INTRODUCTION

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BECAUSE BLOOD FLOW TO MANY tissues is regulated at the microvasculature level, studies of the effect of flow have often been done in small arteries and arterioles using low flow rates. Within the cerebral circulation, however, autoregulation is not confined to the small-diameter vessels. Larger-diameter arteries, such as the middle cerebral artery, possess myogenic properties and may also be involved in the regulation of cerebral blood flow, and little is known about the mechanisms underlying flow responses in larger-diameter vessels. Previously, we studied the effects of laminar flow in cannulated and pressurized middle cerebral arteries from neonatal piglets (34) and found constriction at low flows (0.08-0.2 ml/min) and a nitric oxide (NO)-mediated dilation at higher flows (0.2-1.6 ml/min). However, middle cerebral arteries in adult animals autoregulate over a wider pressure range and have a more negative resting membrane potential (E_m) than arteries of neonates (32); thus their responses to flow and the mechanisms underlying them might be different.

In this study we used isolated, cannulated, and pressurized middle cerebral arteries from adult cats and measured changes in diameter and E_m during flow. Because various studies have shown that the level of vascular tone and/or the luminal shear stress may determine whether a vessel dilates or constricts to flow (31, 36), we also studied the effect of flow in arteries pressurized at the lower end of the autoregulatory scale and in vessels either constricted with a thromboxane mimetic or dilated by hypercapnia. Various studies have indicated roles for endothelium-derived factors (14, 28, 32, 35), free radicals (12, 21, 23), and tyrosine kinases (14, 37) in flow-induced responses. To determine their participation in middle cerebral arteries in the cat, we inactivated the endothelium with an air bubble and inhibited prostacyclin and NO production. Free radical production was inhibited with superoxide dismutase (SOD) and catalase and tyrosine kinase with genistein and tyrphostin-23. Finally, because integrins have been shown to transduce dilatory flow responses (30) and initiate or activate cellular responses (5), we used active and inactive synthetic versions of the extracellular matrix proteins containing the arginine-glycine-aspartic acid (RGD) peptide sequence to bind with the integrins to determine whether they also participated in contractile responses to flow.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Vessel preparation. This study was approved by the Animal Care and Use Committee of the Zablocki Veterans Affairs Medical Center. Forty adult mongrel cats (2.5-4.0 kg) of either sex were premedicated with ketamine hydrochloride (15 mg/kg) and anesthetized with intraperitoneal pentobarbital sodium (30 mg/kg). The animals were exsanguinated by severing the carotid artery, the skull was opened, and the brain was removed. Left and right segments of the middle cerebral arteries were dissected out and placed in cold (4°C) physiological saline solution (PSS) until use.

Membrane potential measurement. Membrane potentials were measured with glass microelectrodes filled with 3 M KCl and having tip resistances between 50 and 80 M. Impalements were made from the adventitial side of the vessel. The E_m was measured during no-flow conditions and at each flow step after pressure and diameter were stable. Turning flow on or off during an impalement dislodged the microelectrode. Criteria for a successful impalement were an abrupt negative drop in voltage when the electrode entered the cell, an immediate return to baseline on withdrawal of the electrode, and no change in electrode resistance.

The pressure/flow control system. Arterial pressure under flow conditions was controlled with a system similar to that used in our previous studies (34, 35). However, in the present study, instead of using a syringe pump connected to the inflow cannula to deliver PSS into the arterial lumen, a microprocessor-controlled roller pump (Masterflex model 7524-10; Cole Parmer) was used to deliver heated PSS gassed with the same mixture as above into the vessel lumen. The pump head has eight rollers, and a pulse damper in line before the inflow pressure transducer eliminates virtually all oscillations. With the use of this system, drugs could be added to the intraluminal perfusate or the gas mixture could be changed.

Protocols. The cerebral arteries were considered to be viable if their diameters decreased during the 90-min equilibration at 100 mmHg and if they contracted when 25 mM KCl was added to the bath. The presence of a functional endothelium was verified by dilation to ACh (1 µM).

Solutions and reagents. The composition of the PSS (in mM) was 145 Na⁺, 4.5 K⁺, 2.5 Ca²⁺, 0.72 Mg²⁺, 126 Cl, 1.7 H₂PO₄, 22.5 HCO₃, and 11 glucose. ACh, N-nitro-L-arginine methyl ester (L-NAME), SOD, catalase, hypoxanthine, and xanthine oxidase (Sigma Chemical, St. Louis, MO) were dissolved in PSS. Stock solutions of genistein, tyrphostin-23, and tyrphostin-1 (Sigma) were prepared in dimethyl sulfoxide and PSS. A stock solution of U-46619 (Biomol Research Laboratories, Plymouth Meeting, PA) was prepared in ethanol and added to PSS. The active and inactive RGD peptides GRGDNP and GRGESP were obtained from GIBCO-BRL (Gaithersburg, MD) and prepared in PSS. All drug concentrations are expressed as the final molar concentrations in the bathing solution and/or intraluminal perfusate.

