Pulsatile Stretch in Coronary Arteries Elicits Release of Endothelium-Derived Hyperpolarizing Factor

A Modulator of Arterial Compliance

From the Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt, Germany.

Correspondence to Dr Rüdiger Popp, Institut für Kardiovaskuläre Physiologie, Klinikum der J. W. Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail r.popp{at}em.uni-frankfurt.de

	Abstract

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Abstract—To date, the release of the endothelium-derived hyperpolarizing factor (EDHF) has been demonstrated only in response to receptor-dependent Ca²⁺-elevating agonists. Since endothelial cells in situ are continuously subjected to rhythmic distension, we investigated the effect of rhythmic stretch on the release of EDHF from isolated porcine coronary arteries. In the combined presence of diclofenac and N^G-nitro-L-arginine (L-NNA), sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) led to simultaneous oscillations in the external diameter of coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. In order to directly demonstrate the release of EDHF, the intraluminal solution from endothelium-intact coronary segments exposed to pulsatile stretch was applied to detector rat aortic smooth muscle cells, the membrane potential of which was continuously measured using the patch-clamp technique. The hyperpolarization of detector cells induced by the intraluminal solution was proportional to the amplitude of the pressure oscillations applied to the donor artery and was attenuated by either preincubation of donor arteries with 17-octadecynoic acid or application of either tetrabutylammonium or iberiotoxin to detector cells. In contrast to the bradykinin-induced release of EDHF, the EDHF synthesized in response to pulsatile stretch did not exhibit any tachyphylaxis. These findings demonstrate for the first time that the synthesis of EDHF in coronary arteries can be mechanically stimulated by rhythmic vessel wall distension and suggest that the continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.

Key Words: mechanotransduction • endothelium-derived hyperpolarizing factor • cytochrome P-450 • transmural pressure • coronary artery

	Introduction

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The vascular endothelium has been shown to synthesize and release vasoactive autacoids such as NO, prostacyclin (PGI₂), and an as-yet-unidentified transferable factor that induces relaxation by the hyperpolarization of vascular smooth muscle cells in response to receptor-dependent and -independent Ca²⁺-elevating agonists. Attributing specific effects and second-messenger pathways to each of these factors is hampered by the fact that all three autacoids may be simultaneously produced in response to a single stimulus. Since the production of the so-called "endothelium-derived hyperpolarizing factor" (EDHF)¹ may be affected by physiological concentrations of NO² and PGI₂, studies involving EDHF synthesis and effector pathways can be performed only under conditions of combined NO synthase/cyclooxygenase blockade. Pharmacological characterization of the EDHF derived from agonist-stimulated porcine coronary and rabbit carotid arteries suggests that this factor displays properties similar to those of cytochrome P-450–dependent metabolites of arachidonic acid.^{3 4 5 6 7 8} Moreover, the production of EDHF in bioassay experiments can be inhibited by the selective cytochrome P-450 inhibitor, 17-ODYA.^{7 9} Although bradykinin, acetylcholine, and the Ca²⁺ ionophore A23187 elicit the release of EDHF, no experimental evidence has been provided to date to suggest that the production and release of EDHF can be stimulated by physiologically more relevant stimuli, such as shear stress or rhythmic vessel distension. The aim of the present study was therefore to investigate, in isolated coronary segments, whether pulsatile changes in intraluminal pressure stimulate the production of EDHF and subsequently modulate vascular tone. We show here that rhythmic changes in intraluminal pressure evoke the synthesis of EDHF, the inhibition of which leads to a marked reduction in vascular compliance. These findings represent the first clear demonstration of a significant role of an endothelium-derived NO synthase/cyclooxygenase–independent factor in the vascular response to a hemodynamic stimulus.

	Materials and Methods

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Materials
Bradykinin was purchased from Bachem-Biochemica; diclofenac (Voltaren), from CIBA-Geigy; and L-NNA and HEPES, from Serva. U46619 was provided by Upjohn; 17-ODYA, nystatin, TBA, iberiotoxin, CHAPS, and all other chemicals were obtained from Sigma.

