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Am J Physiol Heart Circ Physiol 291: H1521-H1528, 2006. First published April 28, 2006; doi:10.1152/ajpheart.00027.2006
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Continuous flow augments reactivity of rabbit carotid artery by reducing bioavailability of NO despite an increase in release of EDHF

Lasse E. Rasmussen, Paul M. Vanhoutte, Boye L. Jensen, and Ole Skøtt

Department of Physiology and Pharmacology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark

Submitted 6 January 2006 ; accepted in final form 24 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experiments were designed to investigate the influence of steady flow and pressure on endothelial function in the rabbit carotid artery. Increases and decreases in isometric force were compared in static rings and perfused (5 or 50 ml/min) segments of the same arteries in the presence and absence of endothelium. The {alpha}1-adrenoceptor agonist phenylephrine and the muscarinic agonist acetylcholine were applied as vasoconstrictor and vasodilator stimuli, respectively. Continuous flow (5 and 50 ml/min) reduced the cGMP content and shifted the concentration-response curve to phenylephrine to the left compared with nonperfused static rings. Removal of the endothelium abolished the differences in cGMP content and the sensitivity to phenylephrine between static rings and perfused segments. No difference in sensitivity to phenylephrine was observed in tissues treated with N{omega}-nitro-L-arginine methyl ester (L-NAME). Acetylcholine-evoked relaxations were increased in perfused segments. L-NAME nearly abolished the acetylcholine-evoked relaxation in static rings, whereas about one-half of the relaxation remained in segments exposed to flow. This remnant relaxation was blocked by inhibition of endothelial small- and intermediate-conductance calcium-activated potassium channels by apamin plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). These experiments demonstrate that continuous flow increases the constriction evoked by {alpha}1-adrenergic activation in the rabbit carotid artery through a reduced influence of basally released endothelial NO and, furthermore, that luminal flow unmasks an ability of the endothelium to release a non-NO, noncyclooxygenase vasodilator, presumably endothelium-derived hyperpolarizing factor.

endothelial shear stress; nitric oxide; endothelium-derived hyperpolarizing factor; conduit arteries; phenylephrine

THE ENDOTHELIUM CONTROLS the tone of the underlying vascular smooth muscle by releasing endothelium-derived relaxing factors [EDRFs, mainly nitric oxide (NO)] and by causing endothelium-dependent hyperpolarization (EDHF-mediated responses). In most large blood vessels, prostacyclin appears to play a minor role as an EDRF (9, 19). The impact of increases in flow and shear stress on the basal and stimulated release of the endothelial factors is uncertain. In the canine femoral artery, a sudden increase in flow augments the basal luminal release of EDRF (27). In the perfused aorta of normotensive and spontaneously hypertensive rats, increments in flow decrease the amount of EDRF released in response to acetylcholine (14). In porcine coronary conduit arteries, the dilatation to acute changes in flow is independent of NO (7, 27). Middle cerebral arteries of the rat constrict when exposed to flow, a response that is attenuated in the presence of the endothelium (5). Likewise, in rabbit femoral arteries, flow causes a constriction (4, 15, 31). The reason for these apparent discrepancies include differences in experimental protocols where little attention has been paid to dissociate flow-induced circumferential stretch (evoked by an increased flow as a result of an increase in perfusion pressure) from the effect of flow (shear stress) per se. Likewise, little attention has been paid to dissociate the effect on the influence of NO- and EDHF-mediated dilatations of exposure to sustained versus acute changes in flow. Indeed, a sudden increase in flow is mediated by an abrupt change in perfusion pressure that could stretch and thereby stimulate the endothelial cells (16, 22, 26). In addition, several studies (14, 17, 27) were carried out in the presence of indomethacin to block the formation of vasoactive prostanoids. Oxyhemoglobin is a scavenger of NO (18, 21), thus in vivo, luminal NO would be of little importance for regulation of downstream tone, and it could be argued that accurate measurement of endothelial function requires the direct study of the underlying vascular smooth muscle.