Statistics. All diameter measurements are expressed as means ± SE of actual diameter or as a percentage of original diameter defined as the stable diameter at zero flow under either control conditions or after an intervention. To determine the differences between groups, Student's paired t-test or ANOVA with single or repeated measures and Fisher's least significant difference test were used as appropriate. A significant value of P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The average diameter of the 57 total arteries used in this study was 807 ± 13 µm at mounting. During equilibration, the arteries developed spontaneous tone, and by the end of the period their diameters averaged 679 ± 14.4 µm, a decrease of 15.7 ± 1.44% (P < 0.05).

Effect of flow on diameter and E_m. As flow was increased from 0 to 4.0 ml/min at a constant transmural pressure of 100 mmHg, artery diameters decreased by 20%, stabilizing at 81.9 ± 0.95% (P < 0.05) of their values at zero flow, and E_m depolarized from 54 ± 0.51 mV to 40 ± 1.26 mV (P < 0.05; Fig. 1). The diameter curves generated during increasing and decreasing flow showed little hysteresis, and the E_m repolarized as flow decreased (Fig. 2). If flow was turned off at 4 ml/min, the artery dilated and repolarized to its original no-flow values in <2 min. Turning on flow to 4 ml/min resulted in constriction and depolarization to a level close to that achieved when flow was increased in steps (data not shown). Flow-induced constriction was abolished by treating the arteries with papaverine (1 µM) or nifedipine (0.1 µM).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of increasing flow on vessel diameter (n = 44 arteries) and membrane potential (Em; n = 19 arteries and 6-30 impalements at each flow) in cannulated middle cerebral arteries in the cat.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of increasing and decreasing flow on diameter and Em in a cat middle cerebral artery.

Functional versus nonfunctional endothelium. In 11 arteries an air bubble was present in the vessel lumen during equilibration. These arteries developed spontaneous tone and responded to KCl, but they either did not dilate to ACh or they constricted. These arteries were thus considered to have a nonfunctional endothelium. However, their response to flow was no different from arteries with a functional endothelium (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A: effect of increasing flow in arteries with a functional endothelium and in arteries whose endothelium had been inactivated with an air bubble (n = 11 arteries). B: effect of inhibiting NO production with N-nitro-L-arginine methyl ester (L-NAME; n = 6 arteries) on arterial diameter change in response to increasing flow. * Significantly different from diameter at same flow under control conditions; P < 0.05.

Effect of shear stress and transmural pressure. Table 1 shows the average wall shear stress,  (in dyn/cm²) ( = 4 µ/ r³, where µ is viscosity in poise, is flow in ml/s, and r is artery radius in cm) throughout the flow range under control conditions. To increase shear stresses, five arteries were constricted with the thromboxane mimetic U-46619 (10⁹ M) to 82.5 ± 1.87% of their original diameter at no flow (P < 0.05). The reduction in diameter was thus close to that achieved by flow at 4 ml/min in untreated arteries. Although shear stresses in the U-46619-treated vessels were increased by over 30% to 65 dyn/cm² at 4 ml/min, arterial diameter still decreased with flow (Fig 4; P < 0.05), and the total constriction to flow was similar in both treated and untreated arteries (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of constriction induced by 109 M U-46619 (n = 5 arteries) on arterial diameter change in response to increasing flow. * Significantly different from control conditions; P < 0.05. Inset: similar percent constriction at 4 ml/min by control and U-46619-treated arteries.

