Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 92: 41-49, 2002;
8750-7587/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (19)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bitar, K. N.
Right arrow Articles by Patil, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bitar, K. N.
Right arrow Articles by Patil, S. B.
Vol. 92, Issue 1, 41-49, January 2002

HSP27 modulates agonist-induced association of translocated RhoA and PKC-alpha in muscle cells of the colon

K. N. Bitar, A. Ibitayo, and S. B. Patil

Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The recruitment of signal transduction molecules to the membrane is crucial for the efficient coupling of extracellular signals and contractile response. The trafficking is dynamic. We have investigated a possible cross talk between agonist-induced association of translocated RhoA and translocated protein kinase C-alpha (PKC-alpha ) and a role for heat shock protein 27 (HSP27) in mediating this interaction. Immunoprecipitation with HSP27 monoclonal antibody followed by immunoblotting with either RhoA antibody or PKC-alpha antibody indicated that acetylcholine induced associations of HSP27-RhoA and HSP27-PKC-alpha in the membrane fraction but not in the cytosolic fraction. Immunoprecipitation with anti-RhoA monoclonal antibody followed by immunoblotting with PKC-alpha antibody indicated that acetylcholine induced a significant complexing of RhoA-PKC-alpha in the membrane fraction but not in the cytosolic fraction. In summary, the data indicate that agonist-induced contraction is associated with 1) association of translocated RhoA with HSP27 on the membrane, 2) association of translocated PKC-alpha with HSP27 on the membrane, and 3) association of PKC-alpha with RhoA on the membrane. The data suggest an important role for HSP27 in modulating a multiprotein complex that includes translocated RhoA and PKC-alpha .

cytoskeleton; contraction; signal transduction; heat shock protein 27; protein kinase C-alpha


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HEAT SHOCK PROTEIN 27 (HSP27) is a member of the mammalian small heat shock proteins family. HSP27 is expressed in a variety of tissues in the presence or absence of stress (16). HSP27 is relatively abundant in all types of cells (42), and it colocalizes with actin filaments in cardiac (28), skeletal (3, 41), and smooth muscle (5, 21). Evidence has shown that HSP27 is important in many cell functions, such as cell survival during stress, apoptosis mediated by Fas/APO-1 receptor, and microfilament organization in response to growth factor or stress, as well as smooth muscle contraction (5, 10). HSP27 has been shown to exhibit chaperone activity in vitro and modulate actin filament dynamics (14, 16). The physiological role of HSP27 is still undetermined. HSP27 becomes phosphorylated in response to heat shock and in response to different stimuli, such as cytokines, growth factors, and peptide hormones (27). HSP27 exists as both large oligomers that are hypothesized to have chaperone-like activity and as smaller oligomers that bind to and cap the barbed end of microfilaments and stabilize them (3, 41). Phosphorylation of HSP27 changes the actin cytoskeleton and actin-dependent events. Thus HSP27 may regulate and stabilize the cytoskeleton.

RhoA belongs to the superfamily of ras-related proteins (38). These proteins function by utilizing a guanine nucleotide-binding and -hydrolyzing cycle (7, 15). The evidence to date indicates that Rho regulates the cytoskeletal system, particularly actin-dependent functions, such as cell motility (37), formation of stress fibers and focal adhesions (30), and smooth muscle contraction (18, 36). However, the mode of action of RhoA in reorganization of the cytoskeleton has not been identified.

Recently, a model has been proposed in which Rho regulates myosin light chain (MLC) phosphorylation through its effectors, Rho-associated kinase (Rho kinase), and myosin-binding subunit (MBS) (2, 24, 36). GTP-bound Rho interacts with both Rho kinase and MBS of myosin phosphatase, resulting in activation of Rho kinase and translocation of MBS. Activated Rho kinase phosphorylates MBS, thereby inactivating myosin phosphatase (24). Concomitantly, Rho kinase phosphorylates MLC at the same site (Ser19) that is phosphorylated by MLC kinase and could play a role in activation of myosin ATPase. Both events appear to be necessary for an increase in MLC phosphorylation, yet data suggest that Rho kinase appears to regulate MLC phosphorylation downstream of Rho in nonmuscle as well as in muscle cells (1).