The present experiments were designed to determine the effects of sustained flow on the ability of the endothelium to influence tone of the underlying vascular smooth muscle with regard to both basal and stimulated release of NO- and EDHF-mediated responses. This was done under conditions where the impact of sustained flow could be dissociated from that of changes in intraluminal pressure. To assess the importance of basally released EDRFs, we determined the consequences of removing the endothelium and inhibiting the NO- and EDHF-mediated components on the responsiveness to {alpha}1-adrenergic stimulation, in view of the dominant role of the sympathetic nervous system in the control of blood pressure (28). The importance of NO was addressed by inhibition of its formation with the NO synthase (NOS) inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) and that of EDHF by inhibition of the endothelial small- (SKCa) and intermediate-conductance calcium-activated potassium channel (IKCa) inhibitors with apamin plus 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), respectively (8, 9, 36). A possible role of prostacyclin was ruled out by inhibition of its formation with the nonselective cyclooxygenase inhibitor indomethacin.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Modified Krebs-Henseleit solution. The composition was (in mM) 115 NaCl, 25 NaHCO3, 1.2 MgSO4, 2.5 K2HPO4, 1.3 CaCl2, and 5.5 glucose. The solution was maintained at 37°C and was continuously equilibrated with 5% (vol/vol) CO2 in air (pH 7.4) through a microfilter-glass candle with a permeable tip (ROBU Glasfilter, Hattert, Germany). The pH was measured with a pH-meter (PHM-38; Radiometer, Copenhagen, Denmark). Osmolality was measured with an osmometer (Advanced Instruments, Norwood, MA) before and after the experiment. Temperature was measured with a NTC resistor connected to a temperature controller unit (Jens Ole Pedersen, University of Southern Denmark, Denmark) connected to a data acquisition and analysis unit (Powerlab/800; AD Instruments, Castle Hill, New South Wales), allowing simultaneous measurement of perfusate temperature and force.

Tissue preparation. Rabbit common carotid arteries were prepared as described previously (25). New Zealand White male rabbits (mean weight: 3,120 ± 65 g) obtained from Harlan AD (France) were killed by a blow to the head and exsanguinated. This procedure conformed to the American Veterinary Medical Association guidelines for euthanizing laboratory animals (1). Animal care followed the guidelines of the National Institute of Health, and permissions were obtained from the Danish Animal Experiments Inspectorate under the Danish Ministry of Justice. The carotid arteries were removed from each side, immersed in cold (1–2°C) modified Krebs-Henseleit solution, and cleaned of fat and connective tissue. The arteries were divided into segments of either 10 or 4 mm in length and used for perfusion or static ring experiments. In some preparations, the endothelium was removed by rubbing the luminal surface with 21-gauge syringe needles that had been bent and rebent to obtain a rough surface. If after this procedure, acetylcholine (10–6 M) resulted in a 10% relaxation or more during contractions to phenylephrine (10–5 M), the tissue was discarded. If the arteries did not contract at least 2 g upon exposure to 10–5 M phenylephrine, they were discarded.

Static preparations. Rings (4 mm wide) of rabbit carotid arteries were suspended by two platinum pins passed through the lumen in a double-jacketed tissue bath filled with 50 ml of modified Krebs-Henseleit solution at 37°C and aerated with 5% CO2 in air. The isometric mechanical tension response was recorded with a strain-gauge transducer (type SG 4–180; Swema, Stockholm, Sweden) connected to the data acquisition unit (Powerlab/800; AD Instruments), which registered the converted signal in grams. After mounting, a tension of 1 g was applied. The segments were washed once with modified Krebs-Henseleit solution during a 60-min equilibration period. After the rings were suspended, there was a drop in tension, which was adjusted three times during the equilibration period to obtain a tension of 1 g in the tissue before the actual experiment. The contraction to 10–5 M phenylephrine was then measured, followed by the response to 10–6 M acetylcholine, to test endothelial viability. Measured values were corrected for the initial (passive) tension in the absence of vasoconstrictor agents to quantify development of active force as increases in tension. After washout, agonists were added cumulatively. Additions were made whenever a steady response was obtained to the preceding concentration. The effect of the agonist was considered to be maximal when at least a threefold increase in concentration failed to cause a further increase in tension. When pharmacological inhibitors were used, an incubation period of 30 min was allowed before starting the cumulative concentration-response curve to the tested agonist.