Effect of free radical scavengers. To determine whether the cat isolated middle cerebral arteries responded to superoxide radicals, four vessels were exposed to hypoxanthine (0.2 mM) for 15 min under no-flow conditions. When xanthine oxidase (0.06 U/ml) was added, arterial diameter decreased by 9.15 ± 0.79% (P < 0.05). The effect of free radical scavengers on the flow response was then investigated. SOD (200 U/ml) or catalase (140 U/ml) was added to the perfusate bathing the artery and to the intraluminal perfusate for 30 min without flow. Arteries exposed to SOD showed no significant change in diameter at zero flow (Fig. 5A). When flow was started and SOD was perfused intraluminally, arterial diameter at first decreased slightly but not significantly. (This was most likely due to the SOD-free PSS in the tubing leading into the artery). When flow was increased to 1.5 ml/min, arterial diameter began to increase, and it had returned to zero flow levels by the time flow was 3 ml/min and remained there (Fig. 5A). Adding 1 mM L-NAME to both the bathing solution and intraluminal perfusate in the presence of SOD reduced the diameter of the vessels at zero flow; however, this reduction was not statistically significant. The addition of L-NAME did not affect the dilatory response to flow (Fig. 5B). Three arteries treated with catalase showed no change in diameter at zero flow and still constricted throughout the flow range to the same extent as before treatment.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of superoxide dismutase (SOD) (n = 6 arteries) (A) and SOD + L-NAME (n = 3 arteries) (B) on arterial diameter change in response to increasing flow. * Significantly different from diameter at same flow under control conditions; P < 0.05.

Effect of tyrosine kinase inhibition. Tyrosine kinase was inhibited with genistein (50 µM). The active receptor tyrosine kinase blockers, tyrphostin-23 or its inactive form, tyrphostin-1 (50 µM), were also used. Arteries exposed to genistein dilated by over 30% at zero flow (P < 0.05) and remained significantly dilated as flow was increased (Fig. 6). The E_m remained at 52 ± 0.5 mV throughout the flow range in contrast with the 38 ± 1 mV measured at 4 ml/min under control conditions (P < 0.05). Arteries exposed to tyrphostin-23 exhibited a smaller, albeit significant, dilation of 5% (P < 0.05) at zero flow, and like the genistein-treated vessels, they also did not constrict (Fig. 7) or depolarize during flow. The behavior of arteries exposed to the inactive analog tyrphostin-1 resembled that of controls (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of genistein (n = 4 arteries) on arterial diameter change in response to increasing flow. * Significantly different from diameter at same flow under control conditions; P < 0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of tyrphostin-23 (n = 4 arteries) and tyrphostin-1 (n = 3 arteries) on arterial diameter change in response to flow. * Significantly different from diameter at same flow under control conditions; P < 0.05.

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View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effect of dilation induced by increased PCO2 on arterial diameter change in response to increasing flow (n = 5 arteries). * Significantly different from diameter at same flow under control conditions; P < 0.05. Inset: similar percent constriction at 4 ml/min by control and hypercapnic arteries.

Effect of synthetic integrin binding peptides. Arteries treated for 30 min with the active synthetic RGD peptide GRGDNP (0.5 mM) showed no change in diameter at zero flow, but during flow they constricted significantly less than under control conditions (P < 0.05; Fig. 9A). Constriction to flow after the peptide was washed out for 30 min was not significantly different from the first control curve (Fig. 9A). The inactive peptide GRGESP had no effect on resting diameter or the constriction to flow (Fig. 9B).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Effect of treatment with active RGD peptide GRGDNP (A) and inactive RGD peptide GRGESP (B) on arterial diameter change in response to increasing flow (n = 4 and 3 arteries, respectively). * Significantly different from diameter at same flow under control conditions; P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, isolated, cannulated, and pressurized middle cerebral arteries in the cat constricted in response to flow, and the constriction was accompanied by membrane depolarization. The constriction was independent of a functional endothelium and appeared to involve the generation of superoxide radicals and tyrosine kinase. These responses appeared to be mediated through integrin signaling. Neither reducing intraluminal pressure nor increasing tone or shear stresses altered the constriction to flow. Constriction during flow did not impede the arteries' ability to dilate to CO₂, and inhibiting flow-induced constriction did not alter contractile responses to other agonists.

Although our result showing constriction to flow in isolated middle cerebral arteries from the cat is in contrast with the finding of dilation by cat pial arteries in vivo (38), it is consistent with other reports of constriction by cerebral arteries in vitro (3, 9, 31, 34, 37) and with the observations that flow-induced constriction in various arteries is most often seen in vitro (1, 2). In many in vitro studies, whether a cerebral artery constricted or dilated during flow has depended on the level of arterial tone. For example, both rabbit (9, 10) and rat (37) pial arteries dilated to flow at low pressure, but at higher pressure they constricted. However, this pattern has not always been consistent. Rabbit middle cerebral artery rings constricted with histamine had only a dilatory response to flow (11). In the cat middle cerebral arteries used in this study, the constrictor response to flow persisted both when tone was increased by constriction with U-46619 and when tone was reduced either by dilating with CO₂ or lowering intraluminal pressure. In other in vitro studies in which vessels were held at constant pressure, constriction or dilation to flow seemed to depend on the flow rate. At 60 mmHg, rat cerebral arterioles in vitro dilated at low flows and constricted at higher flows (31), whereas we found that piglet middle cerebral arteries at 20 mmHg (34) constricted at low flows and dilated at higher ones. The cat middle cerebral arteries used in this study constricted continuously throughout the flow range used.

