INTRODUCTION

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PRESSURE-INDUCED MYOGENIC constriction represents a fundamental property of resistance-size arteries and arterioles and contributes to autoregulation of regional blood flow and maintenance of capillary hydrostatic pressure. It is also an essential component of basal vascular tone, which allows blood vessels to constrict or dilate in response to vasoactive mediators, and to elevations or reductions in transmural pressure, respectively. The myogenic response occurs independently of the endothelium and perivascular nerves and is therefore an inherent property of vascular smooth muscle (VSM) cells (10, 12, 13, 24, 33, 40, 45).

Numerous studies (10, 12, 16, 18, 26, 33, 40, 48) have demonstrated the importance of membrane depolarization, with resultant Ca²⁺ influx through voltage-dependent Ca²⁺ channels and an increase in intracellular free Ca²⁺, as the principal mechanism by which pressure or stretch elicits smooth muscle contraction. In cerebrovascular smooth muscle, L-type Ca²⁺ channels are themselves sensitive to mechanical load (stretch), an observation that adds further complexity to the mechanisms underlying Ca²⁺ elevation induced by intraluminal pressure (29). Some studies (12, 14, 25, 33) have suggested that, in addition to enhanced Ca²⁺ influx, increased smooth muscle Ca²⁺ sensitivity may also contribute to the development of myogenic tone.

Despite considerable progress in our understanding of the mechanisms underlying myogenic tone and reactivity, the major questions of which cellular structures within the vascular wall detect the mechanical load imposed by transmural pressure and of how this signal is transduced into the myogenic response, remain unresolved. In a variety of nonmuscle cell types, the complex of extracellular matrix, transmembrane integrins, and intracellular cytoskeletal structures has been implicated in mechanotransduction (3, 8, 22, 23, 39). Hence, the detection of mechanical deformation, presumably the initial step in the signal transduction pathway, may occur within the cell membrane, cytoskeleton, or both.

Two models have been proposed to explain how cells can sense a mechanical load (3). According to the inside-out signaling model, the cytoskeleton primarily detects mechanical deformation and transfers tension to the mechanosensory integrin-dependent system in the cell membrane. This, in turn, triggers a cascade of biochemical events that subsequently evoke the appropriate cellular response. It is also possible that membrane-associated integrin molecules play a key role in detecting the mechanical load and transmit the mechanical stimulus from the cell membrane to the interior of the cell, which responds by structural reorganization and/or polymerization of one or more cytoskeletal components, an elevation in the intracellular cytosolic Ca²⁺ concentration ([Ca²⁺]_i) or other signaling molecules (the outside-in model) (3, 8, 11, 13, 47, 49).

Thus far, very little is known about the specific role of the cytoskeleton in VSM mechanoreception and mechanotransduction. Together with intermediate filaments and microtubules, actin represents a well-defined and complex cytoskeletal structure in smooth muscle cells (42). A role for actin polymerization in response to the contractile activation of tracheal smooth muscle has been proposed by Mehta and Gunst (30). A study (6) from our laboratory showed that disruption of the actin filamentous system with cytochalasin B resulted in accelerated forced dilatation of cerebral arteries, suggesting a similar role for the actin cytoskeleton in pressure-induced vasoconstriction. On the basis of the latter study, we hypothesized that the actin cytoskeleton might be important in linking the cellular deformation induced by transmural pressure to myogenic vasoconstriction. The actin cytoskeleton of VSM is physically coupled to or part of the contractile machinery (42) and, therefore, any manipulation of actin cytoskeletal structure and dynamics could also influence constriction. Hence, the objective of this study was to examine the effects of actin cytoskeletal disruption on pressure-induced membrane depolarization and Ca²⁺ influx, the aforementioned events that are fundamental to the mechanism underlying the development of myogenic constriction. We used two structurally unrelated compounds, cytochalasin D and latrunculin A, which disrupt actin filamentous structure by binding to filamentous actin (F-actin) and monomeric actin (G-actin), respectively. Despite their different mechanisms of action, both compounds disrupt and disorganize the actin cytoskeletal structure within living cells (2, 19, 35, 43).

	METHODS

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Animals. Adult (16-24 wk old) male Wistar-Kyoto rats (n = 72) were anesthetized by an intraperitoneal injection of methohexital sodium (Brevital; 50 mg/kg) and killed by decapitation. The brain was removed and immersed in a dissection dish filled with physiological salt solution (PSS; composition presented below). The entire posterior cerebral artery, including its branches, was removed and carefully dissected free from surrounding connective tissues under a stereodissection microscope. All experiments were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1985). The experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Vermont.

