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(Stroke. 1998;29:1194-1201.)
© 1998 American Heart Association, Inc.

Original Contributions

High Levels of Myogenic Tone Antagonize the Dilator Response to Flow of Small Rabbit Cerebral Arteries

Nathalie Thorin-Trescases, PhD; John A. Bevan, MD

From the Totman Laboratory for Cerebrovascular Research, Department of Pharmacology, College of Medicine, University of Vermont, Burlington.

Correspondence to John A. Bevan, MD, Department of Pharmacology, University of Vermont, Given Building, Burlington, VT 05405-0068.

	Abstract

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Background and Purpose—Pressure and shear stress exerted by flowing blood are two mechanical forces that play a major role in the regulation of vascular tone. We sought to evaluate the interaction between pressure and flow in isolated rabbit cerebral arteries.

Methods—Responses to intraluminal flow of isolated pressurized rabbit posterior cerebral arteries were investigated at low, medium, and high levels of myogenic tone by setting the luminal pressure at 40, 60, and 80 mm Hg, respectively.

Results—At both low and medium levels of myogenic tone, flow induced dilation. The response was significantly larger at 40 than at 60 mm Hg. At the high level of myogenic tone, the response to flow consisted of a combination of an initial transient dilation followed by sustained constriction. Flow-induced dilation but not flow-induced constriction response was endothelium dependent. Removal of the endothelium inhibited the dilator response by 80%. Flow-induced dilation was inhibited (40%) by N-nitro-L-arginine (100 µmol/L) but not by indomethacin (10 µmol/L). Endothelium removal not only decreased the amplitude of flow-induced dilation but also promoted the appearance of flow-induced constriction at low and medium levels of myogenic tone.

Conclusions—The intraluminal pressure and in consequence the level of myogenic tone at which flow is applied determine the nature of the response of the smooth muscle cells of the blood vessel wall.

Key Words: intraluminal pressure • myogenic responses • posterior cerebral artery • stress, shear • rabbits

	Introduction

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Resistance arteries are characterized by a considerable level of intrinsic muscle tone, variation in which is to a great extent responsible for the autoregulation of blood flow. Pressure and shear stress exerted by flowing blood are two mechanical forces that play a major role in the regulation of vascular tone.^{1 2} To evaluate the interaction between pressure and flow, different in vitro studies have been performed in which pressure and flow can be changed independently. Alteration in intravascular pressure leads to well-established changes in blood vessel diameter: an increase in intraluminal pressure induces myogenic contraction,³ while a decrease results in myogenic dilation. In contrast, vascular responses to flow are more controversial. An increase in flow velocity can promote either dilation,^{4 5 6 7 8} constriction,^{9 10 11} or both dilation and constriction.^{12 13} The dilator response to flow has been found to be mainly endothelium dependent,^{4 5 14} while flow-induced constriction is endothelium independent.^{9 10} Furthermore, in pathological conditions associated with endothelial dysfunction such as atherosclerosis, flow-induced dilation can be reversed to flow-induced constriction.^{15 16}

Flow probably elicits both constrictor and dilator responses simultaneously, the final level of tone resulting from the interaction between the two responses. Our hypothesis is that (1) the response to flow might be a combination of dilator and constrictor components and (2) the level of myogenic tone, determined by the intraluminal pressure at which flow is applied, influences the response to flow. Indeed, if the inter- action between pressure and flow plays a role in the balance of vascular tone and moment-by-moment regulation of blood flow during local changes in pressure, its effectiveness would be expected to vary with the level of tone. Kuo et al⁵ demonstrated that in coronary arteries flow-induced dilation is opposed by myogenic constriction, and Sun et al¹⁷ recently showed that in skeletal muscle arterioles, flow-induced dilation is reduced by increases in perfusion pressure. In cerebral arteries, we previously showed that at a high intravascular pressure, flow induced constriction,^{9 13} suggesting that flow-induced dilation appears to be limited at high pressure. However, only two flow rates were tested, and one was supramaximal, which limited the interpretation. In the present study we examined flow response curves on the same vessel at three different levels of tone. Furthermore, we standardized the stimulus: flow rate was always adjusted to the inner, preflow diameter of the artery to apply comparable shear stresses to arteries of different diameters.