Perfused preparations. Segments (10 mm long) were kept in an isolated organ bath containing 50 ml of modified Krebs-Henseleit solution at 37°C. They were perfused with two heated (37°C) tubes connected via silicone hoses to chambers containing modified Krebs-Henseleit solution, 20–80 cm above the arterial segment. The vertical fluid column height determined the intraluminal pressure. A pump pumping fluid from one chamber to the other created a pressure difference between the chambers whereby the desired flow rate was obtained. At equilibrium, the flow rate through the artery was the same as the flow rate created by the roller pump. Adjustments in flow rate were obtained by changing the speed of the roller pump. During the experiments, segments were exposed to flow from the time of suspension until the end of the experiment (4–6 h). During the experiments, the perfusion pressure was kept at the same level when the blood vessels contracted. For measurement of tension, two 27-gauge stainless steel needles (B. Braun, Melsungen, Germany), were bent and inserted in the lumen through the artery wall. There was no fluid leakage from the artery. One needle was fixed and the other connected to a transducer (type SG 4–180; Swema). Isometric tension was recorded in the same manner as for the static rings. The segments were exposed to flow (5 or 50 ml/min) throughout the entire duration of the experiment, and the responses to phenylephrine and acetylcholine were assessed by adding the drugs to both the perfusate and the superfusate. To investigate whether the flow-mediated effects on endothelial function were reversible, segments were suspended as described above and exposed to sustained flow (50 ml/min) for 2 h, after which the segments were removed from the perfusion setup and suspended in the static ring setup, where the responses to phenylephrine and acetylcholine were measured in the absence and presence of L-NAME.

Pressurized preparations without flow. Pressurized preparations were suspended and treated as the perfused segments, except that the roller pump was stopped. The fluid column height (in cm; 1 cmH2O = 0.74 mmHg) was set to determine the intraluminal pressure to 15, 30, or 55 mmHg. The vasoconstrictor was added only to the superfusate. L-NAME was added to both the perfusate and superfusate. By adding L-NAME to the upper chambers and turning on the roller pump for 10 min, we ensured that the concentration of the drug was homogenous in the entire perfusate volume. After the pump was stopped, the pressurized vessels equilibrated for 30 min before phenylephrine was added again.

Measurement of tissue cGMP. Perfused and static segments with and without endothelium were suspended as described above. After the equilibration procedure and the test for endothelial viability, the arteries were exposed to 10–6 M phenylephrine. When a steady contractile response was obtained, the tissue was transferred to liquid nitrogen and stored at –80°C. To increase yield, tissue from two experiments were pooled and homogenized with the same mortar and pestle in liquid nitrogen. The cGMP content was the measured with a cGMP EIA kit (catalog no. 581021.1; Cayman Chemicals) according to the manufacturer's instructions. Briefly, the pulverized tissue was transferred to a test tube containing 200 µl of 5% trichloroacetic acid in water. The trichloroacetic acid of the supernatant was then extracted with ether, and the remaining cGMP was acetylated. The pellet was then dried to determine the dry weight. The cGMP content was expressed as femtomoles per milligram dry weight.

Drugs. Acetylcholine hydrochloride, apamin, indomethacin, L-NAME, and phenylephrine hydrochloride were purchased from Sigma Chemical (St. Louis, MO). 1-[(2-Chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) was a kind gift of Dr. H. Wulff (University of California, Irvine, CA).