Measurement of smooth muscle Ca²+ in pressurized arteries. To obtain simultaneous measurements of arterial diameter and [Ca²⁺]_i in VSM cells, arteries were loaded with fura 2, a Ca²⁺-sensitive fluorescent dye. Fura 2-acetoxymethyl ester (AM), 2 µl of 1 mM stock dissolved in dimethyl sulfoxide (DMSO), was premixed with an equal volume of a 25% solution of pluronic acid in DMSO and then diluted in PSS to yield a final concentration of 5 µM. The excised posterior cerebral artery, including its branches, was incubated in the fura 2-AM-PSS loading solution at room temperature in the dark for 60 min. Fura 2-loaded arteries were then washed with PSS and kept refrigerated until use. Each arterial segment was cannulated, mounted in an arteriograph and continuously superfused at 3 ml/min with oxygenated PSS (95% O₂-5% CO₂) at 37°C. Fura 2 fluorescence was measured using a photomultiplier system (IonOptix) in which background-corrected ratios of the 510-nm emission from arteries alternatively excited at 340 and 380 nm were obtained at a sampling rate of 5 Hz. Arterial diameter was simultaneously recorded using the SoftEdge Acquisition Subsystem (IonOptix). All experimental protocols were started after a 30-min equilibration period at 10 mmHg.

Measurements of membrane potential. For measurements of membrane potential we used glass microelectrodes filled with 0.5 M KCl having tip resistances of 110-150 M; an Ag-AgCl pellet was used as an indifferent electrode. Microelectrode impalements of VSM cells were made from the adventitial surface of arterial segments. A microelectrode was connected to a motorized micromanipulator (World Precision Instruments) and membrane potential was recorded using a high-input impedance amplifier (Electro 705, World Precision Instruments). Changes in membrane potential and arterial diameter were simultaneously displayed and recorded on a personal computer with the use of an IonWizard data-acquisition program (IonOptix). The following criteria were used for acceptance of membrane potential recordings: 1) abrupt negative change in voltage on impalement of the cells; 2) a sharp return to 0 V after withdrawal of a microelectrode tip; 3) tip potential of <7 mV; and 4) unchanged resistance of microelectrodes after impalement.

Solutions and drugs. PSS was composed of (in mM) 119 NaCl, 4.7 KCl, 24.0 NaHCO₃, 1.2 KH₂PO₄, 1.6 CaCl₂, 1.2 MgSO₄, 0.023 EDTA, and 11.0 glucose equilibrated with a mixture of 95% O₂-5% CO₂, pH 7.4. High-potassium solution (60 mM) was prepared by substituting equimolar amounts of NaCl with KCl. For the fura 2 calibration procedure, we used a solution composed of 140 mM KCl, 20 mM NaCl, 5 mM HEPES, 5 mM EGTA, 1 mM MgCl₂, 5 µM nigericin, and 10 µM ionomycin, pH 7.15.

Calculations and statistical analysis. Arterial wall [Ca²⁺]_i was calculated using the following equation (17): [Ca²⁺]_i = K_d(R  R_min)/(R_max  R), where R is the experimentally measured ratio (340:380 nm) of fluorescence intensities, R_min is the ratio in the absence of [Ca²⁺]_i, R_max is the ratio at Ca²⁺-saturated fura 2 conditions, and K_d is the dissociation constant of fura 2.  is a ratio of the fluorescence intensities at 380-nm excitation wavelength at R_min and R_max. R_min, R_max, and  were determined by an in situ calibration procedure using arteries treated with nigericin (5 µM) and ionomycin (10 µM) as previously described (26). These values were then pooled and used to convert the ratio values into [Ca²⁺]_i. K_d was 282 nM, as determined by using in situ titration of Ca²⁺ in fura 2-loaded posterior cerebral arteries (26). Arterial diameter, pressure, ratio, and membrane potential values were recorded using an IonWizard data-acquisition program and imported into SigmaPlot and SigmaStat programs for graphic representation, calculations and statistical analysis. Data are expressed as means ± SE, where each n is the number of arterial segments studied. Only one vessel per animal was used for a particular protocol. A paired or unpaired Student's t-test or ANOVA were used to determine the significance of differences between sets of data, with P < 0.05 considered significant.

