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Davis Heart and Lung Research Institute, Ohio State University, Columbus, Ohio
Submitted 18 June 2004 ; accepted in final form 20 September 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-adrenergic agonist phenylephrine (0.1–1 µmol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10–60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 µmol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 µmol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.
myogenic response; laser scanning microscopy; cytochalasin D; latrunculin A; phenylephrine
Vascular smooth muscle cells (VSMCs) of small arteries and arterioles are mechanosensitive, constricting in response to elevations in transmural pressure (PTM) (5, 29). This constriction, termed the "myogenic response," contributes to blood flow autoregulation and the generation of basal vascular tone (5, 29). Previous reports have observed impaired constrictor responses to elevations in PTM and to other constrictor stimuli in small arteries after inhibition of actin polymerization (9, 17, 30). Furthermore, PTM elevation has been reported to decrease the VSMC content of G-actin in small cerebral arteries (4). Actin polymerization may therefore be important for VSMC constriction in response to agonist and mechanical stimulation (9, 17, 30). However, the role played by actin polymerization has not yet been clearly defined. Indeed, no previous studies have assessed potential changes in the organization of the actin cytoskeleton during arteriolar constriction. The aim of the present study was, therefore, to determine the role of actin polymerization and changes in the architecture of the VSMC actin cytoskeleton in arteriolar constriction.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Animal procedures were approved by the Ohio State University Institutional Animal Care and Use Committee. Male mice (C57BL6) were euthanized by CO2 asphyxiation. Distal tail arterioles were then rapidly removed and placed in cold Krebs-Ringer bicarbonate solution containing (in mmol/l) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose (control solution) (22). Arterioles were cannulated at both ends with glass micropipettes, secured using 12-0 nylon monofilament sutures, and placed in a microvascular chamber (Living Systems; Burlington, VT). The chamber was superfused with control solution, maintained at 37°C, pH 7.4 (gassed with 16% O2-5% CO2-balance N2), and placed on the stage of an inverted microscope (x20, Nikon TMS-F) connected to a video camera (Panasonic, CCTV camera). The vessel image was projected onto a video monitor, and the internal diameter was continuously determined by a video dimension analyzer (Living Systems) and monitored using a BIOPAC (Santa Barbara, CA) data-acquisition system (22).
Vasomotor Responses
Arterioles were initially maintained at a PTM of 60 mmHg for 60 min, during which time they developed phasic and tonic vasomotor activity (3, 22). Arterioles analyzed in the present study had basal internal diameters of 50–90 µm. To assess myogenic responses, PTM was decreased to 10 mmHg for 30 min and then abruptly increased to 60 or 90 mmHg (22). When the effects of drugs on the myogenic response were assessed, they were administered at a PTM of 10 mmHg, 15 min before elevations in PTM. Unless stated otherwise, constrictor responses to the 1-adrenoceptor (1-AR) agonist phenylephrine were also evaluated at a PTM of 10 mmHg, because this PTM is below the threshold for mechanosensitive signaling in arteriolar VSMCs (22).