Shear stress. Vascular wall shear stress was estimated using the Hagen-Poiseuille equation (Eq. 1), assuming laminar flow and Newtonian behavior:

Formula 1(1)
where {tau}wall is the wall shear stress [in pascals (Pa)], {eta} is the viscosity (Pa·s), Q is the flow rate (m3/s), and r is the radius (m). Pascals were converted to dyn/cm2 as 1 Pa = 10 dyn/cm2.

Calculations and statistical analysis. Data are expressed as means ± SE, and n refers to the number of animals from which arteries were obtained. Log concentration-response curves are plotted. To obtain EC50 values, data are expressed as percentages of the maximally evoked active tension to the {alpha}1-adrenoceptor agonist. Differences between mean values were evaluated using Student's t-test for unpaired observations. In the case of unequal variance between the mean values compared (evaluated with a variance ratio test), a t-test for unequal variance was used. Comparisons among three or more mean values were assessed using one-way analysis of variance followed by Bonferroni's multiple comparison test. The statistical calculations were performed with GraphPad Prism (San Diego, CA). Significance was accepted at the 0.05 level of probability.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Physicochemical environment. Temperature, pH, and osmolality were constant during the experiments (data not shown).

Shear stress. The inner diameter of the rabbit common carotid artery varied from 1.5 to 2.0 mm. Based on the viscosity of water (0.001 Pa·s), a flow rate of 5 ml/min resulted in a calculated shear stress of 1–2 dyn/cm2, and a flow rate of 50 ml/min resulted in a shear stress of 10–20 dyn/cm2.

Endothelium, flow, and resting tension. The decay in tension of the static rings with endothelium was more pronounced than that of those without endothelium, measured as the time-dependent decrease in tension immediately after suspending the preparation (Fig. 1). The decay in tension of segments with endothelium suspended in the perfusion setup and exposed to flow was similar to that of the static ring without endothelium (Fig. 1). The decay in tension was augmented in the absence, compared with the presence, of flow (Fig. 1). L-NAME, indomethacin, and TRAM-34 plus apamin did not cause significant changes in tension in quiescent preparations (data not shown).


Figure 1
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  		Fig. 1. Resting tension of rabbit carotid artery: effect of endothelium and flow. Segments were stretched immediately after suspension, and the tension was measured. The tension immediately after suspension was taken as 100%. Top: static rings with ({circ}) and without (bullet) endothelium, segments with endothelium exposed to flow at 50 ({lozenge}) and 5 ml/min ({blacktriangleup}). Bottom: pressurized segments without flow at 15 ({blacksquare}) and 55 mmHg (bullet) (n = 6–14). Data from top for flow at 50 ml/min ({lozenge}) and static rings ({circ}) are included to facilitate interpretation. The decay in tension was more pronounced in pressurized segments than in segments exposed to flow. *P < 0.05. Flow was initiated immediately after mounting.

 
Tissue cGMP content. The cGMP content per milligram of tissue in the static rings with endothelium was significantly higher than that in segments exposed to flow (5 and 50 ml/min). No difference was observed in the cGMP content between the two levels of flow. Removal of the endothelium decreased the cGMP content to similar low levels in perfused segments and static rings (Fig. 2).


Figure 2
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  		Fig. 2. Tissue cGMP content in static rings and perfused segments with and without endothelium of rabbit carotid artery. Columns represent the tissue mean cGMP content in fmol/mg dry weight of phenylephrine (10–6 M)-contracted arteries. Tissue from 2 experiments was pooled in 1 vial to obtain enough cGMP. Data represent means ± SE of 3–4 vials of tissue from 6–8 animals per condition. *P < 0.05 compared with all others. #P < 0.05 compared with segments with endothelium. NS, nonsignificant difference.

