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Circulation Research. 1999;84:53-63

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(Circulation Research. 1999;84:53-63.)
© 1999 American Heart Association, Inc.

Original Contribution

Nitric Oxide–Independent Relaxations to Acetylcholine and A23187 Involve Different Routes of Heterocellular Communication

Role of Gap Junctions and Phospholipase A2

Iain R. Hutcheson, Andrew T. Chaytor, W. Howard Evans, Tudor M. Griffith

From the Departments of Diagnostic Radiology (I.R.H., A.T.C., T.M.G.) and Medical Biochemistry (W.H.E.), Cardiovascular Sciences Research Group, University of Wales College of Medicine, Cardiff, United Kingdom.

Correspondence to Prof Tudor M. Griffith, Department of Diagnostic Radiology, Cardiovascular Sciences Research Group, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK. E-mail griffith{at}cardiff.ac.uk


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—NO- and prostanoid-independent relaxations are generally assumed to be mediated by an endothelium-derived hyperpolarizing factor (EDHF) that has been postulated to be an arachidonic acid metabolite. Recent evidence also suggests that direct heterocellular gap junctional communication (GJC) between endothelium and smooth muscle contributes to NO-independent relaxations. In the present study we have investigated the contribution of phospholipase A2 (PLA2)-linked metabolites and GJC to EDHF-type relaxations in rabbit mesenteric artery. In isolated rings preconstricted with 10 µmol/L phenylephrine in the presence of NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin, acetylcholine (ACh) and the Ca2+ ionophore A23187 evoked relaxations that were markedly attenuated by the Ca2+-dependent PLA2 inhibitors 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (3 µmol/L) and arachidonyl trifluoromethyl ketone (3 µmol/L), but were potentiated by the sulfhydryl agent thimerosal (300 nmol/L). In intact rings, relaxations to ACh were attenuated synergistically by L-NAME and Gap 27 peptide, an inhibitor of GJC, whereas ACh-evoked relaxations of "sandwich" preparations were unaffected by the peptide but were abolished by L-NAME. In both ring and sandwich preparations A23187-induced relaxations were attenuated by inhibition of PLA2 but were insensitive to L-NAME and Gap 27 peptide. We conclude that EDHF-type relaxations of rabbit mesenteric artery to ACh and A23187 depend on a common pathway that involves activation of PLA2. In the case of ACh, relaxation requires transfer of a factor or factors from the endothelium to smooth muscle via gap junctions, whereas A23187 permits release directly into the extracellular space.


Key Words: EDHF • phospholipase A2 • gap junction • acetylcholine • A23187


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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The endothelium plays a central role in the control of vascular tone through the release of vasoactive autacoids in response to agonist stimulation and shear stress.1 Mediators include NO, prostanoids, and in many vessel types a distinct endothelium-derived hyperpolarizing factor (EDHF).2 3 The chemical identity of EDHF remains controversial, although it seems probable that it mediates smooth muscle hyperpolarization by activating K+ channels (see References 2 and 32 3 for review). Recent reports suggest that epoxyeicosatrienoic acids (EETs), which are cytochrome P-450 monooxygenase metabolites of arachidonic acid, and the endocannabinoid anandamide, which is also derived from arachidonic acid, possess the characteristics of EDHF in certain artery types.3 4 5 6 It has been suggested that the source of arachidonic acid for EDHF production is the Ca2+-dependent cytosolic form of phospholipase A2 (PLA2), since specific inhibitors of this enzyme attenuate NO-independent relaxations to acetylcholine (ACh), histamine, and bradykinin in the perfused rat heart and mesenteric bed.7 8

EDHF has been detected both in cascade bioassay9 10 and in sandwich preparations,11 suggesting that this factor can diffuse freely in the extracellular space. However, recent evidence suggests that NO-independent relaxations in rabbit mesenteric artery and aorta could involve the preferential direct transfer of a mediator through gap junctions, rather than via/by an extracellular route.12 13 Intercellular continuity is facilitated by gap junctions that are assembled when 2 connexon hemichannels supplied by neighboring cells interact and dock. Each connexon consists of 6 connexin protein subunits arranged around a central pore,14 and the characteristic pentalaminar appearance of sections of gap junction plaques has previously been demonstrated in rabbit conduit arteries.15 Connexin 43 is the most prevalent subtype in endothelium-denuded rabbit superior mesenteric artery, and connexins 37, 40, and 43 have been identified in endothelial cells.16 17 Previous studies have shown that the inhibitory Gap 27 peptide (amino acid sequence SRPTEKTIFII), which possesses conserved sequence homology with a region of the second extracellular loop of these connexins, rapidly and reversibly attenuates NO-independent relaxations to the agonists ACh and adenosine triphosphate and also to cyclopiazonic acid.12 Direct heterocellular communication may therefore contribute to EDHF-mediated smooth muscle relaxations evoked by receptor-dependent and -independent activation of the endothelial cell. Although the exact mode of action of Gap 27 peptide remains to be elucidated at the molecular level, possibilities include (1) inhibition or reversal of connexon docking before or after penetration of the peptide into the intercellular gap and (2) induction of a conformational change that results in channel closure.16 18

