Flow-Mediated Dilatation

Transcription

1 697 Flow-Mediated Dilatation of the Basilar Artery In Vivo Kenichiro Fujii, Donald D. Heistad, and Frank M. Faraci large arteries failed to dilate, microvascular pressure (distal perfusion pressure) would fall and, thereby, impair tissue perfusion through a "steal" phenomenon. It has been hypothesized, but not demonstrated, that flow-mediated dilatation of large arteries would decrease resistance of large arteries and restore microvascular pressure toward normal.'1"3 Thus, flow-mediated dilatation of large arteries may be important in preventing a decrease in perfusion pressure. The first goal of this study was to determine whether flow-mediated dilatation occurs in large cerebral arteries in vivo. Our hypothesis was that flow-mediated dilatation may be of large magnitude in the cerebral circulation, because resistance of large arteries is high relative to other vascular beds.14 The second goal of this study was to test the hypothesis that flow-mediated dilatation may contribute to the maintenance of microvascular pressure and, thus, tissue perfusion during increases in blood flow. Several mediators of flow-mediated dilatation have been suggested in the peripheral circulation. In the aorta and iliac arteries, a diffusible vasodilator such as endothelium-derived relaxing factor may be in- Flow-mediated dilatation has been described mainly in peripheral conduit arteries. The goal of this study was to examine mechanisms and functional implications of flow-mediated dilatation in large cerebral arteries in vivo. Vessel diameter and velocity of blood flow through the basilar artery were measured using a cranial window in 45 anesthetized rats. Mean blood flow velocity through the basilar artery increased by 94±8% during unilateral common carotid artery occlusion and 23±13% during bilateral occlusion. Diameter of the basilar artery increased by 1±1% during unilateral common carotid artery occlusion and 29±2% during bilateral occlusion from control diameter of 275+±8,um. Vasodilatation appeared with a delay of 13±1 seconds after the onset of the increase in flow velocity. With systemic arterial pressure maintained at baseline levels, pressure in the basilar artery (servonull) decreased initially during carotid occlusion, and during dilatation of the basilar artery, pressure was restored partially toward normal. Indomethacin (1 mg/kg i.v.), topical application of tetrodotoxin (1-6 M), N Gmonomethyl L-arginine (5 x 1-6 M), tetraethylammonium chloride (1-2 M), glibenclamide (1` M), SKF 525A (3 X1` M), and ouabain (1-' M) had no effect on flow-mediated dilatation. These findings indicate that 1) pronounced dilatation of the basilar artery occurs in response to an increase in blood flow in vivo, 2) dilatation of large arteries attenuates reductions in cerebral microvascular pressure during increases in blood flow, and 3) flowmediated dilatation of the basilar artery does not appear to depend solely on cyclooxygenase activity, formation of nitric oxide, voltage-dependent or ATP-sensitive K' channels, activity of cytochrome P-45-dependent monooxygenase, or sodium pump activity. (Circulation Research 1991;69:697-75) In blood vessels, "flow-mediated dilatation" occurs in response to an increase in blood flow. This response was first described in large peripheral arteries1 and is usually relatively small in amplitude. In general, the response appears to involve an endothelium-dependent mechanism.2-11 A similar mechanism has been described in cerebral blood vessels in vitro, but the response to increases in flow appears to be contraction or relaxation, depending on the initial level of basal tone.12 If blood flow through large cerebral arteries, which have considerable resistance, increased and these From the Departments of Internal Medicine and Pharmacology, Veterans Administration Medical Center and Cardiovascular Center, University of Iowa College of Medicine, Iowa City, Iowa. Presented in part at the 63rd Scientific Sessions of the American Heart Association, Dallas, Tex., November 199. Supported by a Medical Investigatorship and research funds from the Veterans Administration, by National Institutes of Health grants HL-3891, HL-1666, NS-24621, HL-14388, and HL-1423, and by a grant from the Iowa Affiliate of the American Heart Association (1A9-G-5). F.M.F. is an Established Investigator of the American Heart Association. Address for correspondence: Frank M. Faraci, PhD, Department of Internal Medicine, Cardiovascular Center, University of Iowa College of Medicine, Iowa City, IA Received January 9, 1991; accepted May 14, 1991.