 
Flow and phenylephrine-evoked contractions. Phenylephrine (3 x 10–8 M–3 x 10–5 M) caused a concentration-dependent contraction of perfused segments and static rings (Fig. 3A). The phenylephrine concentration-response curve was shifted significantly to the left by continuous flow [at 5 ml/min (EC50 = 6.2 –log M) and 50 ml/min (EC50 = 6.2 –log M)] compared with the static rings (EC50 = 5.6 –log M) (Figs. 3 and 4).


Figure 3
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  		Fig. 3. Flow-mediated differences in phenylephrine-evoked contractions in isolated rabbit carotid artery. A: untreated arteries with endothelium. B: arteries treated with N{omega}-nitro-L-arginine methyl ester (L-NAME; 10–4 M). C: arteries without endothelium. The increase in active tension is represented as the percentage of the maximal response to the agonist to obtain EC50 values. {blacksquare}, Static rings; {triangledown}, segments perfused with flow at 50 ml/min. Data are means ± SE (n = 6).

 

Figure 4
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  		Fig. 4. EC50 values for phenylephrine in perfused segments and static rings of rabbit carotid artery. Columns represent average phenylephrine EC50 values (–log M) as means ± SE. Series: +L-NAME, tissue treated with L-NAME (10–4 M); +L-NAME + Indomethacin, tissue treated with L-NAME (10–4 M) and indomethacin (10–5 M); +L-NAME + Indomethacin + TRAM-34 + Apamin, tissue treated with L-NAME (10–4 M), indomethacin (10–5 M), 1-[(2-chlorophenyl) diphenylmethyl]–1H-pyrazole (TRAM-34; 10–7 M), and apamin (10–7 M); values also are shown for untreated tissue and tissue without endothelium, as well as segments that were suspended in the perfusion setup and exposed to flow for 2 h before being removed and suspended in the setup for static rings (n =4–6 in each series). *P < 0.05 compared with all other experiments. {dagger}P < 0.05 compared with L-NAME-treated tissue. #Not significantly different from untreated static rings.

 
Endothelium and sensitivity to phenylephrine. Inhibition of NO production with L-NAME (10–4 M) or removal of the endothelium abolished the differences in the responses to phenylephrine between perfused segments and static rings (Figs. 3 and 4). In both conditions of flow, the concentration-response curve to phenylephrine-sensitivity was shifted to the left compared with static rings with intact endothelium. Removal of the endothelium resulted in a further leftward shift of the phenylephrine concentration-response curve than did treatment with L-NAME.

After treatment with L-NAME or L-NAME plus indomethacin, the EC50 values for phenylephrine were similar in perfused segments (5 or 50 ml/min) and static rings (Fig. 4). Endothelial removal led to further reduction of EC50 values for the {alpha}1-adrenergic agonist in all three preparations (Fig. 4) compared with treatment with L-NAME. Treatment of perfused segments or static rings with the inhibitors of small and intermediate potassium channels (apamin plus TRAM-34) did not influence the basal tone (not shown) or phenylephrine-evoked contractions when added to tissue treated with L-NAME plus indomethacin (Fig. 4). In L-NAME (10–4 M)-treated segments exposed to flow (50 ml/min) and contracted with the EC50 value for phenylephrine, the administration of apamin (10–7 M) plus TRAM-34 (10–7 M) evoked an increase in tension (Fig. 5), which averaged 8 ± 1.2% (n = 5).


Figure 5
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  		Fig. 5. Original trace showing the contraction evoked by the inhibition of intermediate- (IKCa) and small-conductance K+ channels (SKCa) in a perfused rabbit carotid artery. Segments were perfused (50 ml/min) and treated with L-NAME (10–4 M) before they were contracted with phenylephrine (6 x 10–7 M, EC50). After a steady contractile response was obtained, TRAM-34 (10–7 M) and apamin (10–7 M) were added to block IKCa and SKCa, respectively. This evoked an 8 ± 1.2% increase in tension (n = 5).