In the present study we have used isolated ring and sandwich preparations to evaluate the contributions of gap junctional communication (GJC) and PLA2 to NO-independent relaxations evoked by ACh and the calcium ionophore A23187 in rabbit superior mesenteric artery. Arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of the cytosolic form of PLA219 ; [E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-1] (HELSS), a specific inhibitor of the secretory form of PLA220 ; 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082), an inhibitor of both forms of PLA221 ; 1-(6-{[17ß-3-methoxyestra-1,3,5(10)-triene-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), a specific inhibitor of phospholipase C (PLC)22 ; and thimerosal, an organic mercury sulfhydryl reagent that is known to inhibit the acyl-coenzyme A:lysolecithin acyltransferase23 and to modulate mobilization of intracellular Ca2+ from stores,24 were used to characterize the pathways involved in the mediation of these relaxations. Gap 27 peptide was used to elucidate the predominant diffusion pathway of the putative mediator once formed. Previous studies have shown that this peptide does not directly affect ambient force development in the rabbit mesenteric artery.16


*    Materials and Methods
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 up arrowAbstract
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 *Materials and Methods
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 down arrowDiscussion
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Isolated Ring Preparations
Experiments were performed with superior mesenteric arteries from male New Zealand White rabbits (2.5 kg) that had been sacrificed by injection of sodium pentobarbitone (120 mg/kg IV). The tissues were transferred to cold Holman's solution of the following composition (in mmol/L): NaCl 120, KCl 5, NaH2PO4 1.3, NaHCO3 25, CaC12 2.5, glucose 11, and sucrose 10. Rings (2 to 3 mm wide) were cut and suspended by thread from FT102 force transducers connected to a MacLab/4e (ADInstruments) in 3-mL organ baths containing gassed (95% O2 and 5% CO2, pH 7.4) Holman's solution at 37°C to measure isometric force development. The rings were placed under a resting tension of 0.5g and allowed to equilibrate for {approx}1 hour with frequent readjustment of tension to allow for stress relaxation.

Experimental Protocol
The rings were precontracted with 10 µmol/L phenylephrine, and cumulative concentration-response curves to ACh (1 nmol/L-10 µmol/L) and the Ca2+ ionophore A23187 (0.1 nmol/L-1 µmol/L) were constructed before and after a 45-minute incubation with NG-nitro-L-arginine methyl ester (L-NAME) (300 µmol/L) alone or a combination of L-NAME and 1H-(1,2,4)-oxadiazolo-(4,3a)-quinoxaline-1-1 (ODQ, 10 µmol/L), an inhibitor of guanylate cyclase. Experiments were also performed in the presence of indomethacin (10 µmol/L) to determine the involvement of prostanoid synthesis. The role of GJC was investigated by preincubating for 20 minutes with Gap 27 peptide (300 µmol/L). To analyze the role of PLA2 and PLC in the L-NAME–sensitive and –insensitive components of the relaxations, cumulative concentration-response curves were constructed following a 45-minute incubation with ONO-RS-082 (1 to 10 µmol/L), AACOCF3 (3 µmol/L), HELSS (300 nmol/L), or U-73122 (500 nmol/L), respectively. In other studies, thimerosal (300 nmol/L) was added 20 minutes before construction of concentration-response curves to ACh and A23187 in the absence and presence of L-NAME. These experiments were then repeated in the presence of Gap 27 peptide (300 µmol/L).

To assess the vasoactivity of EETs in this preparation, cumulative concentration-response curves to 5,6-, 11,12-, and 14,15-EET (100 nmol/L to 3 µmol/L) were constructed in the absence and presence of L-NAME. In studies in which relaxation was observed, the experiments were repeated in the presence of Gap 27 peptide. Responses to ACh and A23187 were also investigated before and after a 30-minute incubation with 5 µmol/L of each of the 3 EET isomers.

Sandwich Experiments
In a separate series of experiments, rings of superior mesenteric artery were cut open and small strips of endothelium intact vessel were sutured onto strips of endothelium-denuded artery, with their intimal surfaces in close apposition, to provide composite preparations. These sandwich preparations were allowed to equilibrate at a tension of 0.6 g for {approx}1 hour before precontraction with phenylephrine (10 µmol/L), with tension monitored only in the endothelium-denuded vessel. The transferable nature of endothelium-dependent relaxations to ACh and A23187 were assessed in the absence and presence of L-NAME (300 µmol/L), ONO-RS-082 (3 µmol/L), and Gap 27 peptide (300 µmol/L).