Laser Scanning Microscopy
Arterioles were mounted in specialized "flipper" chambers (Living Systems) that enabled the blood vessel assembly to be rapidly (
1 s) transferred from control solution to paraformaldehyde (3%, 4°C, 30 min). Unless stated otherwise, arterioles were fixed under control conditions at a PTM of 10 mmHg or once the constrictor response to an elevation of PTM or phenylephrine was stable (i.e., after 8 min). After fixation, arterioles were rinsed in PBS (3 x 10 min, 4°C), permeabilized in TTX-100 (0.5%, 4°C) for 15 min, and then rinsed again in PBS (3 x 10 min, 4°C). The arterioles were incubated overnight (4°C) with Alexa 568-phalloidin (to label F-actin, 5 U/ml), Alexa 488-DNAse I (to label G-actin, 9 µg/ml), or Sytox (to label nuclei, 300 nM) (Molecular Probes). After being washed in PBS (3 x 10 min, 4°C), the arterioles were cannulated in a specialized chamber designed for use with an inverted fluorescent microscope (Living Systems). The arterioles were then observed using a Zeiss 510 laser scanning microscope equipped with a x63 water immersion objective (1.2 numerical aperture). Alexa 488-DNAseI or Sytox was excited using the Argon/2 488-nm laser line, and the emission was processed through a DM545 and a 500–530BP filter; Alexa 568-phalloidin was imaged using the HeNe 543-nm line and a DM 545 and LP 560 filter. Images were obtained at an optical zoom of 2.0 or 2.5 (x-y pixel dimensions of 0.14 x 0.14 µm and 0.11 x 0.11 µm, respectively), and all images were obtained using a 512 x 512 frame and line averaging of 4 scans. The resident pixel time was 12.8 µs (scan speed 5). When more than one fluorescent probe was imaged, channels were switched after each frame. Laser power was generally 10% for the Argon/2 488-nm and HeNe 543-nm lasers. Pinhole size (0.39 airy units for F-actin staining) was set to generate optical slices (or "z" resolution) of 0.6 µm, and z stacks were obtained at z steps of 0.6 µm. Detector gain and amplifier offset were set to optimize the dynamic range of the instrument.
When images were to be processed for quantitation of fluorescence, two arterioles (control and test) were studied in parallel. These paired arterioles were processed and imaged at the same time, under identical conditions. Quantitation of fluorescence and analyses of nuclear dimensions were performed using Zeiss laser scanning microscope software and Volocity (Improvision; Lexington, MA), respectively. All images are presented in their original state, and no processing, thresholding, or photoediting has been performed.
Data Analysis
Unless stated otherwise, data are expressed as means ± SE; n is the number of animals from which blood vessels were taken. Alterations in arteriolar diameter were expressed as the percent change in the basal prestimulation level. When global changes in fluorescence intensity of optical slices were analyzed, images were processed at a threshold cutoff of 22 (out of an 8-bit maximal intensity of 255) or 297 (out of a 12-bit maximal intensity of 4,095). This was performed to mask the background and to prevent differences in background area from influencing the results. When the profile of F-actin intensity through individual VSMCs was analyzed, a central transverse line was drawn through the widest part of the cells. Arterioles were first visualized in orthogonal mode, and VSMCs that had the complete transverse profile and the majority (>70%) of their longitudinal profile contained within the z stack were selected for single-cell analysis. The axial lengths of the cells were scanned in orthogonal mode to determine the widest transverse profile of each cell. The x, y, and z coordinates describing a central transverse line (single pixel width) through that profile were then used to localize the line in the x-y (two-dimensional) image for that z slice. The fluorescent intensity of the line in the x-y image was then determined using Zeiss laser scanning microscope "Profile" software. The edges of the cell were defined as the first and final peaks in F-actin intensity. The width of the cell was calculated as the distance between the two peaks. The area under the curve (AUC; fluorescent intensity x distance, in µm) was determined using a Riemann sum/trapezoidal rule calculation. For this calculation, curves were restricted to first peak to final peak with an additional 0.28 µm on either side. Statistical evaluation of the data was performed by Student's t-test for either paired or unpaired observations. When more than two means were compared, ANOVA was used. If a significant F-value was found, Scheffè's test for multiple comparisons was employed to identify differences among groups. Values were considered to be statistically different when P values were <0.05.
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RESULTS |
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Elevation in PTM from 10 to 60 or 90 mmHg caused tail arterioles to initially dilate, reflecting passive dilation, and then constrict, reflecting activation of the mechanosensitive "myogenic response" (Fig. 1) (22). Two inhibitors of actin polymerization were used: cytochalasin D, which acts by binding to the rapidly growing barbed end of actin filaments, and latrunculin A, which binds and sequesters actin monomers (34, 38). Cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l) abolished the myogenic constriction evoked by increasing PTM from 10 to 60 or 90 mmHg (Fig. 1, A and B). In contrast, cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l) had no effect on the diameter of arterioles at a PTM of 10 mmHg and did not affect constriction evoked by the 1-AR agonist phenylephrine (0.01–1 µmol/l), assessed at a PTM of 10 mmHg (Fig. 1C).