	RESULTS

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Cytochalasin D and latrunculin A inhibit pressure-induced constriction but augment associated elevation of [Ca²+]_i. To explore the role of the actin cytoskeleton in mechanotransduction in small cerebral arteries, we studied the effects of actin cytoskeletal disruption with cytochalasin D and latrunculin A on pressure-induced elevations in smooth muscle [Ca²⁺]_i and constriction. In control experiments, after a 30-min equilibration period at 10 mmHg, intraluminal pressure was increased to 60 mmHg. The initial distention of the artery was followed by myogenic constriction (24 ± 3% of maximal diameter) and elevation of [Ca²⁺]_i from 88 ± 8 to 237 ± 10 nM. After stabilization of the myogenic response (10-15 min), pressure was returned to 10 mmHg. A second pressure step from 10 to 60 mmHg was applied 15-20 min later. This time was necessary for the restoration of both arterial diameter and [Ca²⁺]_i level (86 ± 9 nM) to the control values. The extent of constriction and [Ca²⁺]_i elevation in response to the second pressure step were not different from the control values: 25 ± 3% and 234 ± 12 nM, respectively (P > 0.05; n = 14).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effects of cytochalasin D (Cyt-D) on pressure-induced elevation of intracellular cytosolic Ca2+ concentration ([Ca2+]i) smooth muscle in arterial wall and constriction of rat cerebral arteries. A: representative traces showing an increase in arterial wall [Ca2+]i and a decrease in arterial diameter in response to an elevation of intraluminal pressure from 10 to 60 mmHg before (control) and after 30 min of treatment with cytochalasin D (5 µM). B and C: bar graphs summarizing the effects of cytochalasin D on pressure-induced elevation in [Ca2+]i and associated vasoconstriction. *P < 0.05, significant difference. dmax, Maximal diameter.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effects of latrunculin A (Lat-A) on pressure-induced elevation of arterial wall [Ca2+]i and constriction of posterior cerebral arteries. A: original traces illustrating changes in arterial wall [Ca2+]i and diameter in response to elevation of intraluminal pressure from 10 to 60 mmHg before (control) and 30 min after treatment with latrunculin A (3 µM). B and C: bar graphs summarizing the effects of latrunculin A on pressure-induced elevation in [Ca2+]i and vasoconstriction. *P < 0.05, significant difference.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effects of acute application of cytochalasin D and latrunculin A on arterial wall [Ca2+]i and diameter of pressurized rat posterior cerebral arteries. A: original traces showing an increase in arterial wall [Ca2+]i and the appearance of myogenic constriction in response to an elevation of intraluminal pressure from 10 to 60 mmHg. Application of cytochalasin D (5 µM) resulted in an additional increase in [Ca2+]i and dilation of the artery. B and C: bar graphs summarizing the effects of cytochalasin D and latrunculin A on arterial wall [Ca2+]i and pressure-induced constriction. *P < 0.05, significant difference.

Cytochalasin D potentiates pressure-induced [Ca²+]_i elevation in endothelium-denuded arteries. There is some evidence that the actin cytoskeleton is involved in the regulation of vascular endothelial function (50). Therefore, the observed enhancement of the pressure-induced [Ca²⁺]_i response might be due to actin cytoskeletal disruption in endothelial cells, with subsequent modulation of smooth muscle function. To test this possibility, we performed additional experiments using endothelium-denuded arteries (see METHODS). As in intact vessels, cytochalasin D virtually abolished pressure-induced constriction (4 ± 2 vs. 28 ± 6% in control; P < 0.05), but significantly potentiated the associated elevation in [Ca²⁺]_i (295 ± 20 vs. 222 ± 19 nM in control; P < 0.05; n = 4). On the basis of these data, we conclude that the cytochalasin-enhanced elevation in [Ca²⁺]_i and inhibition of myogenic tone are due to specific disruption of the actin cytoskeleton in VSM cells.