Protein kinase C (PKC) isoforms are characterized by an NH2-terminal regulatory domain containing binding sites for Ca2+, phosphatidylserine, and diacylglycerol; a small hinge region; and a COOH-terminal catalytic domain (29). PKC is regulated by multiple interdependent mechanisms, including enzymatic activation, translocation of the enzyme in response to activation, phosphorylation, and proteolysis. The PKC isoforms are divided into three families depending on differences in the regulatory domain (12). The isoforms exert different biological functions, yet in vitro the different PKC isoforms demonstrate little substrate specificity. Therefore, other mechanisms must be responsible for their differential effects. One possibility is directed translocation of PKC isoforms to their respective targets (39). One possible targeting mechanism for PKC is through its association with anchoring proteins that tether the enzyme to cellular structures.

Our laboratory (20) and others (44) have shown that HSP27 is phosphorylated in smooth muscle in response to contractile agonists and that HSP27 phosphorylation in gastrointestinal smooth muscle cells is inhibited by the PKC inhibitor calphostin C (20). Our laboratory has previously shown that PKC-alpha translocates to the membrane with stimulation with the contractile agonist ceramide (21). Our laboratory has also shown (40) that RhoA modulates agonist-induced signal transduction cascades in smooth muscle contraction and that when, examined under confocal microscopy, RhoA colocalizes on the membrane with the actin-binding protein HSP27. Therefore, HSP27 may be of importance in smooth muscle contraction. The studies of its structure-function relationship together with its interaction with other signal transduction pathways, namely RhoA and PKC will greatly complement our knowledge of signal transduction pathways mediating contraction in smooth muscle cells. We hypothesize that HSP27 mediates association of PKC-alpha and RhoA to the membrane during agonist-induced muscle contraction. We have attempted here to identify an agonist-induced association of HSP27 with translocated PKC-alpha and RhoA. The data suggest a mechanism by which PKC-alpha translocation is targeted to the membrane through its association with HSP27 and with RhoA on the membrane with stimulation with either ceramide or acetylcholine.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

C2 ceramide (0.1 µM) was from Matreya (Pleasant Gap, PA); collagenase type II was purchased from Worthington Biochemical (Freehold, NJ). Protein G-Sepharose was from Pharmacia Biotech (Uppsala, Sweden). Polyvinylidene fluoride (PVDF) membranes were from Bio-Rad (Hercules, CA); QiaGen Effectene transfection kit was from QiaGen (Valencia, CA); enhanced chemiluminescence detection reagents were from Amersham (Arlington Heights, IL). Monoclonal mouse anti-HSP27 antibody (2B4-123) was previously described (5). Monoclonal mouse anti-RhoA and polyclonal rabbit anti-RhoA IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); herbimycin A was from Calbiochem (La Jolla, CA). Calphostin C was from Kamiya Biomedical (Thousand Oaks, CA). Wortmannin, soybean trypsin inhibitor, poly-L-lysine, creatinine phosphatase, creatinine phosphokinase, and ATP were obtained from Sigma Chemical (St. Louis, MO). All other reagents were purchased from Sigma Chemical.

Methods

Isolation of smooth muscle cells from rabbit rectosigmoid. Smooth muscle cells from rabbit rectosigmoid were isolated as described earlier (4). Briefly, the internal anal sphincter from anesthetized New Zealand White rabbits, consisting of the most distal 3 mm of the circular muscle layer, ending at the junction of skin and mucosa, was removed by sharp dissection. A 5-cm length of the rectosigmoid orad to the junction was dissected and digested to yield isolated smooth muscle cells. The tissue was incubated for two successive 1-h periods at 31°C in 15 ml of HEPES (pH 7.4) containing (in mM) 115 NaCl, 5.7 KCl, 2.0 KH2PO4, 24.6 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 5.6 glucose containing 0.1% (wt/vol) collagenase (150 U/mg, Worthington CLS type II), 0.01 (wt/vol) soybean trypsin inhibitor, and 0.184 (wt/vol) DMEM. After the end of second enzymatic incubation period, the medium was filtered through 500-µm Nitex. The partially digested tissue left on the filter was washed four times with 10 ml of collagenase-free buffer solution. Tissue was then transferred into 15 ml of fresh collagenase-free buffer solution, and cells were gently dispersed. After a hemocytometric cell count, the harvested cells were resuspended in collagenase-free HEPES buffer (pH 7.4). Each rectosigmoid yielded 10-20 × 106 cells.

