPB. Therefore, although it was possible that p-BPB may be toxic to endothelial cells, it selectively and potently blocked the release of prostacyclin from smooth muscle cells. Forstermann & Neufang (1985) showed that melittin, a bee venom polypeptide (Haberman, 1972) that activates release of arachidonic acid from membrane phospholipids (Schier, 1978) by stimulating phospholipase A2, and acetylcholine both caused endotheliumdependent relaxations and release of prostacyclin in rabbit isolated aortic strips. In each case, both the relaxation response and the release of prostacyclin were blocked by quinacrine and a variety of lipoxygenase inhibitors but only the generation of prostacyclin was inhibited by indomethacin (Forstermann & Neufang, 1985). These results suggest that melittin does not act at some site distal to the release of arachidonic acid such as interaction with calmodulin (Mulsch & Busse, 1991 and see below). F6rstermann and colleagues also demonstrated that thimerosal acted like melittin except that the relaxation response to thimerosal was not blocked by quinacrine whereas that to acetylcholine was (F6rstermann et al., 1986a,b). They suggested that thimerosal may interact with a thiol group, perhaps of LAT, since the relaxation response to thimerosal was completely blocked by thiol compounds like glutathione and 2-mercaptoethanol whilst that to acetylcholine was unaffected (Forstermann et al., 1986b). Thimerosal may also interact with thiol groups other than those on LAT. Thus Hecker et al. (1989) proposed that thimerosal-induced platelet aggregation occurs via oxidation of thiol groups of an internal Ca2l pool which leads to release of Ca2", activation of phospholipase A2 and subsequently release of arachidonic acid. The concentration of thimerosal used in that study, however, was 4-5 times greater than that used in ours. Also, since release of EDRF is thought to depend more on influx of extracellular calcium through a receptor-operated ion channel rather than release

THIMEROSAL AND EDRF RELEASE

from an intracellular store (Long & Stone, 1985; Griffith et al., 1986; Peach et al., 1987; Johns et al., 1987; Lodge et al., 1988; see Angus & Cocks, 1989), it is unlikely that the total block by thimerosal of relaxations to all the EDRF-releasing agonists, including the calcium ionophores, was due to block of release of calcium from an intracellular store. Another possible explanation for the effect of thimerosal on vascular endothelial cells is a direct chemical interaction between EDRF and thimerosal if, as has been suggested, EDRF is a nitrosothiol compound and not simply NO (Myers et al., 1990). The endothelium-dependent relaxation to thimerosal, combined with its total block of relaxations to EDRF-releasing agonists may indicate that if EDRF is such a nitrosothiol compound then it may be stored within endothelial cells as first suggested by Cocks & Angus (1991). Thimerosal could deplete this store by oxidizing the thiol group of EDRF, thereby releasing NO. Since thimerosal had no effect on the contractions to L-NNA and L-NMMA, basal EDRF may represent a different chemical form of EDRF, possibly NO. Others have also suggested that stimulated and basal NO release may be derived from different sources (Randall & Griffith, 1991). Block of the relaxation responses to EDRF agonists by thimerosal, melittin and the calcium ionophores was unlikely to be due to maximal, sustained release of EDRF, even when the tissues had recovered their active force. The marked inhibition of the relaxation curves to SNP and GTN in the continual presence of a maximally-relaxing concentration of GTN or SNP respectively, but with active force restored with ET-1 indicates that sustained activation of guanylate cyclase not only maintains the vessel in a maximally relaxed state which is difficult to overcome with excess amounts of vasoconstrictors, but also when active force can be partially restored, even in the presence of maximal guanylate cyclase activation, relaxation to other nitrovasodilators is blocked. Thus if thimerosal continually released EDRF maximally, it would first be difficult to reverse this relaxation and second the relaxation curve to SNP would be expected to be blocked, both of which were found not to occur. Also thimerosal did not appear to have a toxic effect on the tissue since both the endothelium-dependent contractions to the N0-arginine analogues (L-NNA and L-NMMA) as well as those to the thromboxane-mimetic, U46619 were unaffected by prolonged treatment with thimerosal. Other compounds such as A23187, ionomycin and melittin had an effect comparable to that of thimerosal. Melittin is a potent activator of phospholipase A2 and as such would be expected to cause a build-up of intracellular arachidonic acid. Part of the inhibitory effect of melittin on the release of EDRF may also be explained in terms of its ability to bind to calmodulin (see Mulsch & Busse, 1991). A calmodulin binding site has been discovered on the NOS enzyme (Bredt et al., 1991), which is further evidence that Ca2+ plays a crucial role in the production of EDRF. The divalent cationic ionophore A23187 is also known to inhibit LAT (see Mulsch et al., 1989) and thus, like thimerosal and melittin could cause an increase in intracellular arachidonic acid. It is unknown if the more selective calcium ionophore, ionomycin also inhibits LAT. Like thimerosal, however, all of these compounds released large amounts of EDRF-like material from the coronary artery which after 10-20min was not sustained and the tissue recovered its initial level of active force. From this pattern of activity of the three different classes of compounds (thimerosal, melittin and the calcium ionophores), we suggest that both the relaxation and the recovery phases of each compound's response are caused by first accumulation and then depletion of arachidonic acid from a phospholipid pool sensitive to phospholipase A2.