Cytochalasin D-induced augmentation in [Ca²+]_i in response to pressure elevation is not due to increased wall tension. As already shown, both cytochalasin D and latrunculin A abolished constriction in response to elevated intraluminal pressure. This effect was associated with a substantial (~125% of control) increase in wall tension due to arterial dilation. To test whether the increased wall tension was itself responsible for the enhancement in pressure-induced [Ca²⁺]_i, a different group of arteries was treated with either of two inhibitors of myosin light chain kinase, BDM (5 mM) and wortmannin (5 µM). These compounds have been shown to attenuate force generation in VSM cells and were therefore likely to inhibit myogenic constriction as well (4, 34).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of 2,3-butanedione monoxime (BDM) and wortmannin on pressure-induced changes in arterial wall [Ca2+]i and diameter of rat posterior cerebral arteries. A: original traces demonstrating an increase in [Ca2+]i and constriction in response to an elevation of intraluminal pressure from 10 to 60 mmHg during exposure of artery to BDM (5 mM) or combination of BDM (5 mM) and cytochalasin D (5 µM). B: application of wortmannin (5 µM) did not alter the level of [Ca2+]i but dilated the pressurized artery.

Actin cytoskeletal disruption augments pressure-induced Ca²+ influx through L-type channels. A role for the actin cytoskeleton in regulating Ca²⁺ signaling mechanisms was implicated in a number of different cell types, and effects on both Ca²⁺ influx and Ca²⁺ release from intracellular stores have been demonstrated in earlier studies (23). The next series of experiments were designed to elucidate the mechanism of additional [Ca²⁺]_i elevation induced in pressurized cerebral arteries by actin cytoskeletal disruption. We first tested the effects of nifedipine (3 µM), a dihydropyridine inhibitor of L-type Ca²⁺ channels, on enhanced pressure-induced [Ca²⁺]_i responses in arteries treated with cytochalasin D. As shown in Fig. 5, the application of nifedipine resulted in an immediate decrease in [Ca²⁺]_i with no significant effect on arterial diameter (because the vessel was already relaxed due to cytochalasin D). A second pressure step in the presence of both cytochalasin D and nifedipine resulted only in arterial distention with minimal elevation in smooth muscle [Ca²⁺]_i.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects of nifedipine (3 µM) on pressure-induced elevation of arterial wall [Ca2+]i and diameter of cerebral arteries pretreated with cytochalasin D. A: elevation of intraluminal pressure in the presence of cytochalasin D resulted in an increase in [Ca2+]i that was not associated with arterial constriction. Application of nifedipine was followed by an abrupt decrease in [Ca2+]i. B: original traces demonstrating an inhibition of [Ca2+]i changes in response to elevation of intraluminal pressure in the same artery treated with nifedipine and cytochalasin D. C and D: summary of the effects of cytochalasin D and nifedipine on pressure-induced elevation of [Ca2+]i and constriction of posterior cerebral arteries. *P < 0.05, significant difference.

Cytochalasin D induces additional [Ca²+]_i elevation in pressurized arteries depolarized with high-potassium solution. It is well documented that pressure-induced membrane depolarization stimulates the Ca²⁺ influx in small cerebral arteries (18, 26, 48). Therefore, both a direct potentiating effect on voltage-gated Ca²⁺ channels, as well as indirect augmentation of Ca²⁺ influx due to enhanced pressure-induced depolarization, should be considered as potential mechanisms for the observed effects of cytochalasin D. To test the first possibility, we studied the effects of cytoskeletal disruption in arteries depolarized with high-K⁺ solution, to prevent any changes in the membrane potential of VSM cells. As shown in Fig. 6A, high-K⁺ solution raised the [Ca²⁺]_i to 390 ± 38 nM and constricted arteries pressurized to 60 mmHg. Application of cytochalasin D (10 µM) resulted in an additional increase in [Ca²⁺]_i to 462 ± 40 nM (P < 0.05; n = 7) and almost complete arterial relaxation (11 ± 1% of high-K⁺-induced constriction). When wortmannin (5 µM) was used instead of cytochalasin D in conjunction with 60 mM K⁺, significant arterial relaxation was observed without any change in the level of [Ca²⁺]_i (392 ± 32 nM before and 389 ± 30 nM after, n = 6, Fig. 6B). In four other arteries, nifedipine was added once high-K⁺-induced constrictions and [Ca²⁺]_i increases had stabilized; this was followed by an abolition of constriction and a decrease in [Ca²⁺]_i from 384 ± 31 to 94 ± 11 nM. Subsequent application of cytochalasin D has no effect on arterial diameter or on [Ca²⁺]_i (98 ± 13 nM). These experimental results favor a direct effect (i.e., not mediated by membrane depolarization) of the actin cytoskeleton on voltage-dependent Ca²⁺ channels.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effects of cytochalasin D and wortmannin on K+-induced elevation in arterial wall [Ca2+]i and constriction in rat posterior cerebral arteries. A: original traces showing an additional increase in [Ca2+]i and relaxation of an artery depolarized with high K+ (60 mM) solution in response to the application of cytochalasin D (10 µM). B: relaxation and absence of changes in arterial wall [Ca2+]i in response to the application of wortmannin (5 µM) in an artery treated with high-K+ (60 mM) solution. C and D: bar graphs summarizing the effects of cytochalasin and wortmannin on K+-induced elevation in [Ca2+]i and arterial constriction. *P < 0.05, significantly different from values in the presence of 60 mM of K+.

