ARTICLE |
Correspondence to: Shu Chien, Dept. of Bioengineering, UCSD, La Jolla, CA 92093-0412.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We studied the effects of shear stress on protein kinase C (PKC) in cultured human umbilical vein endothelial cells by use of a flow channel and a monoclonal antibody (MAb 1.3) that recognizes the PKC ß-isozyme. The fluorescence intensity (FI) of the secondary antibody, crystalline tetramethylrhodamine isothiocyanate, was determined by image analysis. The results on each of five shearing experiments were normalized by using the paired stationary control. After 30-min shearing at 2 N/m2, FI per cell increased to 1.6 times that of control, as did the mean FI per unit cell area. The FI per unit stained area and the stained area/cell area ratio were also increased significantly by shearing. The distribution of immunostaining in each cell was determined for its cortical, cytoplasmic, perinuclear, and nuclear regions. The normalized FI per unit area in all four regions and the stained area/cell area ratio in cortical and cytoplasmic regions were significantly higher in the sheared cells than in control; the increases were greatest in the cortical area. Double staining with rhodamine-phalloidin and MAb 1.3 showed the association of actin with the PKC isozyme in both stationary and sheared cells. (J Histochem Cytochem 45:237-249, 1997)
Key Words: actin, endothelial cells, protein kinase C, shear stress
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein Kinase C (PKC) has been shown to serve as a signaling molecule for the transduction of extracellular stimuli into the cell to regulate many physiological processes (
The wall shear stress in the vascular system is maintained at an optimal level by an autoregulatory mechanism (
The responses of ECs to shear stress have usually been studied by the use of cultured human umbilical vein endothelial cells (HUVECs). Such investigations have demonstrated changes in morphology (
Hemodynamic forces, including shear stress, play an important role in the focal nature of atherosclerosis (
PKC is widely distributed in many tissues and cell types. The tissue PKC content varies from high levels in the brain (9 µg/mg protein) and spleen (1.4 µg/mg protein), to a low level in 3T3 fibroblasts (1 ng/mg protein) (
PKC in cells has been determined by indirect immunofluorescence with antibodies, e.g., the monoclonal antibody MAb 1.3, which binds to the ß-isozyme of PKC (
PKC has been shown to associate with cytoskeletal proteins (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents
The mouse anti-PKC monoclonal antibody MAb 1.3, which was prepared and characterized by D. Mochly-Rosen and D.E. Koshland, was purchased from Gibco BRL Life Technologies (Gaithersburg, MD). MAb 1.3 was prepared against highly purified rat brain PKC containing both membrane-associated and cytosolic PKC and was tested to ensure that it was PKC-specific (-, ß-, and
-isozymes were transiently expressed in RAT-1 cells to examine the binding of different PKC MAbs. Binding of MAb 1.3 was observed only in cells expressing the cDNA for the ß-isozyme.
Secondary antibodies conjugated with TRITC and fluorescein isothiocyanate (FITC) were purchased from Sigma Chemical (St Louis, MO). Rhodamine-conjugated phalloidin and SlowFade anti-fade reagent were purchased from Molecular Probes (Eugene, OR).
Cell Culture
Primary HUVECs were harvested from human umbilical veins by collagenase treatment and were plated on glass slides (3 x 1 inch) precoated with 2% gelatin (Sigma Chemical). Cells were grown to confluence on glass slides in an EGM medium (Clonetics; San Diego, CA) containing 20% fetal bovine serum and no ATP (Hyclone Laboratories; Logan, UT).
After 1 day the cells were sparse and subconfluent. The medium was changed and cells were allowed to grow for 3-4 additional days to reach confluence. Fluid flow experiments were conducted no more than 2 days after the cultures had reached confluence. The medium was changed 1 day before the experiment. Paired experiments were conducted by placing HUVECs in two flow chambers, with one kept under a static condition (stationary or control group) and the other exposed to a shear stress of 2 N/m2 (N, Newton = 105 dynes) for 30 min (shear group).
Fluid Flow
The shear stress experiments were conducted in a rectangular flow chamber. The chamber, a reservoir, and a circulation circuit were filled with fresh culture medium. The medium was driven by using a constant-head-flow loop, such that the HUVECs were exposed to well-defined shear stress for 30 min. A roller pump was used to return the outflow from the chamber to the feeding reservoir. The medium was kept at a constant temperature of 37C and was equilibrated with a gas mixture of 95% air and 5% CO2. In the control (stationary) group, the HUVECs were kept in the chamber for 30 min without flow.