Particulate fractions. Isolated smooth muscle cells were counted on a hemocytometer and diluted with HEPES buffer as needed. Cells were then treated with agonists and/or antagonist for the indicated periods. After the treatment, the cells were washed twice with buffer A (in mM: 150 NaCl, 16 Na2HPO4, 4 NaH2PO4, and 1 sodium orthovanadate, pH 7.4) and sonicated in buffer B (1 mM Na3VO4, 1 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 1 mM Na4MoO4, 1 mM dithiothreitol, 20 mM NaH2PO4, 20 mM Na2HPO4, 20 mM Na4P2O7 · 10H2O, 50 µl/ml DNase-RNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain, pH 7.4). The cell sonicates were centrifuged at 100,000 g for 60 min. The supernatant material from the high-speed centrifugation was collected as soluble cytosolic fraction. The pellet material was resuspended by sonication twice for 30 s in the lysis buffer plus 1% Triton X-100 and collected as soluble particulate fraction. The protein content was determined by using Bio-Rad protein assay reagent.

Immunoprecipitation and immunoblotting using monoclonal anti-RhoA antibody. Each sample (400-500 µg protein) obtained as described above was subjected to immunoprecipitation with 1:250 monoclonal anti-RhoA antibody overnight. Then the protein G-Sepharose beads were then added and rocked for 2 h. The beads were washed in Tris-buffered saline twice and boiled in 2× Laemmli sample buffer with 2-mercaptoethonal. The samples were subjected to 12.5% SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. Immunoblotting was performed using mouse monoclonal anti-RhoA antibody (1:100 dilution) as primary antibody. The membrane was reacted with peroxidase-conjugated goat anti-mouse IgG (1:3,000 dilution) for 1 h. The enzymes on the membrane were visualized with enhanced chemiluminescent substrates from Amersham.

Immunoprecipitation using monoclonal antibody. Smooth muscle cells were diluted in HEPES buffer as needed. Cells were washed with buffer A [in mM: 150 NaCl, 16 Na2HPO4, and 4 NaH2PO4 at pH 7.4 (PBS) containing 1 mM Na3VO4]. The cells were then disrupted by sonication in buffer B (1 mM Na3VO4, 1 mM NaF, 2 mM phenylmethlysulfonyl fluoride, 5 mM EDTA, 1 mM Na4MoO4, 1 mM dithiothreitol, 20 mM NaH2PO4, 20 mM Na2HPO4, 20 mM Na4P2O7 · 10 H2O, 50 µl/ml DNase-RNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain, pH 7.4), and centrifuged for 15 min at 14,000 g. Protein G-Sepharose was washed two times with buffer B to make a 50% suspension. Lysate containing 200 µg protein in a total of 500 µl of buffer B was precleared with 50 µl of protein G-Sepharose bead slurry by rocking at 4°C for 30 min. The mixture was spun at 14,000 g for 5 min at 4°C, and 1-2 µg of mouse monoclonal anti-RhoA antibody, mouse monoclonal anti-PKC-alpha antibody, or mouse monoclonal anti-HSP27 antibody were added to the resultant supernatant. The mixture was rocked at 4°C for 1 h followed by the addition of 50 µl of protein G-Sepharose bead slurry. The mixture was further rocked at 4°C for 2 h and spun at 14,000 g for 5 min, and the supernatant was aspirated off. The pellet was washed three times with buffer A and resuspended in 25 µl of 2× sample buffer and boiled for 5 min.

SDS-PAGE and electrophoretic transfer. For one-dimensional SDS-PAGE, the samples were mixed in an equal volume of 2× sample buffer [50 mM Tris, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.1% (wt/vol) bromophenol blue, pH 6.8]. The proteins were separated by 12.5 or 15% SDS-PAGE and transferred onto nitrocellulose or PVDF membranes. Proteins were identified by chemiluminescence. Autoradiography was performed on blots or dried gels using a PhosphorImager.

Western immunoblotting of lysates and immunoprecipitates. Lysates (80 µg) or immunoprecipitates of HSP27, RhoA, or PKC-alpha were size separated by SDS-PAGE and electrophoretically transferred to PVDF membranes. Immunoblotting was performed using a monoclonal anti- HSP27 antibody (1:5,000), a monoclonal anti-RhoA antibody (1:1,000), or a monoclonal anti-PKC-alpha antibody (1:1,000), as primary antibody. The membrane was reacted with peroxidase-conjugated goat anti-mouse IgG antibody (1:2,500 dilution) for 1 h at 24°C. The enzymes on the membrane were detected with luminescent substrates. As a negative control, blots were incubated in the secondary antibody only.