E cell

Agonist

R /

tfree

571

t

~~Ca

e

=

~NO Synthase

_

_

~~~~~~~~Receptor

'Lecithin

2+

N

AA

Lysolecithin)

EDRF

Figure 8 Schema for the proposed mechanism of action of thimerosal and role of arachidonic acid in regulation of endotheliumderived relaxing factor (EDRF) synthesis in endothelial cells (E cell). Abbreviations: free Arg, free levels of intracellular L-arginine; NO synthase, nitric oxide synthase; AA, arachidonic acid; PLA2, phospholipase A2; LAT, lysolecithin acylCoA-transferase.

Since L-NNA and L-NMMA were still able to contract the tissues apparently depleted of arachidonic acid, basal EDRF release does not appear to require release of arachidonic acid. There are at least two different forms of nitric oxide synthase (NOS). One, the so-called constitutive type is dependent on calcium for its activity and is the main type found in endothelial cells as well as platelets and the brain (Forstermann et al., 1991). The other, the inducible form is calcium-independent and is found in a wide variety of cells (see Moncada et al., 1991). Therefore basal EDRF may be synthesized from L-arginine by a different form of NOS from that which mediates agonist and ionophore-stimulated EDRF release (see also Randall & Griffith, 1992). Support for this comes from MUlsch et al. (1989) who demonstrated that native porcine endothelial cells synthesize EDRF from L-arginine via both a Ca2+-sensitive and Ca2'-insensitive

pathway. In conclusion, this study provides indirect evidence, that either arachidonate or one of its non-cyclo-oxygenase products may play an integral role in the regulation of EDRF release. Thimerosal, a known inhibitor of LAT in endothelial cells, blocked the relaxation responses to various EDRFreleasing agents whilst it had no affect on the contractions to the L-arginine analogue inhibitors of NOS, L-NNA and LNMMA and therefore by inference had no effect on basal

EDRF release. We hypothesize that arachidonic acid, possibly generated via activity of the Lands Cycle, either amplifies the activating effect of Ca2+ on NOS or releases more Ca2+ from intracellular stores which in turn stimulates NOS above its basal level of activity to generate NO (see Figure 8). Interestingly, arachidonic acid has been shown to cause endothelium-dependent relaxations in the rabbit aorta (Pinto et al.,1986; for review see Angus & Cocks, 1989). This finding is consistent with our hypothesis on the involvement of arachidonic acid in EDRF release. The results from the present study, however, are also compatible with the idea that stimulated EDRF is derived from a preformed 'pool', possibly as a nitrosthiol (Myers et al., 1991; Ignarro, 1991; Cocks & Angus, 1991) whilst basal EDRF is simply NO. This work was funded in part from an Institute Grant from the National Health and Medical Research Council of Australia and Glaxo Australia. We thank Dr James Angus and Dr Ian Smith for their helpful comments.

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(Received March 23, 1992 Revised June 12, 1992 Accepted June 17, 1992)