	Materials and Methods

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General Preparation
Male New Zealand White rabbits (weight, 2.5 to 3.5 kg) were anesthetized with sodium pentobarbital (29 mg/kg) and heparinized (100 U/kg) before exsanguination. The brain was rapidly removed and placed in cold oxygenated PSS of the following composition (mmol/L): NaCl 119, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ 1.17, NaHCO₃ 24.9, CaCl₂ 1.6, EDTA 0.023, and dextrose 5.5.

The procedures followed in this study were in accordance with institutional guidelines.

Secondary or tertiary branches of the rabbit PCA, 1 to 2 mm long, were carefully dissected and mounted in the automated video perfusion system of Halpern et al,¹⁸ which allows independent control and registration of intravascular pressure and flow, as previously described.^{9 13} After the vessel was mounted on the outflow microcannula (OD adjusted to the size of the vessel, 120 µm), the pressure was raised to 5 to 10 mm Hg to flush and clear the vessel of blood. Then the artery was mounted on the inflow microcannula (OD, 120 µm), and pressure was raised to 20 mm Hg with the pressure-servo system. Potential leaks were checked; any leaking vessel was discarded. After the vessel was cannulated, transmural pressure was raised to 60 mm Hg, and the preparation was warmed slowly to 37°C and allowed to equilibrate for 60 to 90 minutes under no-flow conditions, with a longitudinal stretch of 20% to 30%.⁹ Internal diameter and intravascular pressure were measured continuously throughout the experiment.

Experimental Protocols
Flow Responses at Different Levels of Myogenic Tone
Myogenic tone usually appeared after 30 to 40 minutes of equilibration. When the pressure (60 mm Hg)–induced constriction was stable, responses to flow were studied. Flow rates were changed from 0 to 1 to 50 µL/min in a noncumulative fashion at random (perfusion pump, Harvard syringe infusion pump 22). Each flow rate was maintained for 3 to 5 minutes, which was sufficient time to achieve a steady state response. Then flow was stopped, and the next flow response was assessed after a resting period of 10 minutes. Flow rate was always adjusted to the inner, preflow diameter of the vessel to apply comparable shear stresses to arteries of different diameters. Shear stresses from 1 to 12 dyne/cm² (1, 2, 4, 6, 8, and 12 dyne/cm²) were studied, delivered by flows calculated from the formula =4Q/r³, where is the shear stress (dynes per square centimeter), the viscosity (poises [P]), Q the flow rate (milliliters per second), and r the radius (centimeters). In PSS containing 24.0 mmol/L NaHCO₃, at 37°C, was found to be 0.009 P.¹⁹ The physiological range of in small arteries is from 5 to 25 dyne/cm².²⁰

After this first series of flow responses, intravascular pressure was changed to either 40 or 80 mm Hg. The vessel was then allowed to equilibrate until a steady state diameter was reached. When stable, flow rates corresponding to shear stresses of 1 to 12 dyne/cm² were delivered as described above.

Finally, the intramural pressure was returned to 60 mm Hg luminal pressure without flow, and acetylcholine (1 µmol/L), an endothelium-dependent vasodilator in this vessel, was added to assess the functional integrity of the endothelium.

Responses to Flow After Endothelium Removal
In a separate experimental series, the role of the endothelium in the flow responses was assessed. The artery was denuded of endothelium by insertion of an air bubble in the lumen of the artery, which was then flushed by flowing PSS through the lumen at a low flow rate. Liu et al²¹ have shown that this results in the physical removal of endothelial cells. After endothelium denudation, the artery was allowed to equilibrate for 60 to 90 minutes at a luminal pressure of 60 mm Hg until myogenic tone had developed and stabilized. Flow responses induced by =6 dyne/cm² were then measured at different levels of myogenic tone obtained by changing the intraluminal pressure from 60 to either 40 or 80 mm Hg. The vasoactive function of the endothelium was tested with acetylcholine (1 µmol/L). The failure of acetylcholine to induce dilation was taken as an indication of endothelium removal.