Materials
ACh, phenylephrine, indomethacin, thimerosal, A23187, and L-NAME were obtained from Sigma. U-73122 was obtained from Calbiochem-Novabiochem; ONO-RS-082 was obtained from Alexis Corp; and 5,6-EET, 11,12-EET, 14,15-EET, HELSS, AACOCF3, and ODQ were obtained from Affiniti Research Products Ltd. Gap 27 peptide (SRPTEKTIFII) was synthesized by Severn Biotech Ltd. All drugs were dissolved in Holman's buffer with the exception of U-73122, HELSS, AACOCF3, ODQ, and ONO-RS-082 (DMSO); 5,6-, 11,12-, and 14,15-EET (ethanol); and indomethacin (5% wt/vol NaHCO3 in distilled water).

Statistics
All data are given as mean±SEM, and n denotes the number of animals studied for each data point. Concentration-response curves were assessed by 1-way ANOVA followed by the Bonferroni multiple comparisons test. EC50 and maximal responses were compared by the Student's t test for paired and unpaired data as appropriate. P<0.05 was considered as significant.


*    Results
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 *Results
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Effects of PLA2 and PLC Inhibition on ACh- and A23187-Induced Relaxations in Intact Rings
ACh and A23187 both evoked endothelium-dependent relaxations of mesenteric rings that were maximal at {approx}3 µmol/L and {approx}300 nmol/L, respectively. Addition of L-NAME (300 µmol/L) to the organ bath increased tone by 18.8±1.5% (n=20) and attenuated ACh-induced relaxations, causing an increase in the EC50 value from 0.16±0.02 to 0.76±0.11 µmol/L (P<0.001, n=25) and a reduction in the maximal response (control, 70.9±2.8%; L-NAME, 26.4±2.7%; P<0.001, n=25). The EC50 value for relaxation to A23187 was similarly increased from 50.0±1 to 134.5±30 nmol/L following incubation with L-NAME (n=21), and there was a smaller but significant reduction in maximal relaxation from 73.4±3.4% to 61.1±2% (n=21, P<0.01). These values were obtained by pooling data from all experiments. Addition of ODQ (10 µmol/L) in the presence of L-NAME had no further inhibitory effect on either ACh- or A23187-induced relaxations (Figure 1Down). Similarly, indomethacin was without effect on the EC50 values and maximal relaxations to ACh or A23187 both in the absence and presence of L-NAME (Figure 1Down). Indomethacin was included in the buffer for all remaining studies.



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  		Figure 1. Concentration-response curves for endothelium-intact rings of rabbit mesenteric artery showing that indomethacin (10 µmol/L) did not modulate relaxations to ACh (A) and A23187 (B) either in the absence (n=5 for both agents) or presence (n=4 for both agents) of L-NAME (300 µmol/L). Furthermore, ODQ (10 µmol/L) did not modulate the L-NAME–insensitive relaxations to either ACh (A) or A23187 (B) (n=3 for both agents).

In the absence of L-NAME, inhibition of PLA2 with ONO-RS-082 (3 µmol/L) had no effect on ACh-induced relaxations (Figure 2ADown) in terms of either the EC50 value (control, 0.18±0.05 µmol/L; ONO-RS-082, 0.18±0.09 µmol/L; n=5) or maximal relaxations (control, 73.5±2.4%; ONO-RS-082, 72.7±4.2%; n=5) but increased the EC50 values for A23187 from 50±0.1 to 340±80 nmol/L (P<0.01, n=4, Figure 2BDown), although there was no significant effect on maximal relaxations (control, 70.2±3%; ONO-RS-082, 64.5±3.3%). There was also no effect of AACOCF3 (3 µmol/L) on ACh-induced relaxations in terms of either the EC50 value (control, 0.16±0.01 µmol/L; AACOCF3, 0.13±0.03 µmol/L; n=3 for both) or maximal relaxations (control, 70.8±4.7%; AACOCF3, 72.5±2.1%; n=3 for both), but this agent significantly increased the EC50 value for A23187 from 51±0.3 to 122±29 nmol/L (P<0.05, n=3) and caused a small but not significant reduction in the maximal response from 67.3±2.9% to 53.3±7.9% (n=3). In the presence of L-NAME, ONO-RS-082 almost abolished ACh-induced relaxations, with maximal responses being reduced from 37.7±4.6% to 8.8±3% (P<0.01, n=8, Figure 2ADown). AACOCF3 also virtually abolished relaxations to ACh, with maximal responses being reduced from 26.4±3.2% to 6.6±1.4% (P<0.01, n=4, Figure 2ADown). Furthermore, both ONO-RS-082 and AACOCF3 significantly attenuated the maximal L-NAME–insensitive relaxations to A23187 from 54.2±7% to 23.4±7.2% (P<0.01, n=5, Figure 2BDown) and from 66.5±3.4% to 27.3±7.8% (P<0.05, n=6, Figure 2BDown), respectively. HELSS (300 nmol/L) had no effect on ACh-induced relaxations in the presence of L-NAME either in terms of the EC50 value (control, 0.18±0.03 µmol/L; HELSS, 0.27±0.07 µmol/L; n=4) or maximal response (control, 23.1±5.1%; HELSS, 24.4±6%; n=4). Similarly, HELSS was without effect on the L-NAME–insensitive responses to A23187, with EC50 values (control, 108±21 nmol/L; HELSS, 93.5±32 nmol/L; n=4) and maximal responses (control, 66.5±3.4%; HELSS, 60.5±9.5%; n=4) being unchanged.