Cytochalasin D depolarizes smooth muscle cells in pressurized cerebral arteries. In separate experiments, we measured the changes in membrane potential and arterial diameter of pressurized posterior cerebral arteries before and after treatment with cytochalasin D. Resting membrane potential in VSM cells of arteries at 10 mmHg averaged 67 ± 1 mV (n = 15). An elevation of pressure from 10 to 60 mmHg resulted in membrane depolarization of ~30 mV (to 38 ± 1 mV; n = 10; P < 0.05). This depolarization was associated with the development of myogenic tone (31 ± 3%). Application of 5 µM cytochalasin D resulted in an additional depolarization of 5 ± 1 mV (n = 6), and the appearance of membrane potential oscillations that were associated with a complete arterial dilation (Fig. 7).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effects of cytochalasin D on membrane potential and myogenic tone in pressurized rat posterior cerebral arteries. A: depolarization and appearance of oscillations in membrane potential of smooth muscle cells in response to an acute application of cytochalasin D is associated with the relaxation of an artery pressurized at 60 mmHg. B and C: fragments of intracellular recordings of membrane potential (expanded time scale) shown on A,b and A,c. D: bar graph showing the values of smooth muscle membrane potential in arteries at 10 mmHg, 60 mmHg, and at 60 mmHg in the presence of cytochalasin D. *P < 0.05, significant difference.

	DISCUSSION

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The results of the present study demonstrate that the actin cytoskeleton has a modulatory but not a sensory role in the process of transducing the stimulus of intraluminal pressure into VSM contraction. This conclusion is based on our observation that the disruption of actin cytoskeletal structure did not attenuate but augmented two vital components of the pressure-induced VSM cellular response: membrane depolarization and elevation in [Ca²⁺]_i, fundamental events that precede the development of myogenic tone in small cerebral arteries (11-13, 16, 18, 26, 33, 40, 48).

To disrupt the actin cytoskeleton in VSM cells, we used two structurally unrelated compounds with essentially different mechanisms of action. Cytochalasins are membrane-permeable fungal metabolites that have long served as unique tools for studying the functional role of the actin cytoskeleton in cell motility, growth, and development (9, 19, 23, 27, 30, 32, 35, 37, 43, 44). These compounds are known to bind to the rapidly growing end of actin filaments, thereby inhibiting the association and dissociation reactions at those ends and preventing actin polymerization (9, 43). In contrast, the latrunculins are a novel class of compounds that induce highly specific sequestration of monomeric actin (G-actin). Their net effect is to inhibit polymerization of actin and to promote actin depolymerization (2, 43). Despite these different mechanisms of action, both cytochalasins and latrunculins disorganize and disassemble filamentous actin structures in nonmuscle as well as smooth muscle cells (2, 19, 35, 43).

In the experiments described herein, cytochalasin D and latrunculin A profoundly inhibited the contraction induced by intraluminal pressure or high-K⁺ solution, indicating the loss of VSM force-generating capability. These observations are consistent with the findings of others showing that disruption of the supramolecular organization of the actin cytoskeleton with cytochalasins resulted in an inhibition of smooth muscle contraction (30, 32, 38, 44). The latter does not result from an attenuation of associated Ca²⁺ influx, however, as either no change (32, 38) or an increase (this study) in [Ca²⁺]_i occurs. Cytochalasins bind to the barbed end of actin filaments and compete with actin-binding proteins that provide attachment of actin filaments to the membrane structures (9, 43). Considering this mechanism of action, the inhibitory effect of cytochalasins on contraction can be attributed to the detachment of actin filaments from the membrane, thereby compromising force development in VSM. However, in our study, this effect is probably not responsible for attenuation of VSM force generation because, in contrast to cytochalasin D, latrunculin A (which binds directly to G-actin but does not interfere with F-actin or sites of its attachment to plasma membrane) also inhibits myogenic tone (2, 43).