Vessel Preparation
Porcine hearts were obtained from a local abattoir and placed immediately into cold Hanks' solution (GIBCO). Coronary epicardial artery segments (length, 15 mm; mean external diameter, 2.4 to 2.8 mm) were excised and cleaned of fat and connective tissue, side branches were tied using surgical silk, and vessels were incubated in cold PSS (mmol/L: NaCl 140, KCl 4.7, MgCl₂ 1, CaCl₂ 1.3, HEPES 10, and D-glucose 5, pH 7.4 at 37°C; PO₂, 140 mm Hg) until use.

Diameter Registration
Coronary artery segments were cannulated at both ends and placed into organ chambers containing Tyrode's solution composed of (mmol/L) NaCl 132, KCl 4, CaCl₂ 1.6, MgCl₂ 0.98, NaHCO₃ 11.9, NaH₂PO₄ 0.36, and glucose 10, along with 0.1% BSA, 10 µmol/L diclofenac, and 100 µmol/L L-NNA. The extraluminal solution was gassed with 95% O₂/5% CO₂ to give a PO₂ of >300 mm Hg and perfused through the organ chamber at a rate of 0.5 mL/min; the luminal perfusate was gassed with 20% O₂/5% CO₂/75% N₂ to give a PO₂ of 140 mm Hg (37°C, pH 7.4). Perfusion routes for the chamber and the vessel lumen were separate, and drugs could be administered by either route independently. The transmural pressure in the segment was hydrostatically adjusted to 50 mm Hg by altering the height of the outflow, and vessels were gradually stretched to their in situ length. Thereafter, the vessel lumen was perfused at a rate of 0.5 mL/min, and the outer diameter of carotid artery segments was recorded continuously by a photoelectric device as described previously.¹⁰ After an initial 60-minute equilibration period, the segments were constricted several times with KCl until reproducible responses were obtained. After constriction with U46619 (3 µmol/L), bradykinin was administered intraluminally to assess endothelial integrity and the magnitude of the agonist-induced EDHF release. Thereafter, the vessel was constricted to 80% of the maximal KCl-induced constriction, and sinusoidal volume changes (1.5 Hz), which led to simultaneous sinusoidal pressure and diameter changes, were generated by compressing the inflow tubing to the arterial segment with a flat metal sheet controlled by a motor-driven cam gear. At the end of each experiment, the vasodilator response to bradykinin was determined to ensure the functional integrity of the endothelium.

Rat Aortic Smooth Muscle Cells
Rat aortic smooth muscle cells were isolated and cultured as described.⁹ Experiments were performed using confluent smooth muscle cells grown on glass coverslips between passages 6 and 16.

Porcine Coronary Smooth Muscle Cells
Porcine coronary epicardial arteries were isolated under sterile conditions as described above. Endothelial cells were scraped off with a scalpel, and the adventitia and media were separated mechanically. The media was cut into small pieces, which were placed in PSS-dissociation medium containing collagenase (3 mg/mL) and elastase (2 mg/mL) for 50 minutes. During the incubation period, the tissue pieces were dispersed by being repeatedly passed through a Pasteur pipette. The smooth muscle cells thus isolated were recovered by centrifugation, seeded onto fibronectin-coated glass coverslips, and maintained in MEM containing 10% FCS until use (2 to 4 hours).

Patch-Clamp Bioassay
Endothelium-intact segments of porcine coronary artery were attached to steel cannulas in an organ bath, stretched to their in situ length, and perfused with PSS (1 mL/min) at an intraluminal pressure of 30 mm Hg. Rhythmic changes (1 Hz) in circumferential wall stretch were induced in these segments, after blockade of the outflow, by applying sinusoidal volume oscillations in a magnitude that produced corresponding pressure oscillations between 30, 60, and 90 mm Hg. After 2 minutes of rhythmic vessel distension, 200 µL of the intraluminal fluid was withdrawn, applied upstream from the patch pipette to detector smooth muscle cells grown on glass coverslips, and mounted in a superfusion chamber (volume, 1 mL; flow rate, 1 mL/min).