2 698 Circulation Research Vol 69, No 3 September 1991 volved.1516 In the microcirculation of skeletal muscle, prostaglandins may mediate the response.17 In addition, recent studies suggest a crucial role of K' current within endothelium-dependent18 or Na+-dependent19 mechanisms in smooth muscle cells in flow-mediated dilatation. The third goal of this study was to examine potential chemical mediators and ionic mechanisms that may mediate flow-mediated dilatation in the cerebral circulation. Materials and Methods Animal Preparation Experiments were performed on 45 male Sprague- Dawley rats (35-45 g) anesthetized with pentobarbital (5 mg/kg i.p.). The trachea was cannulated, and the rats were mechanically ventilated with room air and supplemental oxygen. Skeletal muscle paralysis was produced after surgery with gallamine triethiodide (5-1 mg/kg). Because the rats were paralyzed, we evaluated them approximately every 3 minutes for adequacy of anesthesia. When pressure to 'a paw evoked a change in blood pressure or heart rate, additional anesthesia was administered intravenously at a rate of mg. kg`1 * hr-1. Catheters were placed in both femoral arteries to measure systemic arterial pressure and to obtain arterial blood samples. A femoral vein was cannulated for infusion of drugs. Arterial blood gases were monitored and maintained within normal limits throughout the experiment: Pco2 was 39±1 (mean±sem) mm Hg, Po2 was 146± 9 mm Hg, and ph was Rectal temperature was monitored and maintained at 37 C with a heating pad. Both common carotid arteries were exposed through a ventral midline incision in the neck, separated from the vagosympathetic trunks, and loosely encircled with sutures for later occlusion. A craniotomy was prepared over the ventral brain stem as described in detail previously,2-23 and a portion of the dura mater was opened. The cranial window was suffused with artificial cerebrospinal fluid (CSF), warmed to 37 C, and bubbled continuously with a gas mixture of 5% C2-95% N2 to produce normal levels of ph and Pco2. In CSF sampled from the craniotomies, Pco2 was 37±1 mm Hg, Po2 was 91±3 mm Hg, and ph was 7.4±.1. Diameter of blood vessels was measured using a microscope equipped with a television camera coupled to a video monitor and image shearing device (model 98, Instrumentation for Physiology & Medicine, Inc., San Diego, Calif.). The images were recorded on videotape for later analysis. With this system, the standard deviation of 1 consecutive measurements (every 1 seconds) of diameter of a basilar artery with a diameter of -25 gm was <2.,um. Velocity of blood flow through the basilar artery was measured with a method described in detail previously.24 Briefly, a pulsed Doppler crystal (.3x.4 mm) was placed perpendicular to the basilar artery by using a micromanipulator. The crystal was rotated until a maximum Doppler shift was obtained. The Doppler shift is proportional to velocity of blood flow. Pressure in the basilar artery was measured using sharpened micropipettes (2-5-,um tip diameter) filled with.8 M NaCI and coupled to a servonull pressure-measuring device (model 4A, Instrumentation for Physiology & Medicine). The tip of the micropipette was inserted into the lumen of the basilar artery using a Leitz micromanipulator. Experimental Protocol Vessel diameter and velocity of blood flow through the basilar artery were measured under control condition, during unilateral common carotid occlusion (UCO), during bilateral occlusion (BCO), and after recovery from occlusion. Changes in systemic arterial pressure were kept to a minimum by withdrawal of arterial blood. In eight rats, diameter of branches of the basilar artery was also measured. In six rats, pressure in the basilar artery was measured. We were unable to measure microvascular pressure with a pipette and velocity of blood flow with a Doppler crystal simultaneously because of limited space within the neck incision and cranial window. Pressure in the basilar artery was also measured during graded hemorrhagic hypotension to examine the effect of pressure per se on vessel diameter. Dilatation of the basilar artery was reproducible under control conditions (up to four to five times). The response also was examined before and during topical application of 1`6 M tetrodotoxin (TTX), 5 x 1`6 M N-monomethyl L-arginine (L-NMMA), 1`2 M tetraethylammonium chloride (TEA), 1-5 M glibenclamide, 3 xi-5 M SKF 525A, or 1`5 M ouabain. SKF 525A was obtained from Smith Kline & French Laboratories, Philadelphia, Pa. All other compounds were obtained from Sigma Chemical Co., St. Louis, Mo. Glibenclamide was dissolved