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  		Figure 2. Concentration-response curves showing the effects of PLA2 and PLC inhibition on ACh- and A23187-induced relaxations in endothelium-intact rings. A, L-NAME (300 µmol/L) inhibited responses to ACh by {approx}60%, whereas ONO-RS-082 and AACOCF3 (3 µmol/L and n=4 for both) alone were without effect. In the presence of L-NAME, however, ACh-induced relaxations were almost abolished by ONO-RS-082 (n=8) and AACOCF3 (n=4) but were unaffected by HELSS (300 nmol/L, n=4). B, ONO-RS-082, AACOCF3, and L-NAME individually reduced the potency of A23187, but the small reductions in maximal relaxation were not statistically significant. In the presence of L-NAME, ONO-RS-082 (n=5) and AACOCF3 (n=7) markedly attenuated relaxations to A23187, whereas HELSS was again without effect (n=4). C, The PLC inhibitor U-73122 (500 nmol/L) almost abolished the L-NAME–insensitive component of responses to ACh (n=4). D, The L-NAME–insensitive component of A23187-evoked relaxations was unaffected by U-73122 (n=5).

Preincubation with U-73122 (500 nmol/L), a specific PLC inhibitor, in the presence of L-NAME attenuated relaxations to ACh, with EC50 values being increased from 0.36±0.12 to 1.06±0.3 µmol/L and maximal relaxations reduced from 29.6±7.6% to 10.2±3.1% (P<0.05, n=4, Figure 2CUp). U-73122 had no significant effect on the L-NAME–insensitive response to A23187 (Figure 2DUp).

Effects of Gap 27 Peptide on ACh- and A23187-Induced Relaxations in Intact Rings
Preincubation of rings with Gap 27 peptide (300 µmol/L) had no effect on phenylephrine-induced tone (not shown) but significantly attenuated relaxations to ACh, reducing the maximal response from 81.3±4% to 58.8±5.5% (P<0.01, n=8) and causing a rightward shift in the concentration-response curve and an increase in the EC50 value from 200±20 to 480±80 nmol/L (P<0.01, n=8, Figure 3ADown). By contrast, A23187-induced relaxations were completely unaffected by pretreatment with Gap 27 peptide (n=4, Figure 3BDown), with no change in either EC50 values (52±3 to 50±4 nmol/L) or maximal responses (75.9±6% to 67.5±2%). In the presence of L-NAME (300 µmol/L), Gap 27 peptide significantly attenuated the residual relaxation to ACh, causing a further increase in the EC50 value from 281±93 to 428±52 nmol/L (n=4) and a significant reduction in the maximal response from 54±3.2% to 33.3±4.9% (P<0.05, n=4, Figure 3ADown). Gap 27 peptide was without effect on the L-NAME–insensitive component of the relaxations to A23187 either in terms of the EC50 values (54±6 to 58±8 nmol/L, n=4) or maximal response (61.5±1.3% to 61.8±4.1%) (n=4, Figure 3BDown). Figure 3CDown illustrates that for relaxations greater than {approx}20%, the time taken from onset to maximum relaxation was substantially longer for A23187 than for ACh in the presence of L-NAME.



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  		Figure 3. Concentration-response curves showing the effects of L-NAME and Gap 27 peptide on relaxations to ACh and A23187 in endothelium-intact rings. A, Both L-NAME and Gap 27 peptide individually attenuated ACh-induced relaxations, and their inhibitory effects were synergistic (n=4 and n=8, respectively). B, L-NAME caused a small but significant reduction in A23187-induced relaxations, but Gap 27 peptide was without effect (n=4 for both agents). C, Scatter plot of the relationship between magnitude of relaxation and time taken to reach its maximum for different concentrations of ACh and A23187 in the presence of L-NAME. This demonstrates that for equivalent responses, relaxation was up to twice as slow for A23187 than for ACh.

Correlations With the Initial Relaxant Response to ACh
Figure 4Down illustrates that in any given preparation there was an inverse relationship between the magnitude of the initial relaxations to ACh and those subsequently obtained in the presence of L-NAME. Linear regression analysis confirmed a negative correlation between maximal responses to ACh in the presence and absence of L-NAME (P<0.01, n=19, r=0.60). There was a direct relationship between the magnitude of the initial relaxations to ACh and those subsequently obtained in the presence of Gap 27 peptide, with a positive correlation coefficient being confirmed by linear regression analysis (P<0.001, n=8, r=0.94).



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  		Figure 4. A and B, Representative traces demonstrating an inverse relationship between initial ACh relaxations and the subsequent L-NAME–insensitive component of the ACh response. C, Negative correlation between maximal responses to ACh in the absence and presence of L-NAME (P<0.01, n=19, r=0.60). D, Positive correlation between maximal responses to ACh in the absence and presence of Gap 27 peptide (P<0.001, n=8, r=0.94).