The membrane potential of freshly harvested porcine coronary and cultured rat aortic smooth muscle cells was recorded using the slow whole-cell configuration of the patch-clamp technique. The patch pipettes used had an input resistance of 8 to 10 M when filled with standard KCl solution (mmol/L: KCl 140, MgCl₂ 1, CaCl₂ 1.3, HEPES 10, and D-glucose 5, pH 7.4). Gigaohm seals were established by gentle suction. The slow whole-cell configuration was obtained using nystatin (100 µg/mL) in the pipette. This nystatin concentration provides a low resistance access to the cytosol to measure intracellular potentials under current-clamp conditions. The electrical contact with the cytosol was established within 1 to 2 minutes of seal formation. The cell membrane potential, measured in the current-clamp mode, was recorded continuously, and only detector smooth muscle cells that had a stable resting membrane potential for >2 minutes and exhibited no further change in the input resistance were used. The resting membrane potential as determined in the whole-cell configuration was -48±2 mV (n=30) in cultured rat aortic smooth muscle cells and -55.3±4 mV (n=9) in freshly isolated porcine coronary smooth muscle cells. Continuous superfusion with PSS (1 mL/min) did not alter the resting membrane potential.

Measurement of 6-Keto-PGF₁
The release of PGI₂ from porcine coronary arteries was assessed by measurement of its stable metabolite, 6-keto-PGF₁, in the coronary intraluminal solution by radioimmunoassay. Intraluminal samples from coronary artery segments were collected immediately after stimulation with either a single-step increase in intraluminal pressure (P, 90 mm Hg; 2 minutes), pulsatile changes in intraluminal pressure (P, 90 mm Hg; 2 minutes, 1 Hz), or bradykinin (0.1 µmol/L, 1 minute).

Statistics
Unless indicated otherwise, all data in the figures and in the text are expressed as the mean±SEM of n experiments. Statistical evaluation was performed by two-sided Student's t test with a value of P<.05 being considered statistically significant.

	Results

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Effect of Pulsatile Stretch–Induced EDHF Formation on Vascular Compliance
Changes in the amplitude of the pressure-induced oscillations in the external diameter of perfused coronary artery segments were assessed in order to determine the effect of the pulsatile stretch–induced release of EDHF on vascular tone. In the combined presence of L-NNA (100 µmol/L) and diclofenac (10 µmol/L), rhythmic oscillations in intraluminal pressure (P, 40 to 50 mm Hg; 4 minutes, 1.5 Hz) elicited simultaneous changes in vessel diameter (Fig 1A and Table). The amplitude of the diameter oscillations, which is a determinant of the segment compliance, was markedly and reversibly reduced after the addition of the selective K⁺_Ca channel inhibitors, iberiotoxin (10 nmol/L) and apamin (100 nmol/L), to the extraluminal solution (Fig 1A). Coronary artery segments were also exposed to pulsatile stretch (P, 40 to 50 mm Hg; 4 minutes, 1.5 Hz) before and after removal of the endothelium. Endothelial denudation, achieved by perfusion with CHAPS (0.3%, 3 minutes), resulted in a decrease in the amplitude of diameter oscillations. Under these experimental conditions, the application of the K⁺_Ca channel inhibitors failed to affect segment compliance (Fig 1B and Table). The latter effect cannot be attributed to a nonspecific effect of the K⁺_Ca channel inhibitors on vascular tone, since identical results were obtained using segments preconstricted to different levels (data not shown).

View larger version (92K): [in this window] [in a new window]   Figure 1. Original tracings illustrating the effect of pulsatile changes in transmural pressure (P) on the corresponding oscillations in the external diameter (D) of porcine coronary artery segments. Sinusoidal volume changes (1.5 Hz) were applied to an endothelium-intact (A) and an endothelium-denuded (B) coronary artery segment in the absence, presence, and after the removal of iberiotoxin (10 nmol/L) and apamin (100 nmol/L) from the extraluminal solution. Experiments were carried out in the continuous presence of L-NNA (100 µmol/L) and diclofenac (10 µmol/L). The traces shown were recorded at 15-minute intervals and are representative of three additional experiments. Note that because of the asymmetric arrangement of the pen recorder, there is an artificial delay between the truly simultaneous pressure and diameter recordings.

View this table: [in this window] [in a new window]   Table 1. Statistical Summary of Effects of Rhythmic Vessel Distension (P, 40 mm Hg; 4 minutes, 1.5 Hz) on Strain and Compliance of E+ and E- Porcine Coronary Arteries

Patch-Clamp Bioassay of EDHFs
The luminal release of EDHF(s) from porcine coronary artery segments in response to rhythmic distension of the vascular wall was assessed by monitoring the changes in membrane potential of detector vascular smooth muscle cells stimulated with an aliquot of the intraluminal solution withdrawn from segments subjected to rhythmic vessel distension.