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Analysis of VSMC actin polymerization. Laser scanning microscopy generated optical sections through the arterial wall, providing excellent visualization of vascular cells (Fig. 2). Endothelial cells were aligned with blood flow and were characterized by F-actin staining that delineated cell borders (Fig. 2). Endothelial cells had intense staining for monomeric G-actin throughout the cell interior (Fig. 2). Adventitial cells, likely representing adventitial fibroblasts, were identified by nuclear staining and by characteristic high-intensity staining for G-actin but minimal staining for F-actin (Fig. 2). The most prominent cells were VSMCs, which comprised one to two cell layers and were oriented in a circular fashion around the blood vessel, perpendicular to the luminal endothelial cells. VSMs stained intensely for F-actin and G-actin (Fig. 2).
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1-AR agonist phenylephrine (to 70% of baseline diameter, 1 µmol/l) at a constant PTM of 10 mmHg was not associated with significant changes in the intensity of F-actin [0.5 ± 10.9%, n = 3, P = not significant (NS)] or G-actin staining in VSMCs (–7.9 ± 12.6%, n = 3, P = NS).
Analyzing VSMC cytoskeleton architecture.
At a PTM of 10 mmHg, F-actin staining of arteriolar VSMCs resembled a cortical actin structure: intense staining for F-actin was localized at the cell periphery, with greatly reduced staining in the cell interior (Fig. 3A). After an elevation of PTM (to 60 or 90 mmHg), there was a dramatic reorganization of the actin cytoskeleton, with fibers dispersed through the interior of the VSMCs, running parallel to the long axis of the cells (Fig. 3, B and C). To quantify remodeling of the VSMC actin cytoskeleton, the profile of F-actin staining through a central transverse axis of VSMCs was determined from orthogonal z sections (Fig. 4). At a PTM of 10 mmHg, cytoskeleton organization was characterized by two prominent peaks of F-actin staining, delineating the cell periphery, with a dramatic reduction in staining between the two peaks, representing the interior of the cells (Fig. 4). The intensity of F-actin staining in the cell interior was significantly increased at 60 mmHg and further significantly increased at 90 mmHg: PTM elevation from 10 to 60 mmHg increased the AUC (fluorescence intensity x distance, in µm) from 351.3 ± 39.8 to 771.2 ± 81.2 (n = 16 cells, P = 0.0001), and an elevation of PTM from 10 to 90 mmHg increased the AUC from 440.5 ± 52.8 to 1,008.7 ± 51.8 (n = 18 cells, P < 0.0001; P < 0.02 for comparison between 60 and 90 mmHg; Fig. 4). In contrast, constriction of arterioles with the 1-AR agonist phenylephrine (to 70% baseline diameter) at a PTM of 10 mmHg was not associated with changes in the architecture of F-actin staining in arteriolar VSMCs: AUC of 446.7 ± 32.3 and 480.1 ± 30.2 for control and phenylephrine-treated arterioles, respectively (n = 26 cells, P = NS; Figs. 4 and 5).