Indirect Immunofluorescence
After the 30-min experimental period, the HUVECs on the chamber slides were fixed with 1% formaldehyde for 20 min at 37C in PBS. The fixed cells were permeabilized and nonspecific binding was blocked by preincubation with PBS containing 0.3% Triton X-100 and 1% normal goat serum. To visualize PKC, the cells were incubated with anti-PKC MAb 1.3 in PBS containing 0.3% Triton X-100, followed by the TRITC-conjugated secondary antibody. For negative controls, normal goat serum was used and followed by either (a) MAb 1.3 without the secondary antibody or (b) the TRITC-labeled secondary antibody without MAb 1.3. When the cells were double stained to visualize PKC and actin microfilaments, they were stained with an FITC-labeled secondary antibody against MAb 1.3 and rhodamine-conjugated phalloidin, respectively. The slides were mounted with SlowFade anti-fade reagent and viewed with a Nikon microscope (Microphot-FX) with x20, x40, and x100 objectives. The light source was a 75-W Xenon lamp. The rhodamine fluorescence was studied with an excitation filter at 546 nm, a dichroic mirror at 580 nm, and a barrier filter at 580 nm. The FITC fluorescence was studied with an excitation filter at 495 nm, a dichroic mirror at 510 nm, and a barrier filter at 520 nm. Images were recorded on Kodak Ektachrome P800/1600 professional black-and-white film 5020, with exposure times of 20 and 40 sec. Stained specimens were viewed with a Nikon Diaphot microscope equipped with a Bio-Rad MRC 1000 laser scanning confocal imaging system (Bio-Rad; Richmond, CA). A x60 oil immersion objective was used to examine the specimens. By stepping the objective through the depth of the specimen, a z-series (a collection of optical sections) was obtained. Double stained specimens were pseudo-colored separately and merged with CoMos and Confocal Assistant programs. These paired images were transferred to a Power Macintosh 8100/80 computer for further analysis. The paired images were combined with Adobe Photoshop (Adobe System; Mountain View, CA). The resultant RGB images depict each of the F-actin and PKC components as red and green, respectively.
Image Analysis
The fluorescence intensity of the photographic image was measured with an image processing system. The film was backlit by using a light table and scanned with a Panasonic WV-CD camera. The resulting image signal was displayed on a high-resolution color video screen and was also entered via a Gateway 2000 486/33 computer system into a Matrox MVP-AT image processor, where it was digitized into an array of pixels. Intensity values for each pixel ranged from a gray scale of 0 for black to 255 for white. The Image-1 system (Universal Imaging; West Chester, PA) software program was used to analyze the image to derive the intensity profile of the object. The analysis consisted of a standard order of procedures, including spatial calibration, image acquisition, thresholding, and profile extraction.
Measurements were made on 10 microscopic fields evenly spaced along the midline (lengthwise) of the flow chamber, each containing 25 cells. The cell profile features were contoured by using a light pen. For each cell, measurements were made on projection areas (PKC-stained area, whole cell area, and cell regional areas), as well as the fluorescence intensity (gray value of PKC staining). After appropriate corrections for stray light and shading, the image was stored in the computer for further processing.
Statistics
Mean values, together with variances, standard deviations, and standard errors, were calculated for the stationary and shear groups. Analysis of variance was performed on a Power Macintosh computer, using the program SYSTAT Version 5.2 (SYSTAT; Evanston, IL). Student's t-test was performed by using the program SigmaPlot (Jandel Scientific; San Rafael, CA). Statistical graphs were drawn by using the program KaleidaGraph Version 2.0 (Synergy Software; Reading, PA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fluorescence Microscopic Examination
Figure 1 shows the MAb 1.3 immunostaining-positive HUVECs as demonstrated by the TRITC-labeled goat anti-mouse secondary antibody. HUVECs treated with normal goat serum (without MAb 1.3) followed by TRITC-labeled secondary antibody were consistently negative for staining (not shown). In addition, no immunostaining was obtained when the TRITC-labeled secondary antibody was omitted (not shown).
|
In stationary experiments, some of the PKC isozyme immunostaining was in fiber form (Figure 1A and Figure 1C). After 30 min of exposure to shear stress at 2 N/m2 (shear experiments), the cells were still in confluent condition. Stronger immunostaining of anti-PKC MAb 1.3 was observed, and most of the immu-nostaining of cell components appeared as fibers (Figure 1B and Figure 1D).