 
Intraluminal pressure in absence of flow. In pressurized segments without flow, phenylephrine evoked a concentration-dependent contraction similar to that observed in the static rings (Fig. 6A). In pressurized (15, 30, and 55 mmHg) segments without flow as in static rings, L-NAME significantly shifted the phenylephrine concentration-response curve to the left (Fig. 6B). In the presence of L-NAME, no significant differences were observed between the EC50 values for phenylephrine in pressurized segments at 15, 30, and 55 mmHg and static rings (data not shown).


Figure 6
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  		Fig. 6. A: phenylephrine concentration-response curve in pressurized (30 mmHg) segments and static rings of rabbit carotid artery before and after treatment with L-NAME. B: columns represent average shift in phenylephrine EC50 (–log M) values evoked by L-NAME (10–4 M) in pressurized segments, static ring, and perfused segments (flow at 5 and 50 ml/min, respectively; data from Fig. 2). Data are means + SE (n = 6). *P < 0.05 compared with static ring.

 
Acetylcholine. Acetylcholine (10–6 M) relaxed perfused segments and static rings (Fig. 7). The relaxation was dependent on the presence of endothelium (data not shown). The relaxation was significantly increased in perfused segments (at 5 and 50 ml/min) compared with the static ring (Fig. 7). L-NAME almost completely inhibited the acetylcholine-evoked relaxation in the static ring, whereas a significantly larger relaxation to the muscarinic agonist remained in the perfused segments at 5 and 50 ml/min (Fig. 7). Indomethacin did not further influence the response to acetylcholine in tissues treated with L-NAME (Fig. 7). Additional treatment with apamin (10–7 M) plus TRAM-34 (10–7 M) in perfused segments treated with L-NAME plus indomethacin nearly abolished the acetylcholine-evoked relaxation (Fig. 7). Apamin plus TRAM-34 had no significant effect on the already blocked response to acetylcholine in static rings treated with L-NAME plus indomethacin (Fig. 7). In indomethacin (10–5 M) treated segments exposed to flow (50 ml/min) acetylcholine (10–6 M) evoked a 54 ± 2% relaxation, and the additional treatment with apamin (10–7 M) plus TRAM-34 (10–7 M) in these segments did not inhibit the relaxation, which averaged 52 ± 2% (n = 6). In L-NAME (10–4 M)-treated segments without indomethacin and exposed to flow (50 ml/min), acetylcholine evoked a 9 ± 1% relaxation in the presence of apamin (10–7 M) plus TRAM-34 (10–7 M) (n = 5).


Figure 7
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  		Fig. 7. Effect of acetylcholine (ACh) in static rings and perfused segments of rabbit carotid artery. Segments were contracted with phenylephrine (3 x 10–5 M). A and B: time traces of the initial ACh (10–6 M)-evoked relaxation (n = 6), expressed as remaining tension (%) of the phenylephrine-evoked contraction (100%). Arteries were untreated (A) or treated with L-NAME (10–4 M) and indomethacin (Indo; 10–5 M) (B). C: comparison of ACh-evoked relaxations after inhibition of the endothelium-derived vasodilators. Columns represent maximal ACh (10–6 M)-evoked relaxation (%) of the phenylephrine-evoked contraction (100%). Interventions: untreated (n = 6); +L-NAME, segments treated with L-NAME (10–4 M) (n = 6); +L-NAME + Indo, segments treated with L-NAME (10–4 M) plus indomethacin (10–5 M) 30 min before the addition of phenylephrine and ACh (n = 6); +TRAM-34 + Apamin, arteries treated with L-NAME (10–4 M), indomethacin (10–5 M), TRAM-34 (10–7 M), and apamin (10–7 M) 30 min before the addition of phenylephrine and ACh (n = 4). *P < 0.05 compared with untreated tissue. {dagger}P < 0.05 compared with untreated segments perfused at 5 or 50 ml/min. #P < 0.05 compared with segments treated with L-NAME + indomethacin.