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Effect of cytochalasin D. Cytochalasin D (300 nM), administered at a PTM of 10 mmHg, significantly decreased the intensity of F-actin staining observed in VSMCs after an elevation in PTM to 60 mmHg (19.8 ± 1.5% decrease compared with paired, untreated control arteries at a PTM of 60 mmHg, n = 3, P < 0.01), which was equivalent to an 84% reduction in the response to PTM elevation. This was associated with marked deterioration in the architecture of F-actin staining, with fragmentation and disordering of F-actin fibers (Fig. 7). This deterioration in structural organization of the cytoskeleton was associated with irreversible impairment of arteriolar function. As noted in Fig. 1, cytochalasin D (300 nM) abolished the myogenic constriction evoked by an elevation in PTM from 10 to 60 mmHg (Fig. 8A). Subsequent removal of cytochalasin D from the perfusate failed to completely restore the normal constriction to PTM elevation (Fig. 8A): the response to PTM elevation (from 10 to 60 mmHg) was a slight decrease in diameter of 2.2 ± 12.8% before cytochalasin D, an increase in diameter of 87.9 ± 12.1% during exposure to cytochalasin D, and an increase in diameter of 40.4 ± 15.6% after subsequent removal of cytochalasin D (n = 3, P < 0.01 for responses before cytochalasin D and after removal of cytochalasin D; Fig. 8A). In contrast, when arteriolar PTM was maintained at 10 mmHg, an equivalent transient exposure to cytochalasin D (300 nM) did not impair the myogenic constrictor response (Fig. 8B). Likewise, cytochalasin D (300 nM) did not affect the pattern of F-actin staining in VSMCs when arteriolar PTM was maintained at 10 mmHg (data not shown).
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1-AR agonist at a PTM of 10 mmHg (and absence of cytochalasin D) was dramatically reduced (Fig. 8C). Therefore, the irreversible functional impairment associated with pressure-induced disruption of the actin cytoskeleton was evident for both myogenic and 1-AR constriction. When constriction to phenylephrine was assessed at a PTM of 60 mmHg, instead of 10 mmHg, cytochalasin D also dramatically reduced constriction to the agonist (Fig. 8D). |
DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-AR stimulation with phenylephrine. Furthermore, elevations in PTM, but not phenylephrine, increased the intensity of F-actin staining and decreased the intensity of G-actin staining in arteriolar VSMCs. These results suggest that actin polymerization is activated by elevation in PTM and plays an essential and selective role in the pressure-induced myogenic constriction of arterioles. Actin polymerization, however, does not appear to play a general role in arteriole constriction, as judged by the response to phenylephrine. In these experiments, responses to phenylephrine and to myogenic stimulation were both initiated at a low PTM (10 mmHg), which is below the threshold for mechanosensitive signaling in arterioles and so minimizes any indirect effects associated with prior mechanical strain (22). Indeed, when assessed at a PTM of 60 mmHg, inhibition of actin polymerization abolished the constriction evoked by phenylephrine. This likely resulted from indirect depression of the 1-AR response. After inhibition of actin polymerization, the arterioles did not tolerate elevations in PTM, which caused disruption of the VSMC actin cytoskeleton and irreversible impairment of arteriolar constriction. Cytochalasin D is a reversible inhibitor of actin polymerization, and arterioles could be transiently exposed to the agent at a PTM of 10 mmHg without significant alteration in arteriole function. However, combined exposure to elevated PTM and to cytochalasin D impaired constriction to myogenic or 1-AR activation, even after PTM was returned to 10 mmHg and cytochalasin D was removed from the superfusate. Although the present study analyzed the response to only 60 and 90 mmHg, the irreversible impairment in arteriolar function makes it unlikely that inhibition of actin polymerization merely shifted myogenic responsiveness to higher levels of PTM. Previous studies have observed generalized depression in constriction of small arteries/arterioles after inhibition of actin polymerization, with reduced responses to KCl, angiotensin II, or an elevation in PTM (9, 17, 30). However, those previous studies analyzed constrictor responses when the arteries were concurrently exposed to significant mechanical strain (PTMs of 50–70 mmHg) (9, 17, 30). Furthermore, the effects were observed using agents (cytochalasin B) or concentrations of cytochalasin D that cause cleavage of actin filaments rather than inhibition of actin