Image Analysis
The photomicrographs were subjected to image analysis to provide an estimation of the fluorescence intensity of PKC staining. A light pen was used to outline the profile of individual cells (Figure 2A and Figure 2B). The computer then summed the gray scale values (range 0-255) of all pixels within the selected cell outline for the calculation of FI in the cell profile (Table 1). Hematoxylin staining was used to identify the cell nucleus and cytoplasm of sheared cells and stationary cells in paired experiments (Figure 2C and Figure 2D).
|
|
In each shear experiment, 250 cells were randomly chosen for measurement, and a total of 1250 cells were studied in five sheared experiments. The same number of cells were measured in the five paired stationary experiments (Table 1). In each of the five paired experiments, the results obtained on the stationary and sheared cells were normalized by the mean value obtained in the stationary experiment. The normalized values were used to combine the results obtained from the five different paired experiments for the 1250 stationary cells and 1250 sheared cells. The frequency distributions of the normalized values obtained are shown in Figure 3 and Figure 4. The FI was found to be higher in the shear experiments than in the stationary control on either a per cell (Figure 3A) or per unit cell area basis (Figure 3B). The FI per unit PKC-stained area (Figure 4A) and the fractional cell area that shows FI (i.e., PKC-stained area/cell area; Figure 4B) also increased in the sheared cells.
|
|
Table 1 lists the raw data on the cell FI for the five experiments. In four of the five paired experiments, the FI was significantly higher in the sheared cells than in the stationary cells. The normalized FI for all 1250 sheared cells (mean ± SEM 1.667 ± 0.022) was significantly higher (p<0.001) than the 1250 stationary cells (1.000 ± 0.014). In all five paired experiments, the FI expressed as per unit cell area (FI/cell area) was higher in the sheared cells (1.604 ± 0.014).
The normalized FI per unit PKC-stained area was significantly higher in the sheared cells (1.189 ± 0.004) than in the stationary cells for all five experiments (Table 1; p<0.001). The normalized ratio of PKC-stained area to cell area was also significantly higher in the sheared cells (1.390 ± 0.012) than in stationary cells (Table 1; p<0.001).
The distribution of PKC immunostaining in individual HUVECs was analyzed by dividing each cell into four regions, i.e., cortical, cytoplasmic, perinuclear, and nuclear regions (Figure 5). The area of the cell outside of the nucleus (between the nuclear membrane and the cell membrane) is divided into three regions according to the radial distances as follows: the outer one fifth was denoted as the cortical region, the perinuclear one fifth was designated as the perinuclear region, and the remaining three fifths were called the cytoplasmic region.
|
Table 2 compares the normalized FI per unit area in these four different regions between 50 cells in the stationary group and 50 cells in the shear group. For cortical, cytoplasmic, perinuclear, and nuclear regions, the mean values of FI per unit area (normalized to the value for the whole cell) in the stationary group were 0.484, 1.123, 1.413, and 1.210, respectively. The corresponding normalized values in the shear group were 1.034, 1.788, 1.676, and 1.612, respectively. The values of all four regions were significantly higher in the sheared cells than in the stationary cells (p<0.001), but the increase was proportionally greatest in the cortical region. Table 3 compares the normalized ratio of PKC-stained area to cell area in the four regions between 50 cells in the stationary group and 50 cells in the shear group. In the stationary group, the mean values (normalized to the value for the whole cell) for cortical, cytoplasmic, perinuclear, and nuclear regions were 0.546, 1.127, 1.366, and 1.245, respectively. The corresponding normalized values for the shear group were 0.992, 1.437, 1.306, and 1.389, respectively. The values for cortical and cytoplasmic regions were significantly higher in the sheared cells than in the stationary cells, and the relative increase was greater in the cortical region (p<0.001).
|
|
These findings indicate that the PKC per unit area of HUVECs, as demonstrated by immunostaining with antibodies to a PKC isozyme, is increased by shear stress in all four regions of the cell, especially in the cortical region.