Role of L-Arginine/NO Pathway in Response to Flow
In a separate experimental series, the role of the L-arginine/NO pathway in flow-induced dilation was assessed. Flow responses at 4 and 6 dyne/cm² were obtained at 60 mm Hg luminal pressure before and after treatment of the vessel with L-NNA (100 µmol/L) for 30 minutes. Responses to acetylcholine (1 µmol/L) were tested before and after pretreatment with L-NNA.

Role of Prostaglandins in Response to Flow
In another separate experimental series, the role of prostaglandins in flow-induced dilation was studied. Flow responses at 4 and 6 dyne/cm² were obtained at 60 mm Hg luminal pressure before and after treatment of the vessel with indomethacin (10 µmol/L) for 30 minutes.

Flow Responses in Arteries Contracted With KCl
To investigate the mechanisms by which flow may cause constriction, preliminary experiments were conducted: at intraluminal pressure at which flow-induced dilation was mainly observed (60 mm Hg), myogenically constricted vessels were further contracted with a low KCl solution (30 mmol/L). In addition to the KCl-induced tone, flow was applied (shear stress of 4 dyne/cm²). A typical recording of this experiment is shown in Figure 4.

View larger version (6K): [in this window] [in a new window]   Figure 4. Typical recording of the diameter of myogenically constricted rabbit PCA segment exposed to flow at a luminal pressure of 60 mm Hg (left) before and after exposure to KCl (30 mmol/L) (right). Flow-induced dilation is reversed to flow-induced constriction in the presence of KCl. The shear stress applied was similar in both instances (4 dyne/cm2).

At the conclusion of each experiment, the suffusion solution was changed to PSS containing 127 mmol/L KCl to measure the maximum constricted diameter at each pressure step (40, 60, and 80 mm Hg). Finally, the suffusion solution was changed to Ca²⁺-free PSS containing EGTA (2 mmol/L). Vessels were incubated for 15 minutes, and the pressure steps were repeated to obtain the maximum passive diameter at each pressure value.

Measurement of Parameters
The level of myogenic tone developed spontaneously during the equilibration period at 60 mm Hg and then at 40 or 80 mm Hg luminal pressure was calculated according to the formula

where d_Ca-free is the diameter obtained at a given luminal pressure (40, 60, or 80 mm Hg) in Ca²⁺-free PSS containing 2 mmol/L EGTA, d is the steady state diameter reached by the vessel at a given luminal pressure, and d_KPSS is the diameter obtained at a given luminal pressure in PSS containing 127 mmol/L KCl.

Dilator responses to flow are expressed as changes (percentage) in diameter, normalized to the maximum passive diameter. They are calculated according to the formula

where d_Ca-free is the diameter obtained at a given luminal pressure (40, 60, or 80 mm Hg) in Ca²⁺-free PSS containing 2 mmol/L EGTA, b is the diameter reached by the vessel at a given luminal pressure during the steady state of the flow response, and a is the preflow diameter.

Constrictor responses to flow are expressed as changes (percentage) in diameter, normalized to minimum active diameter. They are calculated according to the formula

where d_KPSS is the diameter obtained at a given luminal pressure (40, 60, or 80 mm Hg) in PSS containing 127 mmol/L KCl, b is the diameter reached by the vessel at a given luminal pressure during the steady state of the flow response, and a is the preflow diameter.

We applied flow according to Q=r³/4 (microliters per minute) using r as preflow diameter and as a given stimulus (1 to 12 dyne/cm²), then we calculated the shear stress achieved according to 4Q/r³ (dynes per square centimeter) using r as the steady state diameter during the response to flow. The shear stress achieved is an indicator of the flow response: if it is lower than the applied shear stress, then the response to flow is a dilation; in contrast, if it is higher than the applied shear stress, then the response to flow is a contraction.

Drugs and Statistical Analysis
All salts and chemicals were obtained from Sigma or Aldrich.

The data are presented as mean±SEM of n animals. Differences were considered significant at P<0.05 and were determined by ANOVA followed by Scheffé's test or by paired or unpaired Student's t tests, as appropriate.