polymerization (38). The present study, by analyzing arterial constriction in the absence of significant mechanical strain, by directly analyzing actin polymerization and by utilizing agents that selectively inhibit actin polymerization, demonstrates that actin polymerization is not required for arteriolar constriction but is essential for arterioles to function correctly at an elevated PTM. No previous studies have analyzed the three-dimensional organization of the actin cytoskeleton of VSMCs or its potential modulation during arteriolar constriction. Indeed, the organization of the actin cytoskeleton has not been clearly defined (32, 33). One model, based on histological analyses of chicken gizzard smooth muscle cells, proposes that cytoskeletal elements pervade the cytoplasm extending along the axial plane of the cell, whereas smooth muscle contractile elements are oriented at an angle to the cell axis, coupling to the actin cytoskeleton at dense bodies and dense plaques (32, 33). The present study demonstrates that, at a PTM of 10 mmHg, F-actin staining in arteriolar VSMCs resembled a cortical actin organization, with intense staining at the cell periphery and minimal staining in the cell interior. Indeed, when visualized in orthogonal reconstruction, VSMCs appeared as hollow tubes with intense peripheral bands of F-actin and minimal or low-intensity diffuse staining in the interior. This latter staining may represent thin F-actin filaments that are beyond the resolution of the present images. At this low PTM, VSMC nuclei were localized in areas devoid of F-actin staining, although these areas were not exclusively reserved for nuclei. After an elevation of PTM to 60 or 90 mmHg, transcytoplasmic F-actin fibers were localized running through the cell interior, parallel to the long axis of the cells. The VSMC nuclei were observed compressed between the F-actin fibers in areas that had a reduced fiber density. Indeed, the dramatic change in the organization of the actin cytoskeleton was matched by an equally dramatic elongation of VSMC nuclei. Therefore, although the architecture of the actin cytoskeleton observed at elevated pressure is consistent with the proposed model of VSMCs and with previous histological analyses (19–21, 32, 33), the structural organization observed at low pressure is markedly different.
The emergence of transcytoplasmic F-actin fibers could be observed as early as 15 s after the change in PTM (e.g., Fig. 6), which is too rapid for the fibers to be generated by de novo actin polymerization (6). Indeed, when assessed at the cellular level (e.g., Fig. 4), the pressure-induced increase in F-actin intensity was more striking than changes determined from global quantitation of optical sections. Furthermore, at the cellular level, elevations in PTM from 10 to 60 or 90 mmHg caused a stepwise increase in F-actin intensity, whereas global analyses of optical sections were unable to detect differences between PTMs of 60 and 90 mmHg. Therefore, rather than de novo synthesis of F-actin fibers, these results are more consistent with movement or bundling of existing actin filaments to generate transcytoplamic F-actin cables. Indeed, circumferential stress on the arterioles, imposed by elevations in PTM, would translate into an axial strain on the VSMCs, inducing movement of F-actin fibers to the cell interior. The results with cytochalasin D suggests that actin polymerization plays an essential role in the successful reorganization of transcytoplasmic fibers. Actin polymerization may therefore increase or strengthen attachment of actin filaments to adhesion sites (dense bodies and dense plaques) within the smooth muscle. The elevation of PTM in the presence of cytochalasin D caused disorganization and fragmentation of actin fibers, which was most prominent at the plasma membrane. At the concentrations used in the present study, cytochalasin D acts selectively to cap the fast-growing "barbed" end of actin filaments and inhibit actin polymerization (38). The dramatic effect of cytochalasin to reduce pressure-induced actin polymerization suggests that it occurred predominantly at barbed ends of actin filaments rather than at pointed ends as can occur in striated muscle (7, 13). Actin polymerization at barbed ends could reflect exposure of existing barbed ends by dissociation of high-affinity capping proteins (gelsolin, capping protein, tensin) (6, 14, 28) or the generation of new barbed ends by either filament cleavage (cofilin, gelsolin) (6, 28) or the creation of branch points by the Arp2/3 complex (6, 28). In nonmuscle cells, actin polymerization increases the adhesion between the cytoskeleton and plasma membrane (25).