in dimethyl sulfoxide and diluted with saline to the desired concentration. All other drugs were dissolved in saline. The two vehicles had no effect on baseline diameter or vasodilatation in response to carotid occlusion. Concentrations of the drugs were chosen based on previous experiments in vitro25-33 and our previous studies in vivo.23,3435 Antagonists were superfused over the craniotomy by adding them to the constantly flowing artificial CSF. Responses were also examined before and after intravenous administration of 1 mg/kg indomethacin or replacement of 84% Na+ in artificial CSF by Li+ (low Na+, with the remaining Na+ added as NaHCO3 to maintain normal ph of CSF). Carotid occlusion was started at least 1 minutes after application of each antagonist or change in composition of CSF, except that BCO was performed only 6 minutes after application of TTX. During superfusion of YTX, the response to BCO but not UCO was examined to reduce the time of exposure to ITX, because superfusion of 15TX for longer than 1 minutes produced marked hypotension. It is possible that TTX penetrated into the ventrolateral medulla and acted on

3 Fujii et al Flow-Mediated Dilatation in Brain 699 TABLE 1. Effects of Interventions on Responses of the Basilar Artery to Carotid Occlusion Change in flow velocity Change in diameter Baseline diameter (%) (%) gm Change (%) UCO BCO UCO BCO Tetrodotoxin (n=4) Control 248± ±83 28±7 1,uM... -2± ±6 Indomethacin (n=6) Control 31± ± ±11 27±5 1 mg/kg... -2±2 147±53 24±68 15±4 27±5 L-NMMA (n=5) Control ±53 295±63 18±3 36±6 5,.M * 138±38 284±8 17±3 38±6 TEA (n=4) Control 252± ±7 235±19 12±1 34±5 1 nm * 144±29 276±59 12± Glibenclamide (n=5) Control 267± ±1 188±2 4±1 22±4 11tM... 6±2 87±19 226± ±3 SKF 525A (n=3) Control ±3 237±8 7±1 26±5 3,uM... 1±1 9±23 232±15 9±3 29±8 Ouabain (n=4) Control 249± ±13 24±2 15±4 35±4 1,uM * 84±6 225±41 13±1 33±5 Low Na' CSF (n=5) Control (157 mm) ±47 5±2 26±6 25 mm... -6±4 67±14 191±37 3±2 26±7 Values are mean±sem. UCO, unilateral common carotid occlusion; BCO, bilateral common carotid occlusion; n, number of rats; L-NMMA, NG-monomethyl L-arginine; TEA, tetraethylammonium chloride; CSF, cerebrospinal fluid. *p<.5 vs. control. local neurons to produce hypotension. One or two interventions (antagonists or change in ionic composition) were tested in each rat. Efficacy ofantagonists Superfusion of TTX for longer than 1 minutes produced marked hypotension that was probably due to penetration of TTX into the ventrolateral medulla and effects on local neurons that are involved in autonomic control. This hypotensive response suggests efficacy of TTX. In addition, the concentration of TTX (1`6 M) used in this study is sufficient to abolish neurally mediated action potentials in vitro25 and to inhibit relaxation of cerebral arteries in response to transmural stimulation in vitro.33 We35 have shown previously that the dose of indomethacin used in this study (1 mg/kg) completely blocked dilatation of cerebral vessels to arachidonic acid in vivo. L-NMMA (5x 1-6 M) inhibited steady-state dilatation of the basilar artery in response to 1-5 M acetylcholine (49+±9% versus 5+4%, n=5, p<.5). We34 have shown previously that L-arginine inhibits constriction of the basilar artery in response to L-NMMA and inhibits the effect of L-NMMA on the dilator response to acetylcholine. TEA (1-2 M) produced significant (22%) constriction of the basilar artery in the present study (Table 1), and significantly increased the frequency of spontaneous vasomotion of the basilar artery in a previous study.23 Ouabain (1-` M) produced significant vasoconstriction (17%) in the present study (Table 1) and abolished spontaneous vasomotion of the basilar artery in a previous study.23 These observations suggest that TEA and oubain were efficacious. The concentration of glibenclamide that we used is sufficient to inhibit hyperpolarization of smooth muscle of cerebral vessels in vitro.32 SKF 525A (1`7 M) significantly inhibits endothelium-dependent relaxation in canine coronary arteries in vitro. We used 3 x 1-5 M SKF 525A, because higher concentrations reportedly release nitric oxide and prostacyclin from endothelial cells.3 Statistical Analysis All values are expressed as mean+sem. One-way analysis of variance for repeated observations within each rat was used for comparison of stepwise response. When a significant F value was found, comparisons between mean values were made with Fisher's test for least significant difference. Paired t test was used for comparison of the response before and after interventions. A value ofp <.5 was considered to be significant.