Effects of Thimerosal on ACh- and A23187-Induced Relaxations in Intact Rings
Thimerosal (300 nmol/L) did not affect phenylephrine-induced tone (not shown). Incubation of rings for 20 minutes with this agent, in the absence of L-NAME, enhanced relaxations to ACh (Figure 5ADown), with a significant reduction in EC50 values from 0.11±0.03 µmol/L to 21±0.3 nmol/L (P<0.05, n=5) but no effect on maximal responses (control, 73.5±5.5%; thimerosal, 76.6±5.3%; n=5). Gap 27 peptide (300 µmol/L) had no effect on the thimerosal-induced enhancement in terms of the EC50 value (25±0.4 nmol/L, n=4) but significantly reduced the maximal relaxations to 51.9±2.5% (n=4). Similarly, A23187-induced relaxations were enhanced by thimerosal in the absence of L-NAME (Figure 5BDown), with EC50 values being reduced from 70±9 nmol/L to 33±10 nmol/L (P<0.05, n=4) and the maximal response showing a small, but not significant, rise from 75.1±8.7% to 86.2±4.3% (n=4). In the presence of L-NAME, thimerosal significantly reduced the EC50 values for both ACh (from 0.51±0.13 to 0.13±0.05 µmol/L, P<0.05, n=10) and A23187 (from 0.18±0.06 to 0.083±0.03 µmol/L, n=6, Figure 5CDown and 5DDown). Furthermore, thimerosal increased the maximal response of the residual relaxations to both ACh (from 29.8±8.1% to 42.1±6.1%, P<0.05, n=10, Figure 5CDown) and A23187 (from 56.9±3.5% to 70.4±3.5%, P<0.05, n=5, Figure 5DDown).



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  		Figure 5. Concentration-response curves showing the effects of thimerosal and Gap 27 peptide on ACh- and A23187-induced relaxations in endothelium-intact rings. A and B, Thimerosal (300 nmol/L) significantly increased the potency of ACh (n=5) and A23187 (n=4) but did not affect maximal relaxations. Gap 27 peptide (300 µmol/L) did not reverse the thimerosal-induced increase in potency to ACh but significantly reduced the maximal relaxation. C and D, Thimerosal significantly enhanced L-NAME–insensitive relaxations to both ACh (n=10) and A23187 (n=6). Gap 27 peptide significantly reduced the thimerosal-enhanced relaxations to ACh but not to A23187 (n=4 for both agents).

Concentration-response curves to ACh in the presence of L-NAME (300 mmol/L) and thimerosal (300 nmol/L) showed significant reductions in maximal responses to 18.8±4.9% (n=4) in the presence of Gap 27 peptide (300 µmol/L) although EC50 values remained similar at 0.17±0.05 µmol/L (n=4, Figure 5CUp). A23187-induced relaxations observed in the presence of L-NAME and thimerosal were not significantly affected by Gap 27 peptide in terms of either the EC50 value (35±17 nmol/L, n=4) or maximal response (66.9±2.1%, n=4, Figure 5Up).

Sandwich Preparations
Relaxations of sandwich preparations to ACh exhibited an EC50 of 1.1±0.27 µmol/L and a maximal relaxation of 21.8±2.7% of phenylephrine-induced tone (n=4, Figure 6ADown). In marked contrast to the intact ring studies, ACh-induced relaxations were almost abolished in the presence of L-NAME, whereas preincubation with Gap 27 peptide was without effect (Figure 6ADown). A23187 caused concentration-dependent relaxations of the sandwich preparations, with maximal relaxations constituting 25.4±3.0% of developed tension with an EC50 value of 0.24±0.09 µmol/L (n=8, Figure 6BDown). In contrast to intact ring studies, preincubation with L-NAME had no effect on A23187-induced relaxations, whereas ONO-RS-082 had no effect on the EC50 value (0.16±0.08 µmol/L, n=4) but markedly attenuated the maximal relaxation to 11±5.8% (P<0.05, n=4, Figure 6BDown). As in the case of ACh, Gap 27 peptide was without effect on either L-NAME–sensitive or –insensitive relaxations to A23187 in sandwich preparations (Figure 6BDown).



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  		Figure 6. Concentration-response curves showing the effect of L-NAME, Gap 27 peptide, and ONO-RS-082 on relaxations to ACh and A23187 in sandwich preparations of rabbit mesenteric artery. A, L-NAME, but not Gap 27 peptide, markedly attenuated the relaxations to ACh (n=4). B, ONO-RS-082 significantly reduced A23187-evoked relaxations (n=4), whereas L-NAME, Gap 27 peptide, and their combination were each completely without effect (n=4 for all curves).