In the combined presence of diclofenac (10 µmol/L) and L-NNA (100 µmol/L), rhythmic distension of coronary segments (2 minutes, 1 Hz) elicited the production of a labile factor that induced a transient membrane hyperpolarization in cultured rat aortic smooth muscle cells (Fig 2A). The magnitude of the hyperpolarization of the detector was proportional to the amplitude of the changes in intraluminal pressure applied to the donor and hence to the extent of vessel distension. The membrane hyperpolarization evoked by the luminal incubate from segments subjected to pulse pressure amplitudes of 30 and 60 mm Hg was in the same range as that elicited by the luminal incubate from bradykinin (0.1 and 1.0 µmol/L, respectively)–stimulated segments. Rhythmic pressure oscillations of 90 mm Hg elicited a hyperpolarization that, under the experimental conditions used, was markedly greater than that induced by EDHF released in response to supramaximal concentrations of bradykinin (Fig 2B). The same experiments performed in the absence of L-NNA and diclofenac yielded hyperpolarizations that were also proportional to the magnitude of the change in applied intraluminal pressure but were greater than the hyperpolarizations observed under conditions of combined NO synthase/cyclooxygenase blockade (Fig 2C). The changes in membrane potential were 1.6±0.15 versus 2.2±0.30 mV at P of 30 mm Hg, 3.27±0.20 versus 4.08±0.58 mV at P of 60 mm Hg (P<.05), 6.52±0.40 versus 9.40±0.51 mV at P of 90 mm Hg (P<.01), and 3.10±0.36 versus 4.10±0.20 mV in response to 1 µmol/L bradykinin (P<.05), in the presence versus the absence of L-NNA and diclofenac, respectively. Identical results were obtained using freshly prepared porcine coronary smooth muscle cells as detectors (Fig 3), with a maximal hyperpolarization of 5.7±1 mV being recorded in response to the luminal incubate from rhythmically stretched segments (P, 90 mm Hg; 2 minutes, 1 Hz).

View larger version (34K): [in this window] [in a new window]   Figure 2. Hyperpolarization elicited by a factor released from native porcine coronary endothelial cells in the absence and presence of combined NO synthase/cyclooxygenase blockade. A and B, Original tracings (A) and statistical summary (B) illustrating the hyperpolarization of rat aortic smooth muscle cells induced by the luminal perfusate removed from coronary artery segments subjected to either rhythmic changes in intraluminal pressure (1 Hz, 2 minutes; P, 30 to 90 mm Hg) or the receptor-dependent agonist bradykinin (0.1 or 1 µmol/L). Experiments were carried out in the presence of L-NNA (100 µmol/L) and diclofenac (10 µmol/L) and are expressed as the mean±SEM of five separate experiments. Vm indicates membrane potential. C, Statistical summary of the hyperpolarization of rat aortic smooth muscle cells induced by the luminal perfusate removed from coronary artery segments subjected to either rhythmic changes in intraluminal pressure (1 Hz, 2 minutes; P, 30 to 90 mm Hg) or the receptor-dependent agonist bradykinin (1 µmol/L). Experiments were carried out in the absence of L-NNA and diclofenac and are expressed as the mean±SEM of eight separate experiments.

View larger version (13K): [in this window] [in a new window]   Figure 3. Hyperpolarization of porcine coronary smooth muscle cells by a factor released from native porcine coronary endothelial cells. Original tracings illustrate the hyperpolarization of freshly isolated coronary smooth muscle cells induced by the luminal perfusate removed from coronary artery segments maintained under nonpulsatile conditions (A) and subjected to rhythmic changes in intraluminal pressure (1 Hz, 2 minutes; P, 90 mm Hg) (B). E+ donor indicates endothelium-intact donor artery. Experiments were carried out in the presence of L-NNA (100 µmol/L) and diclofenac (10 µmol/L) and are representative of experiments carried out with nine different cell preparations.