Confocal Imaging of ECs for PKC
The photographs were taken by a confocal fluorescence microscope system, which enables optical slicing of endothelial cells through their depth by the confocal laser scanning system. Image sets of ECs, including PKC immunostaining, the cross-section of nuclear area, and the cell morphology were collected. The immunostaining of PKC was found to localize at the basolateral portion of the cell. These fibers of immu-nostaining were stronger in the shear group (Figure 6J-R) than in the stationary group (Figure 6A-I). The fraction area stained for PKC was larger in the sheared cells than in the stationary cells. Larger composite pictures for these cells are shown in Figure 7.
|
|
Double Staining for F-actin and PKC
Cytoskeletal proteins undergo reorganization in response to shear stress (
Figure 8 shows a set of representative samples from 10 such experiments. As shown in Figure 8A, rhoda-mine-phalloidin staining of F-actin labeling (pseudo-colored red) for the stationary cells was found in the ruffled membranes of lamellipodia and the stress fibers. The fibers were variable in length and distributed as linear, parallel arrays in various cell regions. After exposure to shear stress, the stress fibers increased and became aligned with the long axis of the cell (Figure 8D). Figure 8B and Figure 8E show the PKC staining (pseudo-colored green) of the stationary and sheared cells, respectively. The co-localization of F-actin and PKC staining (yellow) can be seen in the stationary cells (Figure 8C), but the degree of co-localization was enhanced in the sheared cells (Figure 8F).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hemodynamic shear stress arising from blood flow affects endothelial morphology and function. Shear stress reorganizes actin filaments into stress fibers, alters the shape of endothelial cells, and aligns the cells in the direction of flow in vitro (
There is evidence that shear stress-induced changes in ECs are mediated by intracellular second messengers. PKC is an important enzyme involved in the regulation of cell morphology, contractility, secretion, growth and differentiation, and signal transduction, and its activation leads to actin polymerization, network assembly, and modulation of membrane-actin linkages (Wong et al. 1990). A large number of PKC substrate proteins have been found in ECs, and it has been hypothesized that shear stress, by activating PKC, may initiate a variety of responses in these cells.
Shear stress has been shown to activate phospho-inositide turnover, possibly via phospholipase C in endothelial cells, resulting in the production of inositol trisphosphate and diacylglycerol (
PKC is a family of closely related proteins. It has been shown that the enzyme exists as many isozymes, such as , ß,
,
,
,
and
(
Previous biochemical studies have shown that PKC phosphorylates substrates in the cytosol, membrane, cytoskeleton, and nucleus (Nishizuka et al. 1984,1986), and that PKC translocates from the cytosol to the membrane fraction on activation by a number of agents, including phorbol esters, cytokines, and hormones. Immunofluorescence studies have shown that the PKC -isozyme associates with focal contacts in rat embryo fibroblasts (
-, ß-, or
-isozyme) to myofibrils and microfilaments (
-isozyme has been shown to associate with the nucleus on activation with phorbol esters (
In preliminary experiments, we found that the cells sheared for 15 min were not significantly different in their fluorescence intensity compared to the cells sheared for 30 min. The rapid onset of the PKC response to shear suggests that it is not transcriptional in nature. There are several possible alternative explanations, e.g., increased accessibility of the antibody to PKC-ß, release of PKC due to the PKC-binding protein adducins (
Our double staining experiments show the co-localization of PKC and F-actin in some parts of the HUVECs. There is evidence in support of the idea that a PKC isozyme transiently associates with myofibrils and microfilaments after PKC activation (
The changes in cytoskeletal organization are a ubiquitous response to mechanical perturbation and may be involved in the transmission of mechanical forces across the cell surface and the subsequent signal transduction. The transfer of force from integrins to the cytoskeleton may represent a proximal step in an intracellular mechanical signaling cascade that leads to global cytoskeletal rearrangements and mechano-transduction events at multiple locations inside the cell (
The objective of our study was to examine the change in distribution of PKC in HUVECs as a consequence of shear stress. An additional goal was to determine the possible association of PKC with F-actin in the cytoskeletal network. Our results show that shear stress causes an increase in PKC ß-isozyme immunostaining in ECs, especially in the cortical region. Association of the isozyme with cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. An intact microfilament system, with its capacity to form stress fibers and focal contacts, is a critical necessity for maintaining EC adherence under shear stress. Therefore, it is possible that the PKC isozyme is translocated to different intracellular sites, including the cytoskeletal structure. Furthermore, PKC could be activated on the cytoskeletal elements. These interactions between PKC and the cytoskeleton may play a significant role in the modulation of cell function and the rearrangement of cell shape in response to shear stress.
