INTRODUCTION

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AGONISTS THAT ACT VIA THE endothelium, such as acetylcholine (ACh), evoke smooth muscle hyperpolarizations and relaxations that are driven by a primary endothelial hyperpolarization and are independent of nitric oxide (NO) and prostanoid synthesis (13). Passive electrotonic mechanisms may contribute to the smooth muscle response as the endothelium and media are coupled electrically via myoendothelial gap junction plaques that consist of focal clusters of individual gap junctions constructed from connexin proteins (7, 27). Indeed, in arterioles, endothelial hyperpolarization can be detected synchronously in smooth muscle, whether induced by ACh or the injection of electrical current into a single endothelial cell (14). By contrast, in thick-walled vessels, it has been suggested that the endothelium cannot act as a major source of hyperpolarizing current because large differences in the mass of this monolayer and the media result in electrical mismatching (3). An alternative hypothesis, therefore, is that an endothelium-derived hyperpolarizing factor (EDHF) is released into the extracellular space to activate smooth muscle K⁺ channels and mediate relaxation (8, 16, 26).

There is nevertheless evidence that direct intercellular communication via gap junctions also contributes to the EDHF phenomenon in conduit vessels. Synthetic peptides homologous to the Gap 26 or 27 domains of the first and second extracellular loops of connexin proteins, which interrupt intercellular communication in a connexin-specific fashion, and 18-glycyrrhetinic acid (18-GA), a lipophilic aglycone that disrupts gap junction plaques, inhibit EDHF-type responses evoked by ACh in a spectrum of rabbit arteries and veins (4, 9, 10, 12, 17, 19, 28). Furthermore, in "sandwich" preparations of rabbit mesenteric artery, in which there can be no gap junctional communication between the endothelium of the donor tissue and smooth muscle of the detector tissue, relaxations evoked by ACh are mediated entirely by NO (9, 19). By contrast, sandwich experiments have also provided evidence for the release of a relaxant factor, distinct from NO and prostanoids, that diffuses via the extracellular space after administration of the Ca²⁺ ionophore A-23187 in rabbit femoral and mesenteric arteries (19, 25). Observations that EDHF-type relaxations evoked by ACh in the rabbit iliac artery are dependent on elevations in smooth muscle cAMP levels and phosphorylation events mediated by protein kinase A nevertheless suggest that even responses to agonists may not simply be mediated by passive electrotonic mechanisms (29). Because cAMP accumulation is suppressed by interrupting gap junctional communication with connexin-mimetic peptides or 18-GA in these vessels (29), it is possible that chemical signaling contributes to the response to ACh, as in addition to conferring electrical continuity, gap junctions allow direct transfer of signaling molecules <1 kDa in size between coupled cells (6). In the present study, we demonstrate that cAMP similarly underpins the EDHF response to A-23187 in rabbit arteries, despite being independent of heterocellular communication via gap junctions, thereby providing evidence that similar biochemical events may underpin the EDHF phenomenon even when relaxation is effected via fundamentally different signaling pathways.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated ring preparations. Male New Zealand White rabbits (2-2.5 kg) were euthanized with pentobarbitone sodium (120 mg/kg iv), and the iliac artery was removed and transferred to cold Holmans buffer composed of (in mM) 120 NaCl, 5 KCl, 2.5 CaCl₂, 1.3 NaH₂PO₄, 25 NaHCO₃, 11 glucose, and 10 sucrose. The vessels were stripped of adherent tissue, and rings 2-3 mm wide were cut and suspended in organ chambers containing gassed (95% O₂-5% CO₂, pH 7.4) buffer at 37°C. Tension was set at ~0.25 g and during an equilibrium period of 1 h, the tissues were repeatedly washed with fresh buffer and tension was readjusted after stress relaxation. Endothelium-intact rings were incubated for 40 min with N^G-nitro-L-arginine methyl ester (L-NAME; 300 µM) and indomethacin (10 µM) and after constriction with phenylephrine (PE) cumulative concentration-relaxation curves to ACh or A-23187 were constructed. Some preparations were preincubated for 40 min with either 2',5'-dideoxyadenosine (DDA; 200 µM), 3-isobutyl-1-methylxanthine (IBMX, 20 µM), 18-GA (100 µM), glibenclamide (10 µM), the combination of charybdotoxin (100 nM) and apamin (300 nM), or the combination of charybdotoxin (100 nM), apamin (300 nM), and IBMX (20 µM). Concentration-response curves to ACh and A-23187 were also constructed for endothelium-denuded rings in the absence or presence of IBMX (20 µM). In experiments with IBMX, which itself depresses contraction, the concentration of PE used to induce tone was increased from 1 to 3 µM. All reagents were obtained from Sigma and were dissolved in buffer with the exception of A-23187, 18-GA, and IBMX, which were dissolved in dimethyl sulfoxide. Previous studies (10) have shown that this solvent has no effect on EDHF-type responses at the final concentrations employed in the present experiments.