In order to determine whether the production of the hyperpolarizing factor exhibited tachyphylaxis, experiments were performed using repetitively stimulated donor segments (up to five stimulations with an interval of 5 minutes between stimulations). Application of intraluminal solution from mechanically stimulated coronary segments (P, 90 mm Hg; 2 minutes, 1 Hz) to detector smooth muscle cells resulted in transient, but highly reproducible, hyperpolarizations, which displayed no tachyphylaxis (Fig 4A). Similarly, the magnitude of smooth muscle cell hyperpolarization was unaltered by the repetitive application of the intraluminal solution from different arteries (not shown). These observations demonstrate that smooth muscle cells do not become desensitized to the hyperpolarizing factor contained in the intraluminal solution. The hyperpolarizing factor was produced exclusively by the endothelium as treatment of the donor artery with CHAPS (0.3%, 3 minutes), to remove the endothelium, abrogated the hyperpolarization observed in response to either rhythmic vessel distension (Fig 4B) or the receptor-dependent agonist bradykinin (0.1 µmol/L, not shown).

View larger version (12K): [in this window] [in a new window]   Figure 4. Pulsatile stretch–induced release of an EDHF from native porcine coronary endothelial cells. Original tracings illustrate the effects of the intraluminal perfusate removed from coronary artery segments subjected to rhythmic pressure changes on the membrane potential of cultured vascular smooth muscle cells. A, Change in membrane potential observed in response to intraluminal solution removed from an endothelium-intact donor artery (E+ donor) subjected twice to pulsatile stretch (1 Hz, 2 minutes; P, 90 mm Hg) after an interval of 5 minutes. B, Change in membrane potential observed in response to intraluminal solution removed from a donor artery subjected to pulsatile stretch (1 Hz, 2 minutes; P, 90 mm Hg) before and after perfusion with CHAPS (0.3%, 3 minutes) to remove the endothelium. E- donor indicates endothelium-denuded artery. Experiments were carried out in the continuous presence of L-NNA (100 µmol/L) and diclofenac (10 µmol/L), and the tracings shown are representative of six separate experiments.

Pharmacological Characterization of the Diclofenac/L-NNA–Insensitive EDHF
Experiments were designed to determine the half-life of the transferable EDHF synthesized in porcine coronary arteries after stimulation with either bradykinin (0.1 µmol/L) or rhythmic vessel distension (P, 90 mm Hg; 2 minutes, 1 Hz). In the case of bradykinin stimulation, donor segments and detector cells were arranged so that the effluent from perfused coronary arteries superfused detector smooth muscle cells.^{2 9} The transit time between donor and detector cells was varied using polyethylene or glass tubing of various lengths. In the case of rhythmic vessel distension, the luminal incubate was withdrawn from the donor segment and maintained at 37°C for various times before application to the detector cells. Both experimental procedures revealed a half-life of EDHF of 70 seconds.

In order to determine whether the EDHF synthesized and released in response to rhythmic vessel wall distension displayed pharmacological characteristics similar to those released after agonist stimulation, the effects of the P-450 inhibitor, 17-ODYA (3 µmol/L), and the K⁺_Ca inhibitors, iberiotoxin (10 nmol/L) and TBA (3 mmol/L), on the EDHF-mediated hyperpolarization were investigated. Rhythmic vessel distension (P, 90 mm Hg; 2 minutes, 1 Hz) elicited the production of a factor that induced hyperpolarization of detector smooth muscle cells. The magnitude of this hyperpolarization was markedly reduced when donor segments were pretreated with 17-ODYA (3 µmol/L, Fig 5). 17-ODYA did not affect the EDHF-induced hyperpolarization when applied only to the detector cells. The application of either iberiotoxin (10 nmol/L) or TBA (3 mmol/L) to detector cells significantly attenuated the hyperpolarization induced by the incubate from rhythmically stretched porcine coronary arteries (Fig 5). Identical results were obtained when the release of EDHF was elicited by bradykinin (0.1 µmol/L, data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 5. Effects 17-ODYA, iberiotoxin (Ib-Tx), and TBA on the EDHF-induced hyperpolarization of cultured rat aortic smooth muscle cells. Changes in membrane potential (Vm) were assessed in cultured rat aortic smooth muscle cells in response to the application of the luminal incubate from coronary arteries subjected to subjected to rhythmic pressure changes (1 Hz, 2 minutes; P, 90 mm Hg). Experiments were performed in the continuous presence of L-NNA (100 µmol/L) and diclofenac (10 µmol/L) and in the absence (open bar) and presence of 17-ODYA (3 µmol/L, hatched bar) in the luminal incubate and in the absence and presence of either IbTx (10 nmol/L, crosshatched bar) or TBA (3 mmol/L, solid bar) in the detector cell perfusate. Results are expressed as the mean±SEM of four or five separate experiments. **P<.01 and ***P<.001 vs respective control (CTL).