	Results

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Flow Responses at Different Levels of Myogenic Tone
After 30 to 40 minutes of equilibration at a luminal pressure of 60 mm Hg, rabbit PCAs developed spontaneous myogenic tone. When related to the passive diameter measured in Ca²⁺-free PSS (300.8±22.5 µm) and the minimum diameter in KCl 127 mmol/L (76.0±7.2 µm), the arteries constricted to 213.0±15.3 µm (n=18; P<0.05), which is 34.1±3.1% of maximum. When intravascular pressure was decreased to 40 mm Hg, the diameter obtained at steady state was significantly larger than that previously measured at 60 mm Hg luminal pressure, representing a myogenic tone level of 25.1±2.9% (n=13; P<0.05 versus 60 mm Hg). The level of myogenic tone reached at a luminal pressure of 80 mm Hg was 53.8±6.2% (n=13), which is significantly higher than that at 60 or 40 mm Hg (P<0.05).

The response of an isolated rabbit cerebral artery to flow at 40, 60, and 80 mm Hg luminal pressure is shown in Figure 1. At 40 mm Hg, dilation was observed when flow was applied. The diameter returned to control levels within 4 minutes when flow was stopped. Similarly, at 60 mm Hg flow induced dilation (Figure 1). Flow was stopped when the dilation reached a plateau; the diameter returned to its control level within 8 to 10 minutes. Note that during recovery, the diameter achieved a lower value than the preflow diameter before returning to its original level. This transient constriction was not always observed, and the response to flow at 60 mm Hg luminal pressure was predominantly flow-induced dilation. At 80 mm Hg, the response to flow was a combination of dilation and constriction (Figure 1). Initially, flow-induced dilation was observed, which was followed during the flow period by a larger flow-induced constriction component. When flow was stopped, the diameter returned to its original preflow level within 6 to 10 minutes.

View larger version (9K): [in this window] [in a new window]   Figure 1. Typical recording of changes in diameter of a myogenically constricted rabbit PCA in response to flow (shear stress applied of 4 dyne/cm2) at luminal pressures of 40, 60, and 80 mm Hg. At low (40 mm Hg), medium (60 mm Hg), and high (80 mm Hg) levels of myogenic tone, the response to flow was dilation, dilation followed by small constriction, and small dilation followed by constriction, respectively.

From the data obtained at the three levels of myogenic tone, shear stress achieved during the steady state of the flow response was calculated; the flow–shear stress relationships are plotted in Figure 2. At 40 and 60 mm Hg, flow-induced dilation increased with shear stress, reaching a plateau at 6 dyne/cm² (shear stress applied); then the amplitude of flow-induced dilation decreased despite higher shear stresses being applied. The constriction that occasionally appeared when flow was stopped (60 mm Hg) was small in amplitude and not shear stress dependent (data not shown). At 80 mm Hg, flow-induced constriction was observed at every shear stress tested, reaching a maximum at 6 dyne/cm² (shear stress applied). The relation between flow-induced constriction and shear stress is not linear. The dilator component of the flow response observed at different shear stresses (80 mm Hg) was small and not related to the amplitude of the stimulus.

View larger version (18K): [in this window] [in a new window]   Figure 2. Relationship between the response to flow (changes in diameter, expressed as a percentage) and the shear stress achieved during the steady state response to flow. The stimulus (shear stress applied) is represented by the numbers 1, 2, 4, 6, 8, and 12 (dynes per square centimeter). Rabbit PCA segments were pressurized at 40 (, n=7 to 12), 60 (, n=7 to 12), and 80 mm Hg (, n=6 to 12). Flow-induced dilations (positive values) are expressed as changes in diameter normalized to the maximum passive diameter measured in Ca2+-free solution containing 2 mmol/L EGTA. Flow-induced constrictions (negative values) are expressed as changes in diameter normalized to the minimum active diameter measured in PSS containing 127 mmol/L KCl. Data are mean±SEM of n rabbits. *P<0.05 vs flow response observed at 1 dyne/cm2; aP<0.05 vs flow response observed at 40 mm Hg; bP<0.05 vs flow response observed at 60 mm Hg.

Figure 2 also shows that flow-induced dilations were larger at the low intravascular pressure (P<0.05; 40 versus 60 mm Hg for 2 and 4 dyne/cm²; shear stress applied) and that flow-induced constriction dominated at the high intraluminal pressure.

Flow Responses in the Absence of a Functional Endothelium
Removal of the endothelium by air significantly suppressed acetylcholine-induced dilation of myogenically constricted vessels (Table). However, this procedure did not affect the development of myogenic tone during the equilibration period at a luminal pressure of 60 mm Hg (35.2±2.1% [n=13] versus 34.1±3.1%, absence versus presence of endothelium; P>0.05). The denudation procedure did not affect the myogenic responses to changes in intraluminal pressure: the levels of myogenic tone reached at 40 and 80 mm Hg were similar in the presence and in the absence of endothelium (at 40 mm Hg: 24.3±2.4% [n=12] versus 25.1±2.9% [n=13]; at 80 mm Hg: 40.5±1.8% [n=6] versus 53.8±6.2% [n=13], absence versus presence of endothelium; P>0.05). To quantify the intensity of the myogenic responses to changes in intravascular pressure, myogenic indices (MI) were calculated by the formula of Halpern et al²²: MI=100x[(r_f-r_i)/ri]/(P_f-P_i), where the subscripts i and f refer to the initial and final values of the radius (r) and pressure (P), respectively. This index is an indicator of the relative slope of the active pressure-diameter relation for an artery; the more negative the value, the more powerful is the myogenic responsiveness of that vessel. The removal of the endothelium had no influence on the myogenic indices (Table).

View this table: [in this window] [in a new window]   Table 1. Effect of Endothelium Removal on Myogenic Responses

The fact that neither the development of myogenic tone nor the myogenic responses were affected by endothelium removal indicates that vascular smooth muscle cells were not damaged by the denudation procedure. Furthermore, responses elicited by PSS containing 127 mmol/L KCl were identical in vessels with (76±7 µm) and without endothelium (66±9 µm).

The influence of endothelium removal on responses to flow is summarized in Figure 3. We restricted the data to flow responses elicited at a shear stress of 6 dyne/cm², since at this particular shear stress the flow responses were maximal. At a luminal pressure of 40 mm Hg, when flow was applied on a denuded artery, flow-induced dilation was significantly decreased compared with the response obtained in an intact vessel: flow-induced dilation was inhibited by 65% in the absence of endothelium (Figure 3). Concomitant to the decrease in flow-induced dilation was the appearance of a large flow-induced constriction (Figure 3). Similarly, at a luminal pressure of 60 mm Hg, flow-induced dilation was significantly reduced (79% inhibition), and this was associated with the appearance of a large flow-induced constriction (Figure 3). Finally, at a luminal pressure of 80 mm Hg, removal of the endothelium produced a significant reduction of flow-induced dilation (84% inhibition) without affecting the constriction component of the flow response (Figure 3).

View larger version (13K): [in this window] [in a new window]   Figure 3. Flow responses elicited by a shear stress of 6 dyne/cm2 in rabbit PCA segments pressurized at 40, 60, and 80 mm Hg in the presence (+E, n=11 to 13) and in the absence (-E, n=6 to 12) of an intact endothelium. Endothelium was removed by air. Flow-induced dilations (positive values) are expressed as changes in diameter normalized to the maximum passive diameter measured in Ca2+-free solution containing 2 mmol/L EGTA. Flow-induced constrictions (negative values) are expressed as changes in diameter normalized to the minimum active diameter measured in PSS containing 127 mmol/L KCl. Data are mean±SEM of n rabbits. *P<0.05 vs in presence of endothelium.

These results indicate that flow-induced dilation is partly (80%) endothelium dependent. In contrast, the constriction component of the flow response is endothelium independent.

Effect of L-NNA on Flow-Induced Dilation
To investigate the role of the L-arginine/NO pathway in the endothelium-dependent flow-induced dilation responses observed at a luminal pressure of 60 mm Hg, we tested the effect of L-NNA (100 µmol/L). L-NNA, added for 30 minutes on myogenically constricted arteries, caused no significant change of basal diameter (3.7±6.0%). Treatment with L-NNA significantly depressed the dilation induced by acetylcholine (66.3±13.4% versus 10.0±1.6% [n=4], before versus after L-NNA; P<0.05), indicating that the L-arginine/NO pathway was effectively inhibited by the L-arginine analogue. L-NNA induced a significant reduction of flow-induced dilation elicited at =4 and 6 dyne/cm², inhibiting the response by 47.2±14.7% and 40.1±4.4%, respectively. This suggests that NO is partly involved in the endothelium-dependent flow-induced dilation response.

Effect of Indomethacin on Flow-Induced Dilation
To further investigate the mechanisms responsible for the endothelium-dependent flow-induced dilation responses observed at a luminal pressure of 60 mm Hg, the effects of indomethacin (10 µmol/L), an inhibitor of the cyclooxygenase pathway, were assessed. Pretreatment of the vessel with indomethacin caused no change in baseline diameter (3.1±7.3%) and did not affect the flow-induced dilation elicited at =4 and 6 dyne/cm². This suggests that dilator prostaglandins are not responsible, nor did they contribute to the endothelium-dependent flow-induced dilation responses.

Flow Responses in Arteries Contracted With KCl
Exposure to KCl 30 mmol/L produced a significant increase in tone (from 26.1±3.1% to 49.1±10.1%; n=5; P<0.05). On myogenically constricted arteries, flow elicited at 4 dyne/cm² induced dilation (54.9±19.9%; n=5). After exposure to KCl 30 mmol/L, flow-induced dilation was abolished (2.0±2.0%; n=5; P<0.05) and reversed to flow-induced constriction (Figure 4) (-56.9±19.9%; n=5; P<0.05).

	Discussion

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In this report, responses to flow of isolated rabbit PCAs were studied at low, medium, and high levels of myogenic tone by setting the intraluminal pressure at 40, 60, and 80 mm Hg, respectively. Pressure in vivo in rabbit PCA and its excursions during physiological stress is not known. However, the PCAs exhibit pressure-dependent responses, ie, myogenic responses over this pressure range. We chose the pressures 40, 60, and 80 mm Hg to ensure spanning the normal physiological mean pressure. When a similar flow stimulus was used, flow-induced dilation was observed at low and medium levels of myogenic tone, and flow-induced constriction was observed at high levels. We showed that flow-induced dilation is reversed to flow-induced constriction when the intraluminal pressure is raised. We conclude (1) that the response to flow is a combination of dilator and constrictor components and (2) that under physiological conditions, both types of responses may contribute to the regulation of cerebrovascular tone.

Several findings of this study suggest that flow probably elicits both constrictor and dilator responses simultaneously, the final level of tone resulting from the interaction between the two. First, the relationship between shear stress and flow-induced response measured at either the low or medium level of tone was not linear: progressively increasing dilations were observed up to 6 dyne/cm², when further increases in shear stress led to smaller responses. This profile of flow-induced response was recently reported by Ngai and Winn⁸ in rat cerebral arterioles using higher shear stresses than in the present study. The mechanism underlying these phenomena is unknown, but clearly the response to flow is complex and may represent the net result of competition between flow-dependent dilator and constrictor responses. Second, our results suggest that one of the major determinants of the response to flow is the level of myogenic tone at which flow is applied. Flow-induced dilation appears to be the dominant effect at low myogenic tone, flow-induced dilation is lower in amplitude at medium tone levels, and flow-induced constriction is dominant at high levels (Figure 2). Flow-induced responses in isolated blood vessels, at a constant intraluminal pressure, have been observed in various studies. In most of them, flow was applied at a luminal pressure that resulted in the development of a moderate level of myogenic tone (intraluminal pressure of 60 cm H₂O in pig coronary arterioles^{5 23} and intraluminal pressure of 80 mm Hg in rat first-order skeletal muscle arterioles⁴). In accordance with our observations, using isolated pig coronary arterioles, Kuo et al⁵ showed that the magnitude of flow-induced dilation seen when intraluminal pressure was 60 cm H₂O was attenuated when it was higher (100 cm H₂O). Third, endothelial disruption not only reduced dilation but served to augment or reveal constriction in response to flow (Figure 3). Flow-induced contraction was also observed by Kuo et al⁶ on denuded pig coronary venules. The basis of the reversal of flow-induced dilation to flow-induced constriction resulting from endothelium removal was not investigated in that study. Finally, at 60 mm Hg, we observed that even in the presence of an intact endothelium, higher flow rates evoked a slight constriction after flow-induced dilation when flow was discontinued (Figure 1). This suggests that constriction is part of the normal response to flow and that it is unmasked in the absence of endothelium.

One limitation of the present study is that intraluminal pressure was not directly measured. Instead, outflow pressure was monitored. Outflow pressure does not reflect intraluminal pressure since a micropipette tip possesses some resistance. To minimize such resistance, diameters of both pipettes were adjusted to the size of the artery. In our system, the back pressure generated by flow at the rates we used is probably <2 mm Hg (personal communication, W. Halpern). In the absence of flow, an increase in pressure from 40 to 80 mm Hg induces a 40-µm decrease in diameter (from 235±18 to 195±13 µm), which represents 20% of active constricting tone. Pressure would therefore have to increase to 40 mm Hg to account for the flow constriction observed at 80 mm Hg (22±6% at 6 dyne/cm²). Thus, the error resulting from cannular resistance would be small. Indirect observations suggest that the myogenic response does not significantly contribute to the response to flow. First, if flow produced a significant increase in intraluminal pressure, a transient dilation (due to passive distention) followed by an active constriction should be observed. Furthermore, this response would be stimulus dependent, the higher the flow rate the larger would be the increase in intraluminal pressure, and the change would be observed whatever the level of tone. However, we observed nonlinear flow-dilation relationships at 40 and 60 mm Hg. Responses lasted as long as the flow was maintained, and constriction was sometimes observed during the recovery period, when flow was stopped. Flow-induced constrictions were only observed at high levels of tone. Second, we have observed that flow in a maximally dilated vessel (Ca²⁺-free PSS) does not produce any dilation (N.T.-T. and J.A.B., unpublished data), suggesting that the amount of pressure produced by flow does not significantly stretch the blood vessel wall. Third, if flow-induced dilation was a passive response due to passive distention of the blood vessel wall, it should not be affected by antagonists. However, endothelium removal or treatment with L-NNA significantly reduced flow-induced dilation, indicating that this is an active response.

Flow-induced dilation was primarily endothelium dependent, since it was inhibited by 80% by endothelium removal, and it was at least partly mediated by the L-arginine/NO pathway but not by dilator prostaglandins, since 40% of the response was antagonized by L-NNA but not by indomethacin. After endothelium removal, part of the flow-induced dilation remained, indicating that vascular smooth muscle cells might directly respond to changes in shear stress. Flow-induced constriction was endothelium independent (80 mm Hg) (Figure 3). The flow responses observed after endothelium removal and after exposure to L-NNA might be expected to be different. In the absence of endothelium, flow takes place over the internal elastic lamina, while flow takes place over the endothelial surface when the vessel is treated with L-NNA. Therefore, if there is a mechanical link between the shear stress and the response, it may involve different structures in the two circumstances. Using different methods of endothelium removal (rubbing,⁵ chemical¹⁰), others have reached similar conclusions regarding the role of the endothelium in mediating vascular responses to flow.

The reason why flow-induced constriction appears at high levels of myogenic tone is not clear. It is not a myogenic response due to an increase in pressure concomitant with the increase in flow, since the expected change of pressure is too small to account for it. Increasing intraluminal pressure produces a high level of myogenic tone and depolarization²⁴: the higher the intraluminal pressure, the higher is the myogenic tone and the less negative is the resting membrane potential. It would be expected that the open probability time of endothelial potassium channels involved in the flow-induced dilation²⁵ would be decreased, while voltage-activated calcium channels associated with constriction in vascular smooth muscle cells would be increased. Thus, flow-induced constriction observed at a high level of myogenic tone could be related to a shift in membrane potential to a more depolarized state. This explanation is consistent with our observation of the flow response seen when myogenically contracted arteries were exposed to PSS containing a higher concentration of KCl (30 mmol/L). Under these circumstances only flow-induced constriction was observed (Figure 4), suggesting that depolarization indeed inhibits flow-induced dilation and promotes flow-induced constriction.

An understanding of the response to flow at different levels of myogenic tone is essential to assess how both flow and pressure interact to balance vascular tone and regulate blood flow. The complex flow response—dilation at lower pressure and constriction at higher pressure—would be expected to play a role in autoregulation of cerebral blood flow. Dilation observed at 40 and 60 mm Hg intraluminal pressure would be self-facilitating and would serve to allow increased cerebral blood flow with only minimal change in resistance. Constriction observed in tandem with raised pressure (80 mm Hg intraluminal pressure) would be self-limiting and would serve to protect the brain parenchymal circulation from the consequences of sudden and substantial increases in pressure.

In conclusion, this study suggests that the response to an increase in shear stress is a combination of dilation (partly endothelium and NO dependent) and constriction (endothelium independent). The level of vascular tone at which the shear stress increase occurs will determine the pattern of the response: the lower the intraluminal pressure, the greater is dilation; the higher the pressure and the greater the level of myogenic tone, the more dominant is constriction. Pressure- and flow-dependent mechanisms appear to control the vascular tone of arteries of this size, allowing blood flow to be matched to metabolic demands.

	Selected Abbreviations and Acronyms

 

L-NNA = N-nitro-L-arginine NO = nitric oxide PCA = posterior cerebral artery PSS = physiological salt solution

	Acknowledgments

 
This study was supported by a grant from the US Public Health Service (HL-32985).

Received October 15, 1997; revision received February 26, 1998; accepted March 20, 1998.

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Editorial Comment

William J. Pearce, PhD, Guest Editor

Departments of Physiology, Pharmacology, and Biochemistry, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, Calif

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
In all vascular beds, the basal level of contractile tone is a key determinant of in vivo vascular reactivity. It is against this resting tone that vasodilator influences act to reduce vascular resistance and increase tissue perfusion. Resting contractile tone, in turn, is determined by a multitude of factors, which in the cerebral circulation include tonic neurogenic influences^{1 2} ; circulating vasoactive substances, such as angiotensin^{3 4} and epinephrine⁵ ; tonic release of nitric oxide from the neuropil and vascular endothelium^{6 7 8} ; and ambient carbon dioxide tension.^{9 10} Arterial pressure is perhaps one of the most important of these factors by virtue of its effects on stretch-induced or myogenic tone.^{11 12} Blood viscosity and shear-stress are also important, owing to their ability to modulate endothelial release of nitric oxide and other vasoactive compounds.^{13 14 15}

Whereas it is clear that many different factors contribute to basal cerebrovascular tone, their relative importance and the nature of their interactions is much less certain, particularly during integrated cerebrovascular responses such as cerebral autoregulation.^{3 6 11 16} The accompanying article by Bevan et al in this issue addresses this problem by exploring the balance between pressure-induced myogenic tone and the well-documented ability of changes in shear stress to induce changes in flow.^{14 15 17 18} In cannulated preparations of rabbit posterior cerebral arteries maintained at low (40 mm Hg) or moderate (60 mm Hg) hydrostatic pressures, increases in shear stress secondary to increases in luminal flow elicited vasodilatory responses that could be dramatically attenuated by either endothelial denudation or inhibition (100 µM L-NNA) of endothelial nitric oxide production. In contrast, preparations maintained at high (80 mm Hg) hydrostatic pressures exhibited only a transient endothelium-dependent vasodilatation followed by a sustained endothelium-independent vasoconstriction. These findings demonstrate that responses to flow-induced increases in shear stress are balanced between endothelium-dependent vasodilator influences that predominate at low transmural pressures and endothelium-independent vasoconstrictor influences that predominate at high transmural pressures.

Although the mechanisms of the flow-induced vasoconstriction observed at high transmural pressures remain uncertain, it is tempting to speculate that this vasoconstriction contributes significantly to the autoregulatory increases in cerebrovascular resistance observed in response to elevated cerebral blood flow and perfusion pressure. Correspondingly, the flow-induced vasodilatation observed at low transmural pressures might also help meet demand for increased cerebral perfusion at low perfusion pressures and thereby facilitate autoregulatory decreases in cerebrovascular resistance. Overall, the complex nature of the interaction observed between initial myogenic tone and responses to increased flow emphasizes that the relative importance of the mechanisms governing basal cerebrovascular tone and autoregulation can be appreciated fully only when these mechanisms are studied simultaneously and in the context of integrated homeostatic responses.

	Selected Abbreviations and Acronyms

 

L-NNA = N-nitro-L-arginine NO = nitric oxide PCA = posterior cerebral artery PSS = physiological salt solution

Received October 15, 1997; revision received February 26, 1998; accepted March 20, 1998.

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