To our knowledge, in only one other study (4) was E_m measured during flow. In that study, ring segments of rabbit middle cerebral artery (200-250 µm) mounted on a wire myograph were subjected to flows of 20 µl/min delivered from a cannula inserted into the lumen of the stretched ring. At resting E_m more negative than 58 mV, the arteries depolarized with flow, but if the vessels were more depolarized they then tended to hyperpolarize with flow. In contrast, the cat middle cerebral arteries used in the present study were cannulated segments that were pressurized to 100 mmHg and had myogenic tone. The resting E_m averaged 54 ± 0.51 mV, and during flow the E_m always depolarized, eventually reaching 40 ± 1.26 mV at 4 ml/min. As discussed in more detail below, the contractile response to flow appeared to involve activation of tyrosine kinase, an event that is associated with membrane depolarization (22).

The shear stresses generated over the flow range used in this study are consistent with those in other studies of cerebral arteries (3, 31). Increasing shear stresses on endothelial cells has often resulted in an NO-mediated dilation (26, 33), and we had found this to be the case in the piglet cerebral arteries (35). Therefore, we wondered if increasing shear stresses in the cat cerebral arteries would eventually result in a dilatory response to flow. However, when shear stresses were increased by contracting the arteries with U-46619 to approximately the same diameter as with flow alone, constriction to flow persisted. It also persisted in ~400-µm-diameter side branches of the middle cerebral arteries in which shear stresses were >200 dyn/cm². Constriction to flow at high shear stress has also been found in 45-µm-diameter rat cerebral arterioles (31) in which flows of 25 µl/min produced shear stresses over 500 dyn/cm².

Even though flow did not induce an NO-mediated dilation in the cat middle cerebral arteries, basal NO release did seem to modulate arterial diameter. Treating the arteries with L-NAME reduced their baseline diameter so that the overall percent constriction to flow was less than before treatment. However, the total decrease in vessel diameter, i.e., the sum of constriction to L-NAME and to 4 ml/min flow was not different from the decrease in diameter due to flow alone.

We had previously found that cat pulmonary arteries constricted to flow through a mechanism involving endothelin-1 (ET-1; Ref. 35) and that the constriction persisted for 45 to 60 min after flow was stopped. A role for ET-1 in the flow-induced constriction of the cat middle cerebral arteries did not seem likely because the constriction reversed completely within 2 min when flow was turned off, which is not consistent with the slow recovery associated with ET-1 responses, and adding staurosporine, which inhibits the protein kinase C necessary for ET-1 synthesis and activity, did not affect the constriction to flow (data not shown).

In this study, generating superoxide ions directly with xanthine/xanthine oxidase resulted in contraction, and scavenging superoxide with SOD eliminated the constriction to flow. These findings agree with others that have demonstrated effects of free radicals on vascular diameter and responses to flow (7, 12, 21, 37, 38). In canine basilar arteries superoxide anions also caused contraction (16); in rabbit aorta superoxide ions were generated during flow (21); and in rat gracilis muscle arterioles superoxide ions reduced flow-induced dilation (12). Scavenging superoxide with SOD restored flow dilation in the rat gracilis arterioles (12) and eliminated flow-induced contraction in pig pulmonary arteries (23). Scavenging hydrogen peroxide with catalase had no effect on flow responses either in the present study or in rat gracilis arterioles (12). The mechanism underlying the contractile effect of superoxide ions is not certain. There is evidence that vascular smooth muscle cells can produce superoxide ions (16, 28). It has also been suggested that a cyclooxygenase-independent arachidonate oxidation might be activated by superoxide to result in the release of vasoconstrictor prostaglandins (29) or that superoxide through generation of hydroxyl radical inactivates guanylate cyclase (20). Others have postulated that scavenging superoxide with SOD prolongs the life of a relaxing factor, presumably NO (12, 23, 33).