![]() |
Acknowledgments |
---|
We thank Dr Jeff Price for help in the use of the confocal microscope, Dr John Y. Shyy for valuable advice and discussion, and Mr Gerard Norwich for excellent assistance.
Supported by NIH Research Grants from the National Heart, Lung, and Blood Institute HL 19454, HL 44147, and HL 43026, and by the Whitaker Foundation Development Award.
Received for publication March 28, 1996; accepted September 18, 1996.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmad Z, Lee FT, DePaoli-Roach A, Roach PJ (1984) Phosphorylation of glycogen synthase by the Ca2+- and phospholipid-activated protein kinase (protein kinase C). J Biol Chem 259:8743-8747
Ando S, Tanabe K, Gonda Y, Sato C, Inagaki M (1989) Domain- and sequence-specific phosphorylation of vimentin induces disassembly of the filament structure. Biochemistry 28:2974-2979[Medline]
Boscá L, Márquez C, Martínez AC (1989) Lack of correlation between translocation and biological effects mediated by protein kinase C: an appraisal. Immunol Today 10:223-224[Medline]
Burn P (1988) Phosphatidylinositol cycle and its possible involvement in the regulation of cytoskeleton-membrane interactions. J Cell Biochem 36:15-24[Medline]
Burridge K (1981) Are stress fibres contractile? Nature 294:691-692[Medline]
Dewey CF, Jr, Bussolari SR, Gimbrone MA, Jr, Davies PF (1981) The dynamic response of vascular endothelial cells to fluid shear stress. J Biomech Eng 103:177-185[Medline]
Diamond SL, Eskin SG, McIntire LV (1989) Fluid flow stimulates tissue plasminogen activator secretion by cultured human endo-thelial cells. Science 243:1483-1485[Medline]
Dong L, Chapline C, Mousseau B, Fowler L, Ramsay K, Stevens JL, Jaken S (1995) 35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family. J Biol Chem 270:25534-25540
Eskin SG, Ives CL, Frangos JA, McIntire LV (1985) Cultured endo-thelium: the response to flow. ASAIO J 8:109-112
Girard PR, Nerem RM (1993) Endothelial cell signaling and cyto-skeletal changes in response to shear stress. Front Med Biol Eng 5:31-36[Medline]
Girard PR, Stevens VL, Blackshear PJ, Merrill AH, Jr, Wood JG, Kuo JK (1987) Immunocytochemical evidence for phorbol ester-induced directional translocations of protein kinase C in HL60, K562, CHO, and E7SKS cells: possible role in differentiation. Cancer Res 47:2892-2898[Abstract]
Greenberg ME, Ziff EB (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311:433-438[Medline]
Hsieh HJ, Li NQ, Frangos JA (1992) Shear-induced platelet-derived growth factor gene expression in human endothelial cells is mediated by protein kinase C. J Cell Physiol 150:552-558[Medline]
Huang CL, Ives HE (1987) Growth inhibition by protein kinase C late in mitogenesis. Nature 329:849-850[Medline]
Hunter T, Ling N, Cooper JA (1984) Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature 311:480-483[Medline]
Jaken S, Leach K, Klauck T (1989) Association of Type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell Biol 109:697-704[Abstract]
Kamiya A, Togawa T (1980) Adaptive regulation of wall shear stress to flow change in the canine carotid artery. Am J Physiol 239:H14-H21
Keller HU, Zimmermann A, Cottier H (1985) Phorbol myristate acetate (PMA) suppresses polarization and locomotion and alters F-actin content of Walker carcinosarcoma cells. Int J Cancer 36:495-501[Medline]
Kuchan MJ, Frangos JA (1993) Shear stress regulates endothelin-1 release via protein kinase C and cGMP in cultured endothelial cells. Am J Physiol 264:H150-H156
Langille BL, O'Donnell F (1986) Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science 231:405-407[Medline]
Leach KL, Powers EA, Ruff VA, Jaken S, Kaufmann S (1989) Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells. J Cell Biol 109:685-695[Abstract]