Effect of Rhythmic Vessel Distension on 6-Keto-PGF₁ Release From Porcine Coronary Arteries
The liberation of arachidonic acid, the putative precursor of EDHF, was assessed by the accumulation of the stable PGI₂ metabolite, 6-keto-PGF₁, in the effluent from segments exposed to either a single-step change in intraluminal pressure (P, 90 mm Hg; 2 minutes), pulsatile pressure oscillations (P, 90 mm Hg; 2 minutes, 1 Hz), or the receptor-dependent agonist bradykinin (1 µmol/L, 1 minute). A step change in intraluminal pressure failed to increase 6-keto-PGF₁ levels above those detected in unstimulated segments, whereas pulsatile stretch induced a 5-fold increase in 6-keto-PGF₁ accumulation (Fig 6). An increase in 6-keto-PGF₁ equivalent to that induced by rhythmic vessel distension was observed after stimulation of the coronary segments with bradykinin (1 µmol/L).

View larger version (35K): [in this window] [in a new window]   Figure 6. Effects of rhythmic vessel distension on the accumulation of 6-keto-PGF1 in the intraluminal solution from porcine coronary artery segments. Porcine coronary artery segments were incubated in PSS under resting conditions (2 minutes, open bars) and subjected to either a step increase in intraluminal pressure (P 90 mm Hg, 2 minutes), rhythmic pressure changes (P, 90 mm Hg; 2 minutes, 1 Hz) or the receptor-dependent agonist bradykinin (Bk, 1 µmol/L; 1 minute). Results are expressed as the mean±SEM of four separate experiments. **P<.01 vs respective control.

	Discussion

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The results of the present study demonstrate that pulsatile stretch is a potent stimulus for the synthesis and release of EDHF from isolated coronary arteries. Indeed, pulsatile changes in intraluminal pressure within the physiological range were more effective than supramaximal concentrations of bradykinin in eliciting EDHF production. Moreover, in contrast to agonist-stimulated EDHF production, which is frequently subjected to desensitization, the response to pulsatile stretch did not undergo any apparent tachyphylaxis.

Although receptor-dependent agonists have mainly been used to elicit the synthesis of endothelium-derived vasodilator and vasoconstrictor substances and to characterize their actions, mechanical forces such as fluid shear stress and pulsatile stretch are likely to be more physiologically relevant stimuli for the continuous generation of the endothelium-derived autacoids.^{11 12 13 14} Besides the continuously acting fluid shear stress imposed by the streaming blood, the periodic distension and compression of arteries during the cardiac cycle is known to enhance the formation of NO and PGI₂.^{11 15 16 17} Although high concentrations of NO have been reported to directly activate large conductance K⁺_Ca channels in rabbit aortic smooth muscle cells¹⁸ and to induce hyperpolarization in coronary arteries under certain experimental conditions,¹⁹ controversy exists as to whether biologically relevant concentrations of NO are able to induce the hyperpolarization of vascular smooth muscle cells.^{2 20 21 22 23} In the present study, we clearly identified a diclofenac L-NNA–sensitive component of the smooth muscle hyperpolarization induced by the intraluminal solution from coronary arteries exposed to rhythmic vessel distension. Despite this apparent NO- and/or PGI₂-mediated effect, >60% of the hyperpolarization elicited by the intraluminal solution from rhythmically stretched segments was insensitive to combined NO synthase/cyclooxygenase blockade. This residual hyperpolarization cannot be attributed to the lack of complete inhibition of the NO synthase, since we have demonstrated that oxyhemoglobin and a selective soluble guanylyl cyclase inhibitor, in concentrations that abrogated NO-mediated dilation, were unable to modulate the hyperpolarization induced by an EDHF-containing solution from bradykinin-stimulated coronary artery segments.² Indeed, it has recently been reported that the production of EDHF is actually inhibited in the presence of NO and that only under conditions of impaired NO synthesis can a maximal EDHF-like response be demonstrated.^{2 24} These findings, taken together with the pharmacological characterization discussed below, suggest that most of the "endothelium-dependent" hyperpolarization observed in response to mechanical stimulation was elicited by a labile and transferable hyperpolarizing factor chemically distinct from NO and PGI₂.