We interpret the fact that latrunculin A attenuates the pressure-induced vasoconstriction as evidence that cerebrovascular smooth muscle possesses a considerable pool of G-actin, which is in dynamic equilibrium with F-actin. Similar to the observations made in other cell types, it is likely that sequestration of G-actin by latrunculin A shifts the dynamic equilibrium promoting F-actin depolymerization, diminishing amount of F-actin and thereby decreasing force production in VSM (2, 35). Therefore, these experiments suggest that alteration of actin cytoskeletal dynamics might be an important new mechanism regulating myogenic tone of cerebral arteries.

Our results demonstrate that actin cytoskeletal disruption inhibits myogenic tone but not pressure-induced depolarization or Ca²⁺ influx. Taking into consideration that membrane depolarization and Ca²⁺ influx preceded the development of myogenic tone, these data do not support the hypothesis that the actin cytoskeleton acts as a primary system for the detection of a mechanical load (i.e., as a mechanosensor). A role for the two other components of the cytoskeleton, intermediate filaments and microtubules, in mechanotransduction cannot be ruled out. However, in vimentin-deficient mice, elevation of intraluminal pressure resulted in the development of myogenic tone in small mesenteric arteries, where vimentin is a major component of the smooth muscle intermediate filamentous system (20). In a different study, depolymerization of microtubules resulted in Ca²⁺-independent constriction of isolated cremaster arterioles and a preservation of myogenic responses over a wide range of intraluminal pressures (36). Together, these observations do not support a role for the VSM cytoskeletal network in the detection of pressure-induced mechanical deformation in small arteries and therefore argue against the involvement of the actin cytoskeleton in the inside-out signaling model of mechanotransduction.

On the other hand, the results of this study do support a modulatory role for the actin cytoskeleton in smooth muscle Ca²⁺ handling, as both acute application and pretreatment of arteries with cytochalasin D or latrunculin A resulted in a significant enhancement of pressure-induced [Ca²⁺]_i elevation in cerebrovascular smooth muscle. The cytochalasin D-induced effect was also observed in denuded vessels, eliminating a possible causal role of factors released from vascular endothelium due to cytoskeletal disruption.

We utilized several approaches in an effort to better understand the mechanisms responsible for the potentiation of pressure-induced [Ca²⁺]_i increases by cytochalasin D or latrunculin A in small cerebral arteries. Disruption of the actin filamentous system resulted in almost complete dilation of pressurized arteries and, consequently, a significant increase in arterial wall tension. Arterial wall tension is considered to be one of the potential mechanisms for triggering the pressure-induced myogenic responses in small arteries (12, 33, 40) and could therefore be responsible for the additional Ca²⁺ influx associated with cytochalasin D or latrunculin A-induced dilation. However, this does not seem likely because cytochalasin D produced an increase in [Ca²⁺]_i in arteries that were already completely dilated with BDM. In addition, inhibition of myosin light chain kinase with wortmannin (5 µM) resulted in a complete arterial dilation with no significant changes in pressure- or high-K⁺-induced [Ca²⁺]_i responses. Collectively, these data do not support a role for wall tension as the major cause for the additional increases in [Ca²⁺]_i induced by actin cytoskeletal disruption in our study.

A link between cytoskeletal structure and intracellular levels of Ca²⁺ has been suggested in other studies, and a number of underlying mechanisms have been proposed (23, 27, 28, 35, 44, 49). The actin cytoskeleton may itself be part of the intracellular Ca²⁺ store, and actin depolymerization or treadmilling might result in cytoplasmic Ca²⁺ elevation (28). This mechanism probably is not responsible for the cytochalasin D effect in our experiments because there was no change in [Ca²⁺]_i in response to cytochalasin D application in the presence of nifedipine in arteries depolarized with high K⁺. Therefore, an augmentation of the pressure-induced [Ca²⁺]_i response by cytochalasin D or latrunculin A may be the result of the following: 1) a direct modulatory effect on the function of L-type channels, 2) augmentation of pressure-induced membrane depolarization with a subsequent effect on voltage-dependent Ca²⁺ channels, or 3) both. The finding that cytochalasin D further increased the Ca²⁺ level in pressurized arteries depolarized with high-K⁺ solution, suggests that disruption of F-actin eliminates its inhibitory effect on Ca²⁺ channels, resulting in an enhanced Ca²⁺ influx.