Vascular Effects of EETs
The effects of 3 EET regioisomers, 5,6-, 11,12-, and 14,15-EET, were examined in rabbit superior mesenteric arterial ring preparations. Only 5,6-EET evoked concentration-dependent relaxations of endothelium-intact rings that were abolished following endothelial denudation of the vessel (Figures 7Down and 8Down). Addition of either L-NAME (300 µmol/L) or Gap 27 peptide (300 µmol/L) significantly attenuated these endothelium-dependent responses (P<0.05 [n=4], and P<0.05 [n=6], respectively), and in combination L-NAME and Gap 27 peptide abolished 5,6-EET-induced relaxations (n=4, Figures 7Down and 8ADown). Preincubation of ring preparations with either 5,6-, 11,12-, or 14,15-EET (5 µmol/L for each agent) had no effect on either ACh- or A23187-induced relaxations or maximal relaxations (n=4 for each agent, Figure 9Down).



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  		Figure 7. Representative traces demonstrating relaxations to 5,6-EET in endothelium-intact preparations (A), endothelium-denuded preparations (B), endothelium-intact preparations in the presence of L-NAME (300 µmol/L) (C), and endothelium-intact preparations in the presence of Gap 27 peptide (300 µmol/L) (D). Relaxation was slightly attenuated by L-NAME but almost abolished by Gap 27 peptide. Transient constriction after drug administration was attributable to the vehicle (ethanol).



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  		Figure 8. Effects of 3 regioisomeric EETs, 5,6-EET, 11,12-EET, and 14,15-EET, on vascular tone in endothelium-intact rings of rabbit mesenteric artery. A, 5,6-EET (100 nmol/L to 3 µmol/L) evoked concentration-dependent relaxations (n=6) that were almost abolished following endothelial denudation (n=5). L-NAME (n=4) and Gap 27 peptide (n=6) alone significantly attenuated 5,6-EET-induced relaxations (P<0.05 for both), and in combination they reduced relaxation to the level seen in endothelium-denuded preparations (n=4). B, Neither 11,12-EET nor 14,15-EET induced relaxations of endothelium-intact or -denuded rings.



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  		Figure 9. Concentration-response curves showing the effect of EETs on relaxations to ACh and A23187 in endothelium-intact rings. There was no effect of a 30-minute preincubation with any of the EETs studied (5 µmol/L, n=4 for all 3 agents) on relaxations to either ACh (A) or A23187 (B).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
We have used isolated ring and sandwich preparations to characterize the role of GJC in the NO-independent responses of the rabbit mesenteric artery to ACh and A23187. The major findings are that EDHF-type relaxations evoked by ACh are attributable to an agent that preferentially crosses between endothelium and smooth muscle via gap junctions, and that a similar agent also mediates relaxation to A23187, but transfer to smooth muscle then involves diffusion via the extracellular space. The formation and/or mode of action of this mediator(s) is intimately related to the metabolism of phospholipids by a Ca2+-dependent PLA2.

Previous studies have shown that heterocellular GJC between the endothelium and smooth muscle contributes to NO-independent relaxations of rabbit conduit arteries.12 13 The present experiments confirm that Gap 27 peptide, which contains the SRPTEK motif common to the second extracellular loop of connexins present in the vascular wall, attenuates L-NAME–insensitive relaxations to ACh in rabbit mesenteric artery. In ring preparations, {approx}60% of the initial relaxation to ACh was mediated by NO, whereas in sandwich preparations, in which GJC cannot contribute to responses, relaxations were exclusively NO dependent. In marked contrast, only {approx}15% of the relaxation evoked by A23187 was mediated by NO in rings, and in sandwich preparations relaxation was not susceptible to inhibition by L-NAME. Furthermore, Gap 27 peptide was completely without effect on the responses to A23187 either in rings or in sandwich preparations. These findings indicate that, unlike ACh, A23187 mediates relaxation predominantly through the release of a freely diffusible factor, which is not NO, into the extracellular space. This conclusion is supported by comparison of the time course of the relaxations to the 2 agents. At concentrations producing equivalent mechanical responses in the presence of L-NAME, relaxations to A23187 were approximately twice as slow as those to ACh. This is consistent with the idea that the EDHF released by A23187 would have to negotiate 2 cell membranes before exerting a relaxant effect on vascular smooth muscle.

Our findings are consistent with observations by Plane and coworkers, who found that A23187, but not ACh, evokes the release of a diffusible, L-NAME–insensitive factor from the endothelium in sandwich preparations of rabbit femoral artery.11 These authors suggested that the EDHF released from this artery type was stimulus specific. In the present study, however, NO-independent relaxations to ACh and A23187 were both abolished by the structurally unrelated Ca2+-dependent PLA2 inhibitors ONO-RS-082 and AACOCF3 but not by the Ca2+-independent PLA2 inhibitor, HELSS. Although the experimental protocols cannot differentiate between activation of PLA2 within the endothelium or smooth muscle cell, the simplest explanation of these findings is that a closely similar agent mediates EDHF-type responses to ACh and A23187. Alternatively, if different mediators are involved, the production and/or action of both are crucially dependent on activation of PLA2 by Ca2+ ions. Inhibition of the Ca2+-dependent cytosolic form of PLA2 has also previously been shown to attenuate L-NAME–insensitive relaxations to ACh, histamine, and bradykinin in the perfused rat coronary and mesenteric beds.7 8 Cohen and colleagues have suggested that residual NO synthesis in the presence of the NO synthase inhibitors L-NAME and L-NG-nitroarginine could account for endothelium-dependent hyperpolarization.25 We excluded this possibility, however, by demonstrating that the selective inhibitor of guanylate cyclase, ODQ, did not modulate the relaxations to ACh and A23187 observed in the presence of L-NAME. The ability of ONO-RS-082 and AACOCF3 to virtually abolish L-NAME–insensitive relaxations, without affecting NO-mediated relaxations, provides further evidence that L-NAME completely inhibits NO formation in the rabbit mesenteric artery at the concentration used.