4 7 Circulation Research Vol 69, No 3 September 1991 Velocity of Blood Flow (khz) 1. Bilateral 3 r Carotid Carotid Occlusion Occlusion Mean Velocity (khz) 3 Diameter 2: L (PLM ) Arterial Pressure Release of Occlusion,1, JAUMA AWAIAUM.11 FIGURE 1. Recording of velocity of blood flow through the basilar artery (pulsatile and mean), measurement of diameter of the basilar artery every 1-5 seconds, and recording of systemic arterial pressure (pulsatile and mean) under control conditions and during carotid occlusion. The break in the recording during bilateral carotid occlusion is for 1 minute. Mean Arteral Pressure 3 [ 15 _ Results Responses to Carotid Occlusion An example of the effect of carotid occlusion on velocity of blood flow and diameter of the basilar artery is shown in Figure 1. Velocity of blood flow through the basilar artery began to increase with no detectable time delay after UCO or BCO. Diameter of the basilar artery began to increase after a delay of 13 +±1 seconds after the onset of the increase in flow velocity. Increased velocity and diameter were sustained for the duration of the occlusion. After release of carotid occlusion, velocity returned rapidly to preocclusion levels, and diameter returned gradually to preocclusion levels. Changes in systemic arterial pressure were kept to a minimum by withdrawal and reinfusion of arterial blood. Average changes in aortic pressure, velocity of blood flow, and vessel diameter during carotid occlusion are summarized in Figure 2. Systemic arterial pressure was maintained near the control level of 127±4 mm Hg during carotid occlusion. Velocity of blood flow through the basilar artery and diameter of the basilar artery increased in a graded manner during UCO and BCO. Baseline diameter of the basilar artery and of three progressively smaller groups of branches of the basilar artery is shown in Figure 3. Diameter of the basilar artery in this group of eight rats increased by -27% during BCO. In contrast, diameter of the branches of the basilar artery did not change significantly during BCO (Figure 3). Pressure in the Basilar Artery An example of the effect of carotid occlusion on diameter of the basilar artery, pressure in the basilar artery, and systemic arterial pressure is shown in Figure 4. Pressure in the basilar artery fell immediately after UCO or BCO, while systemic arterial pressure remained constant. Pressure in the basilar artery returned toward normal after dilatation of the basilar artery. 2 *2 A Systemic Arterial Pressure uco BCO 3 15 A Velocity of Blood Flow (%) uco BCO 3 15 _ A Diameter (%) 1 *t * UCO BCO FIGURE 2. Bar graphs showing changes in systemic arterialpressure, velocity ofbloodflow through the basilar artery, and diameter of the basilar artery during unilateral carotid occlusion (UCO) or bilateral occlusion (BCO) (n=45 rats). Increases in arterial pressure during carotid occlusion were prevented by withdrawal ofarterial blood. All values are mean+±sem. *p<.5 vs. before occlusion; tp<.5 vs. UCO.