 
Reversibility. After a 2-h exposure to sustained flow, the rabbit carotid artery segments (n = 4) were suspended in the static ring setup. The EC50 value for phenylephrine was 5.63 ± 0.05 –log M. L-NAME augmented the EC50 values for the adrenergic agonist with 0.45 ± 0.05. In the absence and presence of L-NAME, acetylcholine (10–6 M) evoked a relaxation of 43 ± 1 and 10 ± 1%, respectively. These values were not significantly different from values obtained in static rings that had not previously been exposed to flow.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The present results demonstrate that flow per se is able to increase vasoconstrictor function by reducing the functional bioavailability of NO and, furthermore, that it unmasks an EHDF-mediated relaxation of large arteries.

In the present experiments, a flow of 50 ml/min resulted in a calculated level of shear stress similar to that determined in vivo (29, 30). The flow of 5 ml/min resulted in a shear stress 10 times lower. At a flow of 50 ml/min and an intraluminal diameter of 1.5 mm, the Reynolds number is ~1,000, which suggests laminar flow under the experimental conditions used (28). Although it cannot be excluded that local turbulence occurred distally to the measuring site by the tissue holders at 50 ml/min, it is highly unlikely to occur at 5 ml/min, where the Reynolds number is ~100. Because there were no differences in the vascular responses at 5 and 50 ml/min, local turbulence is not likely to contribute to the present observations.

Flow and functional NO availability. The phenylephrine concentration-response curve was shifted to the left and no longer potentiated by L-NAME in segments exposed to sustained flow (5 or 50 ml/min) compared with pressurized segments without flow and static rings. In the presence of the NOS inhibitor, there were no differences between EC50 values for phenylephrine between the different preparations. This indicates that sustained flow decreases the functional bioavailability of NO and thereby increases vascular reactivity in the rabbit carotid artery. This interpretation is supported further by the reduced cGMP content in segments exposed to flow compared with static rings and by the higher cGMP levels in static rings in the presence of the endothelium. This is consistent with an increased influence of endothelium-derived NO on the sensitivity to phenylephrine in the absence of flow and suggests that the endothelial NOS (eNOS or NOS-3) is the main supplier of NO under basal conditions. Washout of NO in the perfused segments may well contribute to the lower functional bioavailability of the endothelial mediator, but this mechanism must have a limited capacity, because the acetylcholine-evoked relaxation was actually augmented and inhibited by L-NAME.

The geometries of the pressurized segments without flow and the perfused segments exposed to flow were similar. However, L-NAME potentiated the effect of phenylephrine only in pressurized segments. Therefore, it is not likely that the altered functional availability of NO is due to differences in the degree of stretch in the absence or presence of flow. The present findings suggest that in pressurized segments without flow, as in static rings, the endothelium releases NO, which in turn evokes a relaxation of the vascular wall. Consistent with this, there was an accelerated endothelium-dependent decay in basal tension of nonperfused arteries in the initial phase after suspension. Addition of indomethacin [to block cyclooxygenases (32)] and apamin plus TRAM-34 [to block EDHF-mediated responses (12)] did not affect further the sensitivity to phenylephrine. Therefore, when flow is absent, NO is solely responsible for counteracting {alpha}1-adrenoceptor-mediated vasoconstriction. Because the inhibitory effect of the endothelium also is seen in nonperfused static preparations, a differential sensitivity of endothelial {alpha}1-adrenoceptors in the absence and presence of flow is unlikely to cause different levels of stimulation of NOS (11). Because the acetylcholine-evoked relaxation was increased in segments exposed to flow and inhibited by L-NAME, sustained flow does not reduce the ability of NOS-3 to be stimulated. In favor of this interpretation is the finding that the flow-mediated effects on endothelial function were reversible, ruling out permanent damage to the endothelial cells.