In rat small cerebral arteries, cytochalasin D (at 5 µmol/l) was reported to inhibit the constriction but not the depolarization or calcium mobilization caused by elevations in PTM (9). Therefore, although actin polymerization and remodeling of the actin cytoskeleton represent early responses of arterioles to elevations in PTM, they are unlikely to contribute to the initial mechanosensation, which is most likely mediated by integrins or other matrix receptor or matricellular protein (15, 39). Even at these and higher concentrations of cytochalasin D, which are sufficient to induce severing of actin filaments (38), the agent does not inhibit agonist-induced phosphorylation of myosin light chains (MLC) or calcium mobilization in vascular or airway smooth muscle (9, 18, 27, 30). Gunst and colleagues (10, 11, 35) have proposed that in airway smooth muscle, actin polymerization and smooth muscle actin-myosin interaction are regulated by two distinct signaling cascades. On the basis of this model, activation of the mechanosensor may initiate two signaling processes within arteriolar VSMCs: actin polymerization and remodeling of the actin cytoskeleton to enable the arteriole to withstand the increase in PTM and the MLC-based signaling pathway to initiate VSMC shortening and blood vessel constriction. Elevation in PTM in arterioles caused a rapid and dramatic increase in c-Src activity in rat mesenteric resistance arteries (26). In VSMCs, c-Src is an important regulator of agonist-induced calcium mobilization (36, 37) and cytoskeletal remodeling, including actin polymerization and bundling of actin filaments (12), and may initiate activation of these parallel pathways after mechanosensitive activation (1, 8).
In addition to VSMCs, optical sectioning of arterioles revealed two other cell types within the blood vessel wall: the endothelial cells lining the lumen and adventitial cells. The adventitial cells were often in close apposition to VSMCs and may represent adventitial fibroblasts or vascular "interstitial cells of Cajal" (ICCs) (23, 24). ICCs are smooth muscle/fibroblast-like cells that are present in the gastrointestinal tract, urinary tract, mesenteric resistance arteries, and portal vein, where they are thought to function as pacemakers, regulating the phasic activity of smooth muscle cells (23, 24). Although mouse tail arterioles display phasic constrictor activity, the mechanism(s) underlying this activity has not been defined. Elevations in PTM did not alter the actin cytoskeleton of adventitial cells or of the endothelial cells, suggesting that they are either not responsive to changes in PTM or that they respond differently from VSMCs. Endothelial cells are mechanosensitive and respond to shear stress or to axial stretch of arteries with reorganization of the actin cytoskeleton (2, 31).
Therefore, the present study, by analyzing arterial constriction in the absence of significant mechanical strain, by directly analyzing actin polymerization and by utilizing agents that selectively inhibit actin polymerization, demonstrates that actin polymerization plays a selective and essential role in the response of arterioles to elevations in PTM. The myogenic constrictor response of mouse tail arterioles was selectively attenuated by two mechanistically distinct inhibitors of actin polymerization: cytochalasin D and latrunculin A. Moreover, elevations in PTM stimulated actin polymerization in VSMCs, which was associated with a dramatic reorganization of the actin cytoskeleton. Actin polymerization and cytoskeletal remodeling did not occur during arteriolar constriction to the 1-AR agonist phenylephrine (at a low PTM), suggesting that it is not an inherent part of the contractile process. However, these results do not rule out an interaction between mechanosensitive and agonist-induced signaling in modulating actin dynamics at higher PTMs. These results are consistent with mediation of the myogenic response by two parallel signaling pathways: remodeling of the actin cytoskeleton, which enables the arteriole to withstand the increase in PTM, and activation of the MLC-based signaling cascade to cause constriction of the arteriole.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2C-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries. Am J Physiol Heart Circ Physiol 278: H1075–H1083, 2000.This article has been cited by other articles:
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, from 10 to 90 mmHg), and an upward deflection represents an increase in diameter. The traces are offset for clarity; neither Lat A nor Cyto D had an effect on baseline diameter of the arterioles at a PTM of 10 mmHg. In B and C, responses are expressed as the percent change in the baseline diameter at 10 mmHg in response to increases in PTM from 10 to 60 or 90 mmHg (B) or in response to phenylephrine (C). Inhibition of actin polymerization by Cyto D (300 nM) or Lat A (100 nM) abolished the myogenic response (A and B) but did not affect the constrictor response to phenylephrine (C; presented as means ± SE for 3 or 4 experiments).