"Sandwich" preparations. Rings of iliac artery 2-3 mm wide were denuded of endothelium, cut into strips, and pierced ~2 mm from each end with the use of a Monoject needle (0.9 mm × 40 mm). These strips were introduced into the lumen of rings of endothelium-intact iliac artery 4-5 mm wide and the tissues were sutured together. The composite preparations were then mounted in a Mulvany Multi Myograph (Danish Myo Technology) with the pierced denuded strips hooked onto the large vessel mountings. Tension was initially set at ~0.25 g and readjusted during an equilibrium period of 1 h. The preparations were then incubated for 40 min with L-NAME (300 µM) and indomethacin (10 µM), constricted with PE (1 or 3 µM), and concentration-response curves were constructed for ACh in the presence and absence of IBMX (20 µM) or A-23187 in the presence and absence of IBMX (20 µM) or DDA (200 µM).

Radioimmunoassay. Multiple rings from the same artery were incubated in oxygenated Holmans buffer containing L-NAME (300 µM) and indomethacin (10 µM) for 40 min at 37°C in the presence or absence of 18-GA (100 µM). PE (1 µM) was added 3 min before the initial control point. After the addition of ACh or A-23187, the rings were frozen in liquid N₂ at time points up to 180 s and stored at 70°C. cAMP and cGMP were subsequently extracted in 6% trichloroacetic acid, followed by neutralization with water-saturated diethyl ether and radioimmunoassay (Amersham). Nucleotide levels were expressed relative to protein content determined by a dye-binding assay (Bio-Rad). Additional experiments were performed with endothelium-denuded rings.