In endothelium-intact coronary arteries, the initiation of pulsatile changes in transmural pressure induced simultaneous oscillations in diameter, the amplitude of which changed in a biphasic manner. The peak and steady-state levels of diameter oscillation, which at a constant pulse pressure can be taken as a measure of arterial compliance, were also evident under conditions of combined NO synthase/cyclooxygenase inhibition. Removal of the endothelium abolished the peak and reduced the amplitude of the steady-state diameter oscillations. These observations suggest that an endothelium-derived factor other than NO or PGI₂ plays a crucial role in the regulation of arterial compliance during rhythmic vessel distension. Indeed, using iberiotoxin and apamin to prevent EDHF-induced K⁺_Ca channel activation abolished the peak and attenuated the steady-state level of diameter oscillations to the same extent as that observed after removal of the endothelium. It is highly plausible that the profile of EDHF release, as intimated by the biphasic change in arterial compliance, is determined by increases in the endothelial [Ca²⁺]_i after the initiation of rhythmic vessel distension and that the peak and steady-state levels of EDHF production mirror the Ca²⁺ response. However, it is impossible to differentiate effects attributed to changes in the production of EDHF and changes in the sensitivity of the smooth muscle cells to this factor using isolated perfused segments. For this reason, we performed experiments to determine whether tachyphylaxis to EDHF occurs using a patch-clamp bioassay and observed that the hyperpolarization of vascular smooth muscle cells to the EDHF-containing perfusate did not exhibit a marked tachyphylaxis. Taken together, these observations spotlight a potentially crucial function of EDHF in vascular dynamics. Indeed, should this be a general phenomenon observed in other vascular beds, this would imply that EDHF could continuously modulate arterial hemodynamics by maintaining an adequate arterial compliance. A definitive demonstration of the effects of EDHF on pulse-wave velocity as well as other parameters of wave propagation is, at the present time, hampered by the difficulties in studying pure EDHF-mediated responses in vivo.

There is now evidence to suggest that a constrictor P-450–dependent metabolite of arachidonic acid, 20-HETE, produced by vascular smooth muscle cells is involved in the development of myogenic tone.²⁵ 20-HETE increases smooth muscle tone by inhibiting large-conductance K⁺_Ca channels, inducing depolarization, and increasing [Ca²⁺]_i, probably by activating L-type Ca²⁺ channels.^{26 27 28} It is tempting to speculate that the net activity of large-conductance K⁺_Ca channels in arterial segments is determined by the balance in the vascular production of 20-HETE and EDHF and that EDHF affects vascular tone by counteracting the 20-HETE–induced inhibition of K⁺_Ca channels.

Although pulsatile stretch was a potent stimulus for the release of EDHF, a single step increase in intraluminal pressure had no effect on the magnitude of the subsequently observed smooth muscle cell hyperpolarization. However, as previously reported, increases in intraluminal pressure from 5 to 40 or 5 to 60 mm Hg significantly increased the duration of the EDHF-induced hyperpolarization elicited by bradykinin.⁹ It is likely that the different effects of a single step change and pulsatile changes in intraluminal pressure can be attributed to the kinetics of the respective Ca²⁺ responses. Indeed, changes in endothelial [Ca²⁺]_i are only transient after a step increase in fluid shear stress,^{13 29 30} whereas oscillatory or pulsatile flow is associated with a maintained increase in [Ca²⁺]_i.²⁹

The EDHF produced by carotid and coronary arteries displays characteristics similar to those of a cytochrome P-450–derived metabolite of arachidonic acid.^{4 5 6 7 8 31} Moreover, induction of cytochrome P-450 enzymes using ß-naphthoflavone enhances the release of EDHF from cultured porcine and human endothelial cells.⁹ The observations that pulsatile stretch enhanced the liberation of arachidonic acid, as assessed by the accumulation of 6-keto-PGF₁, and that 17-ODYA attenuated the pulsatile stretch–induced release of EDHF from donor arteries support this hypothesis. The finding that pulsatile stretch increased 6-keto-PGF₁ is only indirect evidence that this mechanical stimulus increases the liberation of arachidonic acid from membrane phospholipids. However, although this marker is not an ideal indicator of arachidonic acid release, there is no appropriate alternative for assessing its liberation from intact coronary artery segments. We have no reason to suspect that the increase in 6-keto-PGF₁ occurred as a consequence of endothelial cell damage, as assessed by trypan blue exclusion and LDH release, and we have been unable to detect expression of the inducible cyclooxygenase in any of our preparations.