Data analysis. Bands were quantitated using a densitometer (model GS-700, Bio-Rad Laboratories), and band volumes (absorbance units × mm2) were calculated and expressed as a percentage of the total volume. Blotting data are within the linear range of detection for each antibody used.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Translocation of RhoA and association of translocated RhoA with HSP27 in the particulate fraction, in response to contraction induced by acetylcholine or by ceramide. Smooth muscle cells were stimulated with acetylcholine (0.1 µM) or with ceramide (0.1 µM) for 30 s or 4 min, and immunoprecipitates of HSP27 (anti-HSP27 antibody) from 500 µg of particulate fractions of cells were subjected to SDS-PAGE and Western blotted with anti-RhoA antibody (1:100; Fig. 1A). Stimulation with the contractile agonist ceramide (0.1 µM) resulted in a significant increase in the association of RhoA with HSP27 at 30 s and 4 min. Ceramide induced a 20.1 ± 0.6 and a 15.7 ± 1.8% increase in HSP27-RhoA association at 30 s and 4 min, respectively (Fig. 1B). Significant and sustained increases (12.9 ± 1.3%) in the association of HSP27 with RhoA were seen in cells stimulated with acetylcholine (0.1 µM) (Fig. 1B) compared with control (8.8 ± 2.1%). In parallel experiments, there was no detectable association of HSP27 with RhoA in the cytosolic fraction of resting or unstimulated smooth muscle cells or with stimulation of the cells with either ceramide or acetylcholine (Fig. 1C), indicating that the association of the proteins is predominantly in the particulate fraction.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1.   Coimmunoprecipitation of heat shock protein 27 (HSP27) with RhoA in response to agonist stimulation in rabbit colon smooth muscle cells. Cells were either untreated or stimulated with ceramide (0.1 µM) or ACh (0.1 µM) for 30 s or 4 min. The immunoprecipitates of HSP27 (IP: anti-HSP27 antibody) from 500 µg of particulate fractions were subjected to SDS-PAGE and Western blotted with anti-RhoA antibody (1:100; IB: anti-RhoA antibody). A: representative blot of 4 independent experiments showing increase in association of RhoA with HSP27 with ceramide stimulation at 30 s and 4 min. B: densitometric analysis indicating that stimulation with the contractile agonist ceramide (0.1 µM) resulted in a significant (P <=  0.001) 2-fold increase in the association of RhoA with HSP27 at 30 s and 4 min. Significant (P <=  0.001) and sustained increases in the association of HSP27 with RhoA were also seen in cells stimulated with ACh (0.1 µM) compared with control. C: 500 µg of cytosolic fractions were immunoprecipitated with anti-HSP27 antibody and were subjected to SDS-PAGE and Western blotted with anti-RhoA antibody (1:100). Representative of 3 blots showing no association of RhoA with HSP27 in the cytosolic fraction of resting unstimulated smooth muscle cells or with stimulation of the cells with ACh. H, heavy; L, light.

Translocation of PKC-alpha and Association of Translocated PKC-alpha With HSP27 in the Particulate Fraction in Response to Contraction Induced by Acetylcholine or by Ceramide