Statistical analysis. All data are given as means ± SE, where n denotes the number of animals studied for each data point. Concentration-relaxation curves and nucleotide accumulation were assessed by one-way analysis of variance, followed by Bonferroni's multiple-comparisons test. Fifty percent effective concentration (EC₅₀) values were compared by the Student's t-test for unpaired data. P < 0.05 was considered as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated rabbit iliac artery rings. EDHF-type relaxations to ACh and A-23187 were both maximal at concentrations of 3 µM (Figs. 1 and 2). In endothelium-intact rings, IBMX (20 µM) potentiated these maximal responses from 36.0 ± 5.0 to 65.5 ± 4.5% with ACh (P < 0.05, n = 14 and 10; Fig. 1A) and from 50.2 ± 5.6 to 73.0 ± 8.5% with A-23187 (P < 0.05, n = 18 and 9; Fig. 1B), with corresponding leftward shifts in the EC₅₀ values from 555 ± 121 to 119 ± 21 nM and from 328 ± 123 nM to 196 ± 139 nM, respectively (P < 0.05 in each case). Incubation with DDA (200 µM) almost abolished ACh-evoked relaxations, with maximal responses reduced to 11.0 ± 1.9% (P < 0.05, n = 4; Fig. 1A), whereas with A-23187 there was a significant increase in EC₅₀ value to 644 ± 221 nM (P < 0.05) and maximal relaxations were nonsignificantly depressed to 40.3 ± 8.3% (n = 9, Fig. 1B). Endothelial denudation abolished relaxations to both ACh and A-23187 (n = 8 and 5, respectively; Figs. 1, A and B). Incubation of endothelium-denuded rings with IBMX (20 µM) did not unmask relaxations to ACh (n = 4; Fig. 1A), whereas A-23187 induced a relaxation equivalent to 21.7 ± 3.6% of PE-induced tone (P < 0.05, n = 6; Fig. 1B). In a separate series of experiments, the combination of charybdotoxin (100 nM) plus apamin (300 nM) effectively abolished ACh-induced relaxations, which were reduced from a maximum of 32.2 ± 3.9 to 4.0 ± 1.8% (P < 0.05, n = 5; Fig. 1C), and markedly attenuated responses to A-23187 with maximal relaxation being reduced from 50.0 ± 5.8 to 14.6 ± 5.7% of PE-induced tone (P < 0.05, n = 3; Fig. 1D), with a rightward shift in EC₅₀ values from 291 ± 128 to 784 ± 417 nM (P < 0.05). The presence of IBMX (20 µM) reduced the effectiveness of the apamin plus charybdotoxin combination with maximal relaxations to ACh and A-23187 being increased to 16.4 ± 2.6 and 22.0 ± 5.9% of PE-induced tone, respectively (P < 0.05, n = 4 and 3; Fig. 1, C and D). Incubation with glibenclamide (10 µM) did not significantly affect EDHF-type relaxations evoked by ACh or A-23187 (n = 5 and 9, respectively; Fig. 1, C and D).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Concentration-response curves showing endothelium-derived hyperpolarizing factor (EDHF)-type relaxations evoked by ACh (A and C) and A-23187 (B and D). Relaxations to both agents were potentiated by 20 µM 3-isobutyl-1-methylxanthine (IBMX) (n = 10 and 9, respectively; A and B) and abolished by endothelial denudation (n = 8 and 5, respectively; A and B). ACh-evoked relaxations were essentially abolished by 2',5'-dideoxyadenosine (DDA; 200 µM) and responses to A-23187 were attenuated (n = 4 and 9, respectively; A and B). No relaxation to ACh was evident in endothelium-denuded rings incubated with IBMX (20 µM), whereas A-23187 induced a small relaxation (n = 4 and 6, respectively; A and B). Preincubation with glibenclamide (10 µM) did not affect relaxations evoked by ACh or A-23187 (n = 5 and 9, respectively; C and D). The combination of charybdotoxin (CTX; 100 nM) plus apamin (300 nM) virtually abolished control relaxations (n = 5 and 3; C and D) and markedly attenuated the potentiating effects of IBMX (n = 4 and 3, respectively; C and D).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Representative traces (A and B) and concentration-response curves (C and D) showing abolition of EDHF-type ACh-evoked relaxations by 100 µM 18-glycerrhetinic acid (GA), but no effect on corresponding responses to A-23187 (n = 5 and 8, respectively). In endothelium-intact rings incubated with 300 µM NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin (10 µM), ACh (3 µM) evoked a transient peak in cAMP levels at 15-30 s (n = 4; E), which was abolished by 18-GA (100 µM, n = 5) and endothelial denudation (n = 4). Accumulation of cAMP evoked by A-23187 (3 µM) peaked at 40-50 s (n = 6; F) and was unaffected by 18-GA (100 µM, n = 6) but abolished by endothelial denudation (n = 4). cGMP levels were unaltered by ACh or A-23187 (n = 4 in each case; E and F). *P < 0.05 cf. control; P < 0.05 cf. initial value in the presence of 18-GA.