Although concern has been expressed regarding the nonselective effects of some P-450 inhibitors (ie, clotrimazole and econazole) on K⁺_Ca channel activity^{32 33 34} and endothelial Ca²⁺ signaling,³⁵ it should be stressed that 17-ODYA exhibits neither of these effects and has been shown to selectively inhibit the production of EDHF rather than interfere with its action on smooth muscle cells.⁹ The hyperpolarization of vascular smooth muscle cells elicited by EDHF was inhibited after the preincubation of detector cells with iberiotoxin and TBA, findings that are consistent with reports that EDHF increases the K⁺ conductance of vascular smooth muscle cells by activating K⁺_Ca channels. Moreover, since EDHF activated K⁺_Ca channels in cell-attached membrane patches,⁹ it would appear that this factor activates K⁺ channels in an indirect manner possibly involving membrane-associated second-messenger pathways.

It is likely that the term EDHF encompasses more than one factor, since the hyperpolarizing factor released from the rabbit aorta,³⁶ guinea pig carotid,³⁷ and rat mesenteric and hepatic arteries^{33 38} or the rat portal vein³⁴ exhibits pharmacological characteristics very different from those of the "coronary EDHF." Recently, a specific (CB1) cannabinoid receptor antagonist has been shown to attenuate EDHF-induced relaxation of isolated rat mesenteric vessels, whereas anandamide (arachidonoylethanolamide), an endogenous ligand for central cannabinoid receptors,^{39 40} elicited "EDHF-like" relaxations.⁴¹ However, although anandamide and the CB1 agonist, HU 210, induced relaxation of mesenteric vessels by a cyclooxygenase-dependent mechanism, these compounds did not induce the EDHF-like (L-NNA/diclofenac–insensitive) dilation of either porcine coronary arteries or rabbit carotid and mesenteric arteries (authors' unpublished data, 1997). Despite our failure to observe any direct cyclooxygenase-independent effect of anandamide, the hypothesis that this endogenous cannabinoid is identical to EDHF remains attractive and does not necessarily exclude a role for cytochrome P-450 monooxygenases in the synthesis of this factor. Indeed, anandamide can be enzymatically synthesized from, and may well prove to be a carrier of, arachidonic acid,⁴² the putative precursor of EDHF. Moreover, anandamide can be metabolized by cytochrome P-450, and the induction of P-450 enzymes results in the increased formation of several anandamide metabolites.⁴³

In summary, we have demonstrated that pulsatile stretch/rhythmic vessel distension elicits the release of an EDHF that exhibits a half-life in physiological solution and pharmacological characteristics identical to that of the EDHF released after stimulation with the receptor-dependent agonist bradykinin. Moreover, the release of EDHF, as assessed by the magnitude of the hyperpolarization of detector smooth muscle cells, was much greater in response to pulsatile stretch than in response to supramaximal concentrations of bradykinin and did not exhibit any apparent tachyphylaxis. Thus, mechanical stimulation of arteries during the course of the cardiac cycle is likely to ensure the continuous release of this potent endothelium-derived autacoid, which may contribute not only to the local regulation of blood flow but also to an adequate arterial compliance.

	Selected Abbreviations and Acronyms

 

EDHF = endothelium-derived hyperpolarizing factor K+Ca channel = Ca2+-dependent K+ channel 6-keto-PGF1 = 6-ketoprostaglandin F1 L-NNA = NG-nitro-L-arginine 17-ODYA = 17-octadecynoic acid P = intraluminal pressure change PGI2 = prostaglandin I2 PSS = physiological salt solution TBA = tetrabutylammonium U46619 = 9,11-dideoxy-11,9-epoxymethanoprostaglandin F2

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (Bu 436/6-1 and Po 521/1) and the Commission of the European Communities (BMH4-CT96-0979). The authors are indebted to Isabel Winter and Michaela Stächele for expert technical assistance.

Received August 29, 1997; accepted December 31, 1997.

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