5 Fujii et al Flow-Mediated Dilatation in Brain 71 3 Baseline Diameter (,im) 3 A Diameter (%) A Basilar Arterial Pressure 3 A Diameter (%) I_ 15 * o -1 Basilar Branches Basilar Branches Artery of Artery of Basilar Basilar FIGURE 3. Bar graphs showing baseline diameter of the basilar artery and branches of the basilar artery (left panel) andpercent change in diameter of these vessels during bilateral carotid occlusion (right panel) (n=8). Filled, hatched, and dotted bars in each panel indicate three progressively smaller groups of branches of the basilar artery. All values are mean + SEM. *p <. 5 vs. before occlusion. Changes in pressure and diameter of the basilar artery during BCO and hemorrhagic hypotension are shown in Figure 5. Pressure in the basilar artery decreased by 8+2 mm Hg (n=6, p<.5) under steady-state conditions after BCO, while systemic arterial pressure was maintained at control levels (change= 1 ± 1 mm Hg, p=ns). Similar or larger decreases in pressure in the basilar artery induced by withdrawal of blood did not significantly alter diameter of the basilar artery (Figure 5), which suggests that an autoregulatory response to the small reduction in basilar pressure during carotid occlusion cannot explain the marked dilatation of the basilar artery. To examine the relation between dilatation of the basilar artery and pressure in the basilar artery, we analyzed changes in systemic arterial pressure and pressure in the basilar artery (Figure 6). Systemic arterial pressure was maintained near the control level 3 - Diameter 25 3 (gm) seconds ~~~~~~~~~~~~~~~~~~~~... * -4 oi,, REME Bilateral Hypotension Bilateral Hypotension Carotid Carotid Occlusion Occlusion FIGURE 5. Bar graphs showing changes in pressure in the basilar artery and diameter of the basilar artery during bilateral carotid occlusion and hemorrhagic hypotension. Filled and hatched bars indicate two levels ofgraded reductions in basilar artery pressure. All values are mean ±SEM (n=6-1). *p<.5 vs. control. of 117±5 mm Hg during carotid occlusion. Pressure in the basilar artery decreased by 9±1 mm Hg immediately after UCO (before the onset of dilatation of the basilar artery) from a control level of 16±5 mm Hg. Under steady-state conditions during UCO, the decrease in pressure in the basilar artery was only 5±2 mm Hg. Similarly, after BCO, the decrease in pressure was larger (14±2 mm Hg) initially than during steadystate conditions (8±2 mm Hg). The results suggest that the increase in pressure gradient from aorta to basilar artery during carotid occlusion was attenuated by dilatation of the basilar artery and large arteries upstream from the basilar artery. Effect ofantagonists The effects of several interventions on baseline diameter and changes in velocity of blood flow and vessel diameter during carotid occlusion are shown in in A Sytemic Arterial Pressure A Basilar Arterial Pressure 2 L Unilateral Bilateral Carotid Carotid Basilar Occlusion Occlusion Arterial 15 - Pressure 1 ja 1 -L. T L. -1 Arterial Pressure 3 r- 15 L- FIGURE 4. Measurement of diameter of the basilar artery every 2-3 seconds, and recordings of pressure in the basilar artery and systemic arterial pressure under control conditions and during carotid occlusion. -1-2' early SS eady SS eady SS early SS UCO BCO UCO BCO FIGURE 6. Bar graphs showing changes in systemic arterial pressure (left panel) and pressure in the basilar artery (right panel) immediately after (early) unilateral carotid occlusion (UCO), during steady state (SS) after UCO, immediately after bilateral occlusion (BCO), and during steady state after BCO (n=6). All values are mean +SEM. *p<.5 vs. before occlusion; tp<. 5 vs. immediately after UCO or BCO.