Several possibilities may be advanced to explain why A23187-induced relaxations were not susceptible to inhibition by Gap 27 peptide. First, it is possible that incorporation of A23187 into the plasmalemma alters local membrane structure and phospholipid metabolism, allowing easier access of an EDHF linked to the activity of a Ca2+-dependent PLA2 to the extracellular space. Second, there is evidence that molecules of A23187 aggregate in lipid bilayers to form channels, as well as behaving as a carrier ionophore with selectivity for Ca2+ ions,26 that could potentially allow egress of an EDHF into the extracellular space and its subsequent diffusion to adjacent smooth muscle, thereby "bypassing" gap junctions. Third, A23187 has been shown to cause closure of gap junctions between endothelial cells,27 a mechanism that could promote the egress of an EDHF via the membrane. In other cell types, ACh has also been reported to promote closure of gap junctions,28 but clearly this cannot be an important factor in our experiments, as Gap 27 peptide was highly effective in reducing relaxations to ACh. Conventionally, increases in [Ca2+]i have been thought to mediate closure of gap junctions.29 However, a G-protein/tyrosine kinase-dependent mechanism linked to activation of receptors has recently been shown to promote the closing of gap junctions in cells expressing connexin 43, independently of elevations in local [Ca2+]i.30 Conversely, other evidence suggests that elevated [Ca2+]i may also open gap junctions. In cardiac myocytes, which express connexin 43 as the dominant connexin protein, channel permeability increases monotonically as a function of [Ca2+]i over the physiological range of 0.1 to 1 µmol/L, and decreases only for [Ca2+]i within the supraphysiological range.31 Further experiments are therefore necessary to elucidate the principal mechanisms gating heterocellular GJC between endothelial and vascular smooth muscle cells.

Cascade bioassay experiments have demonstrated that NO inhibits the formation and/or release of EDHF by endothelial cells.32 This inverse relationship between the activities of the 2 mediators may explain why ONO-RS-082 and AACOCF3 had no effect on ACh-evoked relaxations in ring preparations in the absence of L-NAME but reduced the potency of A23187. Under these conditions, ACh-induced responses were substantially more dependent on NO synthesis than those evoked by A23187, thus suggesting that NO masks the effects of PLA2 inhibition by suppressing EDHF activity. In the presence of L-NAME, however, EDHF activity will be enhanced, and relaxations to ACh then become susceptible to ONO-RS-082 and AACOCF3, as observed experimentally. The positive correlation between the magnitude of the ACh-induced relaxations obtained in the presence and absence of Gap 27 peptide is also consistent with a dynamic interaction between NO and EDHF, with inhibition of GJC having a smaller effect in the presence of high levels of NO release. A new observation was that in preparations exhibiting large relaxations to ACh in the absence of L-NAME, the subsequent L-NAME–insensitive response to this agonist was small. This suggests that EDHF-type relaxations are chronically diminished in preparations exhibiting high NO activity, although the mechanisms underlying this downregulation remain to be determined.

Activation of PLC following agonist stimulation results in generation of inositol trisphosphate (IP3) and release of Ca2+ from internal stores. The resulting increase in [Ca2+]i activates the Ca2+-dependent cytosolic form of PLA2 resulting in cleavage of arachidonic acid from membrane glycerophospholipids.33 The specific PLC inhibitor U-7312222 abolished L-NAME–insensitive relaxations to ACh but did not affect responses to A23187. The probable explanation for these findings is that EDHF-type relaxations to ACh require activation of PLC and IP3 formation to release Ca2+ from intracellular stores and activate PLA2, whereas the ionophore properties of A23187 increase [Ca2+]i by directly facilitating Ca2+ influx into the cell, as well as Ca2+ release from stores through second-messenger–independent mechanisms.34 Activation of PLC also generates diacylglycerol (DAG), which can be hydrolyzed by a DAG lipase to provide a second source of arachidonic acid.35 However, inhibition of NO-independent relaxations to ACh by U-73122 is unlikely to reflect loss of DAG as a substrate for arachidonic acid production, as the DAG lipase inhibitor, RHC-80267, has no effect on EDHF-type responses in the perfused rat heart and mesenteric prearteriolar bed.7 8