In support of the present findings that sustained shear stress does not increase the functional bioavailability of basal endothelial NO, in the rabbit aorta perfused ex vivo, shear stress for 24 h does not increase expression and phosphorylation of eNOS (37). Flow-induced remodeling of arteries in vivo occurs despite inhibition of the NO pathway (6, 33). The present experiments may seem at variance with experiments in the human forearm. The reactive hyperemia that follows human forearm ischemia results in a dilatation of the brachial artery, and this dilatation presumably is evoked by an acute increase in shear stress (13). However, if flow is stopped at the wrist instead of the forearm, no dilatation is observed despite an eightfold increase in brachial artery flow (3), suggesting that an increase in shear stress does not necessarily induce an endothelium-dependent dilatation. Responses to shear stress are caused by the transfer of a mechanical into a chemical signal mediated by deformation of endothelial cells (34). Shear stress is not the only factor responsible for such endothelial deformation. Thus, under certain conditions, circumferential stretch is more important than flow for eNOS activation (24), and the normal force exerted on endothelial nuclei protruding into the vascular lumen may predominate over shear stress (35).

In the present study, drugs were applied to both the perfusate and the organ chamber solution (except for the pressurized segments, where the {alpha}1-adrenergic agonist was applied only to the outside). Therefore, the observed changes in tension did not occur as a result of differences in tissue concentrations of the vasoactive drugs.

Flow and EDHF. In the static rings, dilatation to acetylcholine was almost completely blocked by L-NAME, suggesting a predominant role of NO. However, in the perfused vessels, the vasodilator response to acetylcholine was increased and L-NAME inhibited the response only partially, suggesting the participation of an endothelium-dependent hyperpolarizing mechanism (EDHF) under flow conditions. EDHF-mediated relaxation is the result of activation of SKCa and IKCa on the endothelial cells (10). Addition of the blockers of SKCa and IKCa, apamin and TRAM-34 (8, 36), respectively, further inhibited the dilation to acetylcholine in the presence, but not in the absence, of L-NAME. Because apamin and TRAM-34 led to a small contraction of contracted perfused segments with blocked NOS but did not reduce the acetylcholine-evoked relaxation when cyclooxygenase was blocked but NOS was operative, flow must only release EDHF when NOS is inhibited. The present findings thus suggest that the endothelium of the rabbit carotid artery adjusts the release of EDHF only when subjected to flow in vitro. The present findings also support the concept that NO inhibits the EDHF-mediated relaxation (2, 23), because the influence of endothelial NO was increased in static rings, where EDHF-mediated relaxations were not observed. This contrasts with findings in the porcine coronary artery in which bradykinin releases EDHF in the absence of flow (17). This apparent discrepancy may be related not only to differences in receptor subtype and coupling or to the very different experimental setups and conditions at which the experiments were done but also to vascular heterogeneity. Indeed, the coronary circulation is derived from progenitor cells that have a different origin from the rest of the vascular tree (20), and bradykinin but not acetylcholine is a vasodilator in coronary arteries (20).

In conclusion, in the rabbit carotid artery, sustained flow augments vasoconstrictor responsiveness by reducing the functional bioavailability of NO, and sustained flow unmasks an ability of the endothelium to relax the underlying vascular smooth muscle cells through a non-NO, noncyclooxygenase vasodilator, presumably endothelium-derived hyperpolarizing factor.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Danish Cardiovascular Research Academy, the Danish Heart Foundation (03-1-2-26-22078), the Danish Medical Research Council (22-02-0210), the Novo Nordisk Foundation, and Fabrikant Vilhelm Pedersen og Hustrus Mindelegat.


   ACKNOWLEDGMENTS
 
Preliminary reports of these results were presented in abstract form at Experimental Biology 2004, Washington, DC, and Experimental Biology 2005, San Diego, CA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Skøtt, Dept. of Physiology and Pharmacology, Univ. of Southern Denmark, Winsloewparken 21, DK-5000 Odense C, Denmark (e-mail: oskott{at}health.sdu.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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