6 72 Circulation Research Vol 69, No 3 September 1991 Table 1. Treatment with L-NMMA, TEA, and ouabain decreased baseline diameter. Dilatation in response to topical application of nitroglycerin, however, was not altered significantly by these interventions. Dilatation in response to 1`6 M nitroglycerin was 37+7% under control conditions and 31+5% during application of 1 mm TEA (n=3, p=ns), and 38+±8% under control conditions and 37+9% during application of 1-5 M ouabain (n=3, p=ns). We have shown previously that dilatation in response to nitroglycerin is not altered during application of L-NMMA.34 Other interventions summarized in Table 1 had no effect on baseline diameter of the basilar artery. The increase in velocity of blood flow and diameter of the basilar artery during UCO or BCO was not altered by any of the interventions shown in Table 1. Discussion There are several major new findings in the present study. First, the basilar artery dilates during carotid occlusion in response to an increase in blood flow. The response is large relative to that observed in other vascular beds. Second, cerebral microvascular pressure tends to be restored toward normal during dilatation of the basilar artery. The finding implies that flow-mediated dilatation of large arteries attenuates the fall in pressure that occurs along large cerebral arteries during increases in blood flow and, thereby, preserves distal perfusion pressure. Third, flow-mediated dilatation of the basilar artery does not appear to depend solely on production of nitric oxide, cyclooxygenase activity, activity of cytochrome P-45-dependent monooxygenase, voltage-dependent or ATP-sensitive K' channels, or activity of Na+,K+-ATPase. Mechanism of Increase in Velocity of Blood Flow During Carotid Occlusion Pressure in the basilar artery fell during carotid artery occlusion when systemic arterial pressure remained constant or was maintained at control levels. Nevertheless, velocity of blood flow through the basilar artery increased. The pressure difference between upstream and downstream vessels is a determinant of flow velocity. Therefore, even when upstream pressure decreases, velocity of blood flow can increase if there is a larger reduction in downstream pressure. Reductions in downstream pressure almost certainly occurred in the circle of Willis during carotid occlusion, so that velocity of flow increased despite a reduction in pressure in the basilar artery. The mechanism of increase in velocity of flow during carotid occlusion appears to be similar to that observed in previous studies 335,6,1117,36,37 in which increases in flow velocity were obtained by distal vasodilatation induced pharmacologically,6,11,36 by an arteriovenous shunt,3,5,6'36 or by occlusion of parallel arterioles or arteries.1737 Mechanism of Vasodilatation in Response to Carotid Occlusion There are several possible mechanisms that could account for dilatation of the basilar artery in response to carotid occlusion. These include neurogenic, myogenic, metabolic (diffusion of a vasodilator substance from an ischemic area), ascending dilatational (propagated dilatation within the vessel wall), or flow-mediated mechanisms. Studies of the femoral artery36 and large coronary arteries4 suggest that neurogenic mechanisms, including reflex vasodilatation, do not play a major role in dilatation during increases in flow. The studies demonstrated that transsection of the artery distal to the recording site, or adrenergic or ganglionic blockade, did not impair the dilatation. In the present study, TTX, which selectively blocks sodium channels and can thereby abolish neurally mediated responses,25 had no effect on dilatation of the basilar artery during bilateral carotid occlusion. Adrenergic, cholinergic, and nonadrenergic, noncholinergic vasodilatator mechanisms all are inhibited by TTX.33 This finding suggests that neural mechanisms that require sodium channel activity for excitation have little, if any, role in dilatation during carotid occlusion. Pressure in the basilar artery decreased during carotid occlusion with systemic pressure constant. Similar or larger decreases in pressure induced by hemorrhage did not alter the diameter of the basilar artery, which suggests that the vessel does not autoregulate very well and that an autoregulatory (myogenic) response to the small reduction in pressure in the basilar artery cannot explain the pronounced dilatation of the basilar artery during carotid occlusion. Diameter of branches of the basilar artery did not increase during carotid occlusion. These vessels probably are not exposed to an increase in blood flow. This observation provides some evidence against a metabolic mechanism or a role for ascending dilatation of the basilar artery. If the