cAMP accumulation. In the absence of pharmacological intervention, basal cAMP levels were 4.5 ± 0.4 pmol/mg protein in endothelium-intact preparations and were not significantly altered by preincubation with L-NAME (300 µM) and indomethacin (10 µM), followed by PE (1 µM) (n = 9), whereas basal cGMP levels were reduced from 1.84 ± 0.66 to 0.30 ± 0.07 pmol/mg protein (P < 0.05, n = 3 and 8, respectively). Subsequent exposure to either ACh or A-23187 elevated cAMP levels to statistically similar maxima of 6.8 ± 1.5 and 6.9 ± 0.8 pmol/mg protein (n = 4 and 6, respectively), whereas neither agent significantly affected cGMP levels (n = 4 in each case; Fig. 2, E and F). The nucleotide response to A-23187 was initially slower in onset than that evoked by ACh, peaking at 40-50 s compared with 15-30 s, but was more sustained with elevations in cAMP levels being apparent for >60 s only with the ionophore (Fig. 2, E and F). In endothelium-denuded rings, basal cAMP levels were 3.97 ± 0.49 pmol/mg protein (n = 8) and did not change significantly after administration of either ACh or A-23187 (n = 4 in each case; Fig. 2, E and F).

Effects of 18-GA. The gap junction inhibitor 18-GA (100 µM) effectively abolished EDHF-type relaxations evoked by ACh with maximal relaxations being reduced from 25.0 ± 3.8% to 2.4 ± 3.6% of PE-induced tone (P < 0.05, n = 5; Fig. 2, A and C). By contrast, 18-GA (100 µM) exerted no significant effect on EDHF-type relaxations to A-23187 (n = 8; Fig. 2, B and D). In rings with endothelium, 18-GA (100 µM) abolished the rise in cAMP levels evoked by ACh (3 µM; n = 5; Fig. 2E), but had no significant effect on nucleotide accumulation induced by A-23187 (3 µM, n = 6; Fig. 2F).

Sandwich preparations. ACh failed to evoke EDHF-type relaxations either in the presence or absence of IBMX (20 µM, n = 5 in each case; Fig. 3, A and B). By contrast, A-23187 stimulated relaxations with a maximal response of 52.0 ± 8.0% of PE-induced tone and an EC₅₀ value of 240 ± 40 nM (n = 5; Fig. 3, A and C). Responses to A-23187 were attenuated by DDA (200 µM) with maximal relaxation reduced to 18.5 ± 9.2% of PE-induced tone with a rightward shift in EC₅₀ to 1,250 ± 240 nM (P < 0.05, n = 5; Fig. 3, A and C) and were potentiated by IBMX (20 µM) to a maximum of 70.0 ± 6.50% with a leftward shift in EC₅₀ to 120 ± 80 nM (P < 0.05, n = 5; Fig. 3, A and C).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Representative traces (A) and concentration-response curves (B and C) in sandwich preparations incubated with L-NAME (300 µM) and indomethacin (10 µM). ACh failed to induce relaxation either in the presence or absence of IBMX (20 µM, n = 5 in each case; A and B). By contrast, A-23187 evoked relaxations that were attenuated by DDA (200 µM) and potentiated by IBMX (n = 5 in each case; A and C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study has highlighted similarities and differences in the mechanisms that contribute to EDHF-type relaxations evoked by ACh and the Ca²⁺ ionophore A-23187 in the rabbit iliac artery. The major finding is that the endothelium mediates NO- and prostanoid-independent relaxations to both agents by elevating smooth muscle cAMP levels, although the underlying signaling pathways involve myoendothelial gap junctions in the case of ACh and transfer of a diffusible factor via the extracellular space in the case of A-23187.

In experiments with endothelium-intact rings, ACh and A-23187 both evoked EDHF-type relaxations that were attenuated by inhibition of adenylyl cyclase, with the P site agonist DDA effectively abolishing responses to ACh and causing a significant rightward shift in the concentration-response curve for A-23187. Conversely, relaxations to both agents were potentiated by inhibition of cAMP hydrolysis with IBMX. Although this phosphodiesterase inhibitor also unmasked a small concentration-dependent direct relaxant response to A-23187 in rings without endothelium, presumably by weakly stimulating the Ca²⁺-activated type VIII adenylyl cyclase isoform (31), submaximal relaxations of endothelium-intact rings to A-23187 were amplified by up to to threefold in the presence of IBMX. Responses to ACh and A-23187 were inhibited by the combination of apamin plus charybdotoxin, even when relaxation was potentiated by IBMX. This is a hallmark of the EDHF phenomenon and is thought to reflect the opening of apamin-sensitive small conductance channels (SK_Ca) and charybdotoxin-sensitive large and intermediate conductance channels (BK_Ca and IK_Ca) located on the endothelium (13). Experiments with glibenclamide excluded a role for cAMP-dependent activation of ATP-sensitive K⁺ channels in EDHF-type relaxations evoked either by ACh or A-23187.

Evidence that mechanical responses were dependent on elevations in smooth muscle cAMP levels was obtained by radioimmunoassay. In rings incubated with L-NAME and indomethacin, concentrations of ACh or A-23187 that induced maximal relaxations were associated with transient, endothelium-dependent ~1.5-fold increases in cAMP levels, which in other cell types are sufficient to elicit near-maximal biological responses (15). Incubation with L-NAME significantly decreased basal cGMP levels and no subsequent elevations in levels of this nucleotide were detected after administration of ACh or A-23187. This confirms blockade of NO synthase and demonstrates that IBMX, which inhibits phosphodiesterases that hydrolyse both cGMP and cAMP (23), did not potentiate relaxation by amplifying the biochemical consequences of residual NO activity (11). Although the peak elevations in cAMP levels induced by ACh and A-23187 were of equal magnitude, the nucleotide response to A-23187 was slower in onset but more sustained. Differences in the time course of the cAMP response may therefore explain why maximal reductions in PE-induced constrictor tone evoked by A-23187 were ~15% greater than those evoked by ACh and presumably also why DDA was less effective as an inhibitor of relaxations induced by A-23187 than ACh in ring preparations.

Experiments with 18-GA nevertheless provided evidence that the signaling pathways activated by ACh and A-23187 were fundamentally different, as this gap junction inhibitor abolished ACh-induced relaxations and associated cAMP accumulation, but did not affect the corresponding responses evoked by A-23187. Analogous mechanical observations have been made in the rabbit superior mesenteric artery in which EDHF-type relaxations to ACh, but not A-23187, are inhibited by connexin-mimetic peptides that interrupt gap junctional communication (19). The present findings with A-23187 indicate that 18-GA does not inhibit cAMP synthesis nonspecifically. Confirmation that the absolute cAMP content of the endothelial monolayer is small and contributes negligibly to nucleotide measurements in intact rings was therefore provided by the finding that ACh did not elevate cAMP levels in endothelium-intact rings incubated with 18-GA. Observations that 18-GA failed to attenuate A-23187-evoked relaxations in intact rings, whereas these responses were inhibited by apamin and charybdotoxin, additionally indicate that 18-GA does not depress the hyperpolarizing response that is central to the EDHF phenomenon in a nonspecific fashion (13).

Consistent with the differential effects of 18-GA on ACh and A-23187-induced responses, bioassay experiments with sandwich preparations provided evidence that an endogeneous vasodilator may transfer across the extracellular space after stimulation of the endothelium with A-23187 under conditions of combined NO synthase and cyclooxygenase blockade. By contrast, no transferable factor could be detected after administration of ACh, even in the presence of IBMX, which might have been expected to unmask the functional effects of subthreshold release of a freely diffusible mediator. Electrophysiological support for the hypothesis that A-23187 promotes the extracellular release of an EDHF has been reported in the porcine coronary artery on the basis that the SK_Ca channel inhibitor d-tubocurarine attenuates the endothelial hyperpolarization evoked by A-23187 to a greater extent than either mechanical relaxation or smooth muscle hyperpolarization (30). In the present study, concentration-relaxation curves for A-23187 in sandwich preparations were depressed and shifted to the right by DDA, confirming that cAMP synthesis was central to the associated mechanical response, as in endothelium-intact rings. This apparently more effective inhibition of A-23187-evoked relaxations by DDA in composite sandwich preparations than intact rings might reflect differences in luminal versus abluminal release of a diffusible factor and the time course of the subsequent cAMP response in the detector tissue. As in intact rings, relaxations were potentiated by IBMX in sandwich preparations, particularly at intermediate concentrations of A-23187 where direct effects on smooth muscle tone were small.

One mechanistic explanation for the contrasting findings with ACh and A-23187 is that a chemical mediator, synthesized within the endothelium, transfers preferentially to smooth muscle via gap junctions after stimulation with ACh, whereas A-23187 induces an "overspill" of the same factor, thereby elevating smooth muscle cAMP levels via an extracellular route. Such a factor would also be expected to promote cAMP formation within the endothelium and might therefore contribute to the pronounced extracellular release of cAMP from the endothelium that is detectable in the effluent from the buffer-perfused rabbit ear and rat mesentery preparations after administration of ACh or A-23187 (1, 29). In the case of ACh, it is possible that diffusion of cAMP from the endothelium into the media via gap junctions contributes to the elevations in smooth muscle nucleotide levels, at least in part (6, 29). The factor mediating relaxations to A-23187 cannot, however, simply be cAMP derived from the endothelium as ACh-evoked efflux of this nucleotide does not modulate perfusion pressure in isolated rabbit ear preparations if gap junctional communication is interrupted by 18-GA, presumably reflecting its low efficacy as an extracellular vasorelaxant (29). We have previously provided evidence that EDHF-type relaxations of rabbit arteries evoked by ACh and A-23187 both require mobilization of arachidonic acid by a Ca²⁺-dependent phospholipase A₂ (19, 20). In theory, this would be consistent with the hypothesis that epoxyeicosatrienoic acid (EET) metabolites of arachidonic acid function as freely diffusible EDHFs (8). Indeed, these compounds are synthesized by the endothelium, activate hyperpolarizing smooth muscle K⁺ channels, and elevate cAMP levels in cardiac myocytes and monocytes (8, 32, 33). However, in rabbit mesenteric arteries, EDHF-type relaxations evoked by direct activation of phospholipase A₂ with the polypeptide melittin are mediated via a mechanism that involves gap junctional communication (20). Furthermore, 5,6-EET evokes relaxations that possess characteristics identical with ACh in that they are endothelium, gap junction, and cAMP dependent, and other EET regioisomers are inactive (19, 29). These observations suggest that arachidonate metabolism within the endothelium may be an important initiating step in the EDHF phenomenon in rabbit arteries, but provide no support for the idea that the factor released by A-23187 is an EET. The role of alternative hyperpolarizing arachidonate products such as the dihydroxyeicosatrienoic acids in EDHF-type relaxations of rabbit arteries remains to be determined (22).

In conclusion, we have demonstrated that EDHF-type relaxations evoked by ACh and A-23187 both depend on smooth muscle cAMP accumulation in rabbit arteries, but involve different intercellular communication pathways. Although the multiple actions of cAMP encompass the diverse characteristics of the EDHF phenomenon reported in the literature, such as hyperpolarization mediated by K_Ca channels and Na⁺-K⁺-ATPase (13), it remains to be established if responses to ACh and A-23187 involve an identical chemical signal. Furthermore, in the case of ACh, there may be complex interactions between chemical and electrotonic signaling mechanisms as cAMP could in theory enhance relaxation by increasing the electrical conductance of gap junctions (2), thereby facilitating electrotonic spread of endothelial hyperpolarization into the media. Conducted endothelial hyperpolarization might also itself contribute to the smooth muscle cAMP accumulation evoked by ACh, even though the mammalian adenylyl cyclase is not thought to be regulated directly by membrane potential (29). EDHF-type relaxations are associated with closure of L-type voltage-operated Ca²⁺ channels, resulting in a marked reduction in smooth muscle intracellular [Ca²⁺] (5) that might activate the Ca²⁺-inhibited type V and VI adenylyl cyclase isoforms that can be closely coupled to L-type Ca²⁺ channels and are expressed in vascular smooth muscle (21, 24). Alternatively, reductions in intracellular [Ca²⁺] could suppress the type I phosphodiesterase, which is stimulated by Ca²⁺, thereby reducing cAMP hydrolysis and elevating cAMP levels (18).