If superoxide were derived only from the endothelium and scavenging superoxide prolonged the activity of NO, then in the present study we would have expected the following: 1) the arteries without a functional endothelium would not constrict to flow, which was not the case; and 2) adding L-NAME to SOD-treated arteries would attenuate the dilation to flow. L-NAME added to SOD-treated arteries did tend to reduce arterial diameter at zero flow, although this was not statistically significant. However, when flow was turned on, the arteries still dilated to the same extent as before L-NAME treatment. This finding of a lack of NO involvement in the dilation to flow was somewhat surprising but not without precedent. High doses of N-monomethyl-L-arginine did not suppress flow-induced dilation in cat femoral arteries (27), rat basilar arteries (8), or rat cremaster arterioles (19). Release of vasodilator prostaglandins did not seem likely because adding indomethacin to SOD-treated arteries did not affect the dilation to flow (data not shown), and SOD has been shown to interfere with dilation induced by arachidonic acid metabolites (6). Other dilators that could have contributed to the flow-induced dilation include substance P, bradykinin, and ATP, or possibly K⁺ channel activation (13).

The physiological function of superoxide-mediated flow-induced constriction in the middle cerebral arteries is not immediately evident, although it may help regulate blood flow to brain tissue in smaller-diameter vessels as blood flow increases. Alternatively, it has been suggested to have a pathological effect in some disease states or after angioplasty (21). In our study, constriction to flow did not appear to interfere with the ability of the artery to dilate to CO₂, suggesting that the constriction was not irreversible or pathological. Direct measurement of free radical production in the vessels and/or removal of the endothelium without damaging the smooth muscle cells would help clarify any participation of the endothelium in the flow-induced constriction and in generation of free radicals. In addition, using blood that has free radical scavengers rather than PSS might result in a different effect.

Tyrosine kinase has been shown to be important in constrictor responses of cerebral arteries (17). Cross talk between the tyrosine kinase pathway and the myosin light chain kinase pathway in vascular smooth muscle cells appears to be part of the contractile process (15). Free radicals have also been shown to activate tyrosine kinase (14). Therefore, tyrosine kinases activated by superoxide ions generated in response to flow may explain the constriction in the cat middle cerebral arteries. This hypothesis is supported by the finding in rat basilar arteries that inhibition of tyrosine kinase affected flow-induced constriction (37). Tyrosine kinases can activate L-type calcium channels in vascular smooth muscle cells to produce vasoconstriction (22), and tonic phosphorylation by tyrosine kinases may maintain calcium channels in an available state for activation by depolarization (22). In the present study, arteries treated with the tyrosine kinase inhibitor genistein did not depolarize during flow although they did under control conditions.

Tyrosine kinase appears to be active even under no-flow conditions. In this study of middle cerebral arteries in the cat and in the work of Matsumoto et al. (25) in rat cerebral arteries, the vessels dilated after treatment with genistein and active tyrphostin analogs. In addition, the cat middle cerebral arteries did not constrict to flow. However, inactivating tyrosine kinase did not inhibit the ability of the cat middle cerebral arteries to constrict to U-46619, nor did it inhibit the ability of rat cerebral arteries to constrict KCl and phenylephrine (25). These data suggest that signaling pathways and mechanisms of constriction to other agonists may differ from those responsible for flow-induced contraction. Although a role for protein kinase C in flow-induced contraction has also been postulated (39), it did not seem to play a role in the response of the arteries in this study because, as mentioned above, staurosporine had no effect on the cerebral artery constrictor response to flow.

How arteries transduce the shear stress of flowing fluid on their cells into a mechanical response is the subject of intense investigation. Bevan (1) postulated that shear forces on the endothelial cells extend mechanically to subendothelial tissues to cause conformational changes in glycosaminoglycans by extending them from a randomly coiled aggregated state to a more elongated condition parallel to the line of flow. Recently, it has been shown that flow-induced shear stresses on endothelial cells are mechanically transduced by integrins (30). Integrins form attachments to the extracellular matrix and serve as receptors for signaling molecules that activate pathways such as tyrosine phosphorylation (5). Our finding of inhibition of flow-induced contraction by the synthetic integrin binding peptide GRGDNP and its lack of attenuation by the inactive form of the peptide suggests that integrins are involved in the process. Previously, integrin involvement has been shown only in flow-induced dilation (30).

Some of the disparities in results of the flow studies reported in the literature may be attributed to factors such as animal species, location of the vascular bed, differences in experimental preparation, or whether the study was done in vitro or in vivo. In the present in vitro study, the use of an isolated artery eliminated confounding effects of other neural and humoral mediators. However, isolating an artery may unmask behavior that is not normally seen in vivo (24). This might suggest that constrictor responses to flow seen in an in vitro preparation could be modulated or even opposed in vivo by mediators of flow-induced dilation. Thus, although in vitro studies may be helpful in determining physiological and/or possible pathophysiological responses, it also suggests that the results should be interpreted with caution.