dilatation were due to diffusion of vasodilator substances from cerebrum, for example, dilatation would be expected to occur in small branches as well as the basilar artery. It is not possible to distinguish definitely between flow-mediated and propagated responses.38,39 In the dog femoral artery, propagation of a signal along the vessel wall probably does not account for vasodilatation, because dilatation was preserved after transsection of the artery distal to the recording site.36 In the present study, absence of dilatation in branches suggests that dilatation of the basilar artery is not due to propagated dilatation within the wall. Based on these observations, we conclude that dilatation of basilar artery during carotid occlusion occurs primarily in response to an increase in blood flow. There was always a time delay between the onset of increase in velocity of blood flow and the onset of increase in vessel diameter. A similar delay has been

7 observed in flow-mediated responses in other vascular beds in vivo51,13'36'37 and in vitro.4 The cause for this delay is not clear but could reflect the time required for the increase in flow to trigger the production or release of mediators or for the mediators to produce detectable dilatation. Endothelial cells appear to sense changes in shear stress or blood flow,41'42 but the role of endothelium in flow-mediated dilatation is controversial. Endothelium appears to play a crucial role in the response to an increase in flow in dog femoral artery,3,5-8 dog coronary arteries,2,4,9 and rat cremaster arterioles.1 In contrast, a major component of flow-mediated relaxation still occurs after removal of endothelium in the rabbit ear artery studied in vitro.4 Mediators of Flow-Mediated Dilatation Several mediators of flow-mediated dilatation have been identified or proposed in the peripheral circulation. In the aorta and femoral arteries, a diffusible vasodilator similar to endothelium-derived relaxing factor may be involved.7"15"16 In rat cremaster microcirculation, inhibition of synthesis of nitric oxide (a possible endothelium-derived relaxing factor) did not inhibit the dilator response of arterioles during increases in blood flow.17 In the rabbit ear artery studied in vitro, hemoglobin, which completely reversed acetylcholine-induced relaxation, had an inconsistent effect on relaxation during increases in blood flow.4 In the present study, L-NMMA had no effect on flow-mediated dilatation, which suggests that this mechanism in the basilar artery does not require production of nitric oxide. Several studies suggest that flow-mediated dilatation is not attenuated by inhibitors of cyclooxygenase.34,7'134 Arteriolar dilatation in the microcirculation of skeletal muscle, however, is inhibited by indomethacin and meclofenamate.17 In this study, indomethacin had no effect on flow-mediated dilatation, suggesting that flow-mediated dilatation of the basilar artery is not dependent on cyclooxygenase products. The response also was not blocked by SKF 525A, a cytochrome P-45-dependent monooxygenase inhibitor.27'29 Ionic Mechanisms Recently, it has been proposed that activation of a hyperpolarizing K' current within endothelium may produce dilatation in response to an increase in flow.18 A study16 in isolated rabbit iliac arteries also suggests that activation of K' channels is the transducer and endothelium-derived relaxing factor is the effector of flow-mediated dilatation. In our study, TEA, which blocks voltage-dependent K' channels,26 and glibenclamide, which blocks ATP-sensitive K' channels,31'32 did not affect flow-mediated dilatation of the basilar artery. Many potassium channels have been identified or proposed to exist,43 and we cannot exclude an important role of other K' channels. In resistance arteries in vitro, replacement of 85% of total Na+ ions by Li+ abolished flow-mediated Fujii et al Flow-Mediated Dilatation in Brain 73 dilatation, suggesting that sodium-dependent mechanisms may be important in mediating flow-mediated dilatation.19 In our study, replacement of normal CSF by low Na+ CSF did not affect the flow-mediated dilatation of the basilar artery. We cannot, however, exclude a role of sodium-dependent mechanisms, because blood flowing through the basilar artery has a normal concentration of Na+. Ouabain, which inhibits activity of Na4,K+-ATPase,28 had no effect on flow-mediated dilatation. Functional Significance of Flow-Mediated Dilatation Pressure in the basilar artery decreased after carotid occlusion, and this reduction in pressure was restored toward normal as the basilar artery dilated. A major determinant of pressure in the basilar artery is the ratio of upstream resistance (from aorta to basilar artery) to downstream resistance.14 During carotid artery