Cells were stimulated with ceramide (0.1 µM) or acetylcholine (0.1 µM) for 30 s or 4 min, and immunoprecipitates of HSP27 (anti-HSP27 antibody) from 500 µg of particulate fractions of cells were subjected to SDS-PAGE and Western blotted with anti-PKC-alpha antibody (1:200) (Fig. 2A). Stimulation with ceramide (0.1 µM) resulted in a significant increase in the association of HSP27 with PKC-alpha at 30 s and 4 min (11.5 ± 1.4 and 11.4 ± 0.7%) compared with control (8.8 ± 1.2%) (Fig. 2B). Similar significant and sustained increases (12.9 ± 1.3%) in the association of HSP27 with PKC-alpha were seen in cells stimulated with acetylcholine (0.1 µM) (Fig. 2B). In parallel experiments, there was no detectable association of HSP27 with PKC-alpha in the cytosolic fraction of resting/unstimulated smooth muscle cells or on stimulation of the cells with either ceramide or acetylcholine (Fig. 2C), indicating that the association of the proteins is predominantly in the particulate fraction.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   Coimmunoprecipitation of HSP27 with protein kinase C-alpha (PKC-alpha ) in response to agonist stimulation in rabbit colon smooth muscle cells. Cells were either untreated or stimulated with ceramide (0.1 µM) or ACh (0.1 µM) for 30 s or 4 min. Immunoprecipitates of HSP27 (IP: anti-HSP27 antibody) from 500 µg of particulate fractions were subjected to SDS-PAGE and Western blotted with anti-PKC-alpha antibody (1:200; IB: anti-PKC-alpha antibody). A: a representative blot of 5 independent experiments showing an increase in the association of PKC-alpha with HSP27 with stimulation with either ceramide or ACh. B: stimulation with the contractile agonists ACh and ceramide (0.1 µM) resulted in a significant (P <=  0.05) increase in the association of PKC-alpha with HSP27 at 30 s and 4 min compared with control. C: 500 µg of cytosolic fractions were immunoprecipitated with anti-HSP27 antibody and were subjected to SDS-PAGE and Western blotted with anti-PKC-alpha antibody (1:200). Representative of 3 blots showing there was no detectable association of HSP27 with PKC-alpha in the cytosolic fraction of resting unstimulated smooth muscle cells or with stimulation of the cells with ACh.

Association of Translocated RhoA With PKC-alpha in the Particulate Fraction in Response to Contraction by Acetylcholine or Ceramide

Cells were stimulated with ceramide (0.1 µM) or acetylcholine (0.1 µM) for 30 s or 4 min, and immunoprecipitates of RhoA (anti-RhoA antibody) from 500 µg of particulate fractions were subjected to SDS-PAGE and Western blotted with anti-PKC-alpha antibody (1:200; Fig. 3A). Stimulation with acetylcholine (0.1 µM) resulted in a significant increase in the association of RhoA with PKC-alpha at 30 s and 4 min (13.2 ± 2.6 and 13.0 ± 2.2%, respectively) compared with control (7.3 ± 2.2%; Fig. 3B). Similar significant and sustained increases (12.9 ± 1.3) in the association of RhoA with PKC-alpha were seen in cells stimulated with ceramide (0.1 µM; Fig. 3B). Preincubation of smooth muscle cells with the PKC inhibitor calphostin C significantly inhibited acetylcholine-induced RhoA-PKC-alpha association at 30 s and 4 min (6.4 ± 1.03 and 8.9 ± 1.6%, respectively, P < 0.05), suggesting that PKC activation is necessary for its translocation to the membrane (Fig. 3C). In parallel experiments, there was a barely detectable background association of PKC-alpha with RhoA in the cytosolic fraction of resting/unstimulated smooth muscle cells or with stimulation of the cells with either ceramide or acetylcholine (Fig. 3, D and E), suggesting that the association of the proteins is predominantly in the particulate fraction.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 3.   Coimmunoprecipitation of RhoA with PKC-alpha in response to agonist stimulation in rabbit colon smooth muscle cells. Cells were either untreated or stimulated with ceramide (0.1 µM) or ACh (0.1 µM) for 30 s or 4 min. Immunoprecipitates of RhoA (IP: anti-RhoA antibody) from 500 µg of particulate fractions were subjected to SDS-PAGE and Western blotted with anti-PKC-alpha antibody (1:200; IB: anti-PKC-alpha antibody). A: representative blot of 3 independent experiments showing an increase in association of PKC-alpha with RhoA with ceramide or ACh stimulation. B: translocation and association of RhoA with PKC-alpha in the particulate fraction in response to contraction by ACh or ceramide. Cells were stimulated with ACh (0.1 µM) or ceramide (0.1 µM) for 30 s or 4 min in presence or absence of calphostin C, and immunoprecipitates of RhoA from 500 µg of particulate fractions were subjected to SDS-PAGE and Western blotted with anti-PKC-alpha antibody (1:200). Stimulation with ACh resulted in a significant (P <=  0.05) increase in the association of RhoA with PKC-alpha at 30 s and 4 min. Similar significant (P <=  0.05) and sustained increases in the association of RhoA with PKC-alpha were seen in cells stimulated with ceramide. C: calphostin C significantly inhibited RhoA-PKC-alpha association at 30 s and 4 min of ACh stimulation (6.4 ± 1.03 