Levesque MJ, Liepsch D, Moravec S, Nerem RM (1986) Correlation of endothelial cell shape and wall shear stress in a stenosed dog aorta. Arteriosclerosis 6:220-229[Abstract]
Mackie K, Lai Y, Nairn AC, Greengard P, Pitt BR, Lazo JS (1986) Protein phosphorylation in cultured endothelial cells. J Cell Physiol 128:367-374[Medline]
Mochly-Rosen D, Henrich CJ, Cheever L, Khaner H, Simpson PC (1990) A protein kinase C isozyme is translocated to cytoskeletal elements on activation. Cell Regul 1:693-706[Medline]
Mochly-Rosen D, Koshland DE, Jr (1988) A general procedure for screening inhibitory antibodies: application for identifying anti-protein kinase C antibodies. Anal Biochem 170:31-37[Medline]
Morita T, Kurihara H, Maemura K, Yoshizumi M, Nagai R, Yazaki Y (1994) Role of Ca2+ and protein kinase C in shear stress-induced actin depolymerization and endothelin 1 gene expression. Circ Res 75:630-636[Abstract]
Murti KG, Kaur K, Goorha RM (1992) Protein kinase C associates with intermediate filaments and stress fibers. Exp Cell Res 202:36-44[Medline]
Nerem RM, Levesque MJ (1983) Fluid dynamics as a factor in the localization of atherogenesis. Ann NY Acad Sci 416:709-719[Medline]
Nishikawa M, Hidaka H, Adelstein RS (1983) Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. J Biol Chem 258:14069-14072
Nishizuka Y (1986) Studies and perspectives of protein kinase C. Science 233:305-312[Medline]
Nishizuka Y (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308:693-698[Medline]
Nollert MU, Eskin SG, McIntire LV (1990) Shear stress increases inositol triphosphate levels in human endothelial cells. Biochem Biophys Res Commun 170:281-287[Medline]
Nollert MU, Hall ER, Eskin SG, McIntire LV (1989) The effect of shear stress on the uptake and metabolism of arachidonic acid by human endothelial cells. Biochim Biophys Acta 1005:72-78[Medline]
Osada S, Mizuno K, Saido TC, Akita Y, Suzuki K, Kuroki T, Ohno S (1990) A phorbol ester receptor/protein kinase, nPKC, a new member of the protein kinase C family predominantly expressed in lung and skin. J Biol Chem 265:22434-22440
Patskan GJ, Baxter CS (1985) Specific stimulation of histone H2B and H4 phosphorylation in mouse lymphocytes by 12-O-tetradecanoylphorbol 13-acetate. J Biol Chem 260:12899-12903
Peppers SC, Holz RW (1986) Catecholamine secretion from digitonin-treated PC12 cells: effects of calcium, ATP, and protein kinase C activators. J Biol Chem 261:14665-14669
Scholz J, Schaefer B, Schmitz W, Scholz H, Steinfath M, Lohse M, Schwabe U, Puurunen J (1988) Alpha-1 adrenoceptor-mediated positive inotropic effect and inositol trisphosphate increase in mammalian heart. J Pharmacol Exp Ther 245:327-335[Abstract]
Shyy JY, Li YS, Lin MC, Chen W, Yuan S, Usami S, Chien S (1995) The shear stress-mediated gene expression and the cis-elements involved. J Biomechan 28:1451-1457[Medline]
Starksen NF, Simpson PC, Bishopric N, Coughlin SR, Lee WMF, Escobedo JA, Williams LT (1986) Cardiac myocyte hypertrophy is associated with c-myc protooncogene expression. Proc Natl Acad Sci USA 83:8348-8350[Abstract]
Sugden D, Vanecek J, Klein DC, Thomas TP, Anderson WB (1985) Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes. Nature 314:359-361[Medline]
Uratsuji Y, Dicorleto PE (1988) Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endo-thelial cells. J Cell Physiol 136:431-438[Medline]
Wang N, Butler JP, Ingber DE (1993) Mechanotransduction across the cell surface and through the cytoskeleton. Science 260:1124-1127[Medline]
Wolf M, Cuatrecasas P, Sahyoun N (1985) Interaction of protein kinase C with membranes is regulated by Ca2+, phorbol esters, and ATP. J Biol Chem 260:15718-15722
Wong MKK, Gotlieb AI (1990) Endothelial monolayer integrity: perturbation of F-actin filaments and the dense peripheral band-vinculin network. Arteriosclerosis 10:76-84[Abstract]