Further evidence for the common identity of the EDHF released by ACh and A23187 was obtained with the sulfhydryl agent thimerosal, which has previously been reported to hyperpolarize porcine coronary arterial smooth muscle in an endothelium-dependent manner36 and enhance the release of an EDHF from canine carotid artery in cascade bioassay.10 In the present study, low concentrations of thimerosal enhanced relaxations to ACh and A23187 both in the absence and presence of L-NAME and indomethacin. Importantly, the potentiation of EDHF-type relaxations to ACh in the presence of L-NAME remained susceptible to inhibition by Gap 27 peptide, whereas those to A23187 were not. The effects of thimerosal can consequently be attributed to factors other than alterations in the pathways allowing transfer of EDHF between the endothelium and smooth muscle by ACh and A23187. Two known mechanisms of action of thimerosal could contribute to an apparent increase in EDHF activity. First, thimerosal inhibits the acyl-coenzyme A:lysolecithin acyltransferase,23 thereby elevating free levels of arachidonic acid within the cell,33 an action that would be consistent with the inhibition of relaxation observed with ONO-RS-082 and AACOCF3. Second, nanomolar concentrations of thimerosal equivalent to those used in the present study, are also known to sensitize the IP3 receptor and thereby promote Ca2+ release from intracellular stores.24 This mechanism could potentially contribute to a secondary activation of the Ca2+-dependent PLA2 and enhancement of EDHF activity, which, like NO, is known to be Ca2+ dependent.37 While receptor-independent Ca2+ influx induced by cyclopiazonic acid also stimulates NO-independent relaxations, in marked contrast to A23187, these responses are dependent on GJC.12 A dominant effect of Ca2+ mobilization by thimerosal therefore seems less likely than its potential effects on arachidonate metabolism. Furthermore, A23187 does not require IP3-mediated release of Ca2+ from stores to evoke NO-independent relaxations, as demonstrated by the lack of effect of U-73122, yet the responses to A23187 were enhanced by thimerosal. It remains to be determined whether thimerosal exerts additional, as yet unknown, effects such as sensitization of vascular smooth muscle to the action of EDHF, but these would not detract from the logic of our argument.

Several groups have suggested that EDHF could be an EET metabolite of arachidonic acid, synthesized by an endothelial cytochrome P-450 epoxygenase, that evokes smooth muscle relaxation through activation of K+ channels and subsequent membrane hyperpolarization.3 4 5 38 39 Other workers, however, have reported that relaxation is observed only with micromolar concentrations of EETs, and EETs do not induce hyperpolarization and relaxation in all artery types.40 41 A number of groups have demonstrated that inhibitors of cytochrome P-450 attenuate EDHF-mediated relaxations,3 4 5 consistent with the involvement of an EET, but it has become apparent that such inhibitors can also directly inhibit K+ channel activity.42 Furthermore, highly specific cytochrome P-450 inhibitors appear to have little effect on NO-independent relaxations in rat mesenteric arteries.43 In the present study with rabbit arteries, micromolar concentrations of 11,12-EET and 14,15-EET were found to be devoid of vasorelaxant activity, and relaxation to 5,6-EET was a strictly endothelium-dependent phenomenon. Other studies have also shown significantly more potent dilator effects of 5,6-EET than 14,15-EET in rat intestinal and cat cerebral microvessels.44 45 5,6-EET may promote Ca2+ influx and stimulate NO release from endothelial cells,46 a mechanism that seems to operate in rabbit mesenteric arteries, as its effects were partially susceptible to inhibition by L-NAME. A surprising new finding, however, was that L-NAME–insensitive relaxations induced by 5,6-EET were abolished by Gap 27 peptide. In a recent study, Weintraub et al41 reported that preincubation with EETs potentiates endothelium-dependent relaxations to bradykinin in porcine coronary arteries. In the present study we repeated this experiment and failed to demonstrate potentiation with 5,6-, 11,12-, or 14,15-EET after preincubation for 30 minutes at a concentration of 5 µmol/L. Taken together, our findings therefore provide no evidence that the biologically active EDHF in rabbit arteries is an EET.

We conclude that ACh and A23187 both stimulate the production of an EDHF in rabbit superior mesenteric artery the vasodilator activity of which crucially depends on GJC in the case of ACh and on extracellular diffusion in the case of A23187. The similarity of the effects of 2 structurally dissimilar Ca2+-dependent inhibitors of PLA2 and thimerosal on relaxations obtained to ACh and A23187 point to the operation of common mechanistic pathways. The primary signaling mechanism for relaxation appears to be chemical rather than electrical in nature. The exact identity of the EDHF(s) has yet to be established, and it remains to be determined at which point distal to activation of PLA2 an active agent is mobilized and within which cell type. Since the central pore of gap junctions is aqueous and thought to allow preferential diffusion of small, water-soluble charged molecules, a role for hydrophobic arachidonic acid derivatives, such as EETs, might be challenged on a purely physiochemical basis.


*    Acknowledgments
 
This work was supported by grants 9307148 and 9305117 from the Medical Research Council.

Received June 3, 1998; accepted October 10, 1998.


*    References
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 *References
 

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