Application of cytochalasin D also resulted in an additional depolarization of VSM cells in the wall of pressurized (60 mmHg) cerebral arteries (Fig. 7), indicating that the actin cytoskeleton can modulate the activity of ion channels different from L-type Ca²⁺ channels. A role of the actin cytoskeleton in the regulation of ion channel activity has been demonstrated in a number of cellular types, and both channel inhibition and activation have been reported (1, 5, 15, 31, 35, 37, 41, 49). Disruption of the actin cytoskeleton with cytochalasins resulted in significant potentiation of Cl channel activity responsible for volume regulation in different cell types (1, 5, 21, 41) and an induction of Na⁺ channel activity, but an inhibition of K⁺ channel currents in A6 epithelial cells (5). In some studies (21, 23, 41), stabilization of the actin cytoskeleton with phalloidin, which prevents depolymerization, abolished the ion channel effect of cytochalasins. Chaetoglobosin C, a cytochalasin that does not depolymerize actin, failed to affect ion channel activity (41). These observations argue in favor of a specific actin cytoskeleton-mediated effect of cytochalasins on ion channels. Mechanically gated ion channels are difficult to activate in a whole cell configuration, but actin cytoskeletal disruption resulted in the acceleration and considerable enhancement of currents through mechanosensitive channels (27, 46, 49). These observations suggest that intact cortical cytoskeleton normally protects plasma membrane from undergoing significant increases in tension during mechanical deformation.

There is limited information on the role of the actin cytoskeleton in the regulation of ion channel function in smooth muscle cells. The effect of actin cytoskeletal disruption on the function of L-type Ca²⁺ channels has been studied in smooth muscle cells isolated from the ileum and in cultured cells from the aorta; either no effect (50 µM cytochalasin B) (32) or inhibition (100 µM cytochalasin D) (31) has been reported. Differences between these findings and our observations in this study may be explained in part by the fact that voltage-dependent Ca²⁺ channels in cerebral artery VSM cells are sensitive to mechanical load, and this mechanism might contribute to the pressure-induced elevation in intracellular Ca²⁺ (29). Disruption of the actin cytoskeleton might increase the mechanosensitivity of these Ca²⁺ channels that, if present, cannot be observed on the single cell level.

There is also evidence that the actin cytoskeleton inhibits the activity of K⁺ channels in a smooth muscle cell line (DDT₁ MF-2), and that depolymerization of actin with cytochalasin B disinhibits K⁺ channel activity, resulting in membrane hyperpolarization (15). However, it seems that this mechanism cannot explain our findings because actin filament disruption was clearly associated with depolarization rather than hyperpolarization of smooth muscle in small cerebral arteries (Fig. 7).

A recent study (48) showed that activation of nonselective cation channels is involved in the pressure-induced VSM depolarization of rat posterior cerebral arteries. Therefore, additional depolarization induced by cytochalasin D in pressurized vessels might result from the potentiation of the activity of these channels by actin cytoskeletal disruption. Both inhibition of K⁺ channels and activation of nonselective cation channels are potential mechanisms for explaining the cytochalasin D-induced depolarization observed in this study.

Mechanisms mediating actin cytoskeletal regulatory effects on ion channels are not well understood. Negative regulation of Ca²⁺ influx through mechanosensitive cation channels by actin filaments has been shown in fibroblasts, an effect that was mediated by integrin-induced actin polymerization and enhanced ABP-280 protein-dependent cross-linking of actin filaments (27, 49). Cipolla et al. (7) recently showed that a similar mechanism might operate in small cerebral arteries where an elevation of intraluminal pressure resulted in actin polymerization evidenced by a substantial decrease in VSM cell G-actin content. Furthermore, our present data suggest that pressure-induced actin filament reorganization might not only be important in determining actin-myosin interactions and force production, but may also serve to modulate Ca²⁺ influx into VSM cells. Whether this effect is a purely mechanical, i.e., effected by actin anchoring or cross linking, or mediated by diffusible second messenger molecules such as tyrosine and RhoA kinases, protein kinase C, or cAMP-dependent protein kinases, has yet to be resolved.

In conclusion, our data demonstrate an important role for actin cytoskeletal dynamics in the regulation of pressure-induced Ca²⁺ influx, membrane depolarization, and myogenic tone in pressurized small cerebral arteries. Modulation of actin structural organization in VSM cells under physiological and pathophysiological conditions might therefore be an additional and novel mechanism for controlling cerebrovascular resistance and blood flow.