occlusion, resistance in downstream vessels would be expected to decrease, in response to a reduction in perfusion pressure, relative to resistance of upstream vessels. Consequently, pressure in the basilar artery would decrease. A decrease in pressure during increases in blood flow also was observed in the femoral artery during increases in blood flow produced by distal vasodilatation induced pharmacologically36 or by an arteriovenous shunt.3'36 In the present study, dilatation of upstream vessels (presumably flow-mediated dilatation) would decrease resistance of large upstream vessels and restore pressure in the basilar artery toward normal. Thus, restoration of pressure in the basilar artery toward normal during increases in blood flow suggests that there may be dilatation of large arteries, such as the vertebral artery, upstream from the basilar artery. Flow-mediated dilatation appears to minimize the drop in perfusion pressure that occurs along large cerebral arteries during increases in blood flow. This finding provides direct evidence to support the hypothesis"1"13 that flow-mediated dilatation helps to preserve microvascular pressure, or distal perfusion pressure, and thereby protects against a steal phenomenon. The magnitude of the increase in diameter that was observed in the basilar artery in response to increases in blood flow is large relative to other vascular beds. For example, the increase in vascular diameter in response to a similar increase in blood flow is 5-1% in large coronary arteries4,1113 and 2-1% in the femoral artery.3'6'8'36 In peripheral blood vessels, the magnitude of flow-mediated dilatation appears to increase as the size of the artery or arteriole decreases,3"17'37 which suggests that the response to increased flow is somehow dependent on vessel tone or diameter. A change in diameter that is relatively small in magnitude is magnified, in terms of vascular resistance. The large magnitude of the response in the basilar artery suggests that flow-mediated dilatation is especially important in the cerebral

8 74 Circulation Research Vol 69, No 3 September 1991 circulation, where resistance of large arteries is high relative to other vascular beds.14 In summary, prominent dilatation occurs in the basilar artery in response to an increase in blood flow in vivo. Flow-mediated dilatation of the basilar artery does not appear to be dependent on production of nitric oxide, cyclooxygenase activity, cytochrome P-45-dependent monooxygenase activity, Na+,K+- ATPase activity, or several voltage-dependent or ATPsensitive K' channels. It remains possible that redundant mechanisms participate in this response and that some combinations of these factors have additive or synergistic effects that cannot be attenuated by a single inhibitor. Thus, the cellular mechanisms and mediators that account for this striking response in cerebral vessels in vivo remain unidentified. References 1. Schretzenmayr A: Uber kreislaufregulatorische Vorgange an den grossen Arterien bei der Muskelarbeit. Pfiugers Arch 1933;232: Holtz J, Giesler M, Bassenge E: Two dilatory mechanisms of anti-anginal drugs on epicardial coronary arteries in vivo: Indirect, flow-dependent, endothelium-mediated dilation and direct smooth muscle relaxation. Z Cardiol 1983;72(suppl 3): Hull SS Jr, Kaiser L, Jaffe MD, Sparks HV Jr: Endotheliumdependent flow-induced dilation of canine femoral and saphenous arteries. Blood Vessels 1986;23: Hintze TH, Vatner SF: Reactive dilation of large coronary arteries in conscious dogs. Circ Res 1984;54: Smiesko V, Kozik J, Dolezel S: Role of endothelium in the control of arterial diameter by blood flow. Blood Vessels 1985;22: Pohl U, Holtz J, Busse R, Bassenge E: Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 1986;8: Rubanyi GM, Romero JC, Vanhoutte PM: Flow-induced release of endothelium-derived relaxing factor. 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9 Fujii et al Flow-Mediated Dilatation in Brain Bevan JA, Joyce EH, Wellman GC: Flow-dependent dilation in a resistance artery still occurs after endothelium removal. Circ Res 1988;63: Lansman JB: Endothelial mechanosensors: Going with the flow. Nature 1988;331: Davies PF: How do vascular endothelial cells respond to flow? News Physiol Sci 1989;4: Castle NA, Haylett DG, Jenkinson DH: Toxins in the characterization of potassium channels. Trends Neurosci 1989;12: KEY WORDS * basilar artery * blood flow * carotid occlusion * microvascular pressure * endothelium-derived relaxing factor

10 Flow-mediated dilatation of the basilar artery in vivo. K Fujii, D D Heistad and F M Faraci Circ Res. 1991;69: doi: /1.RES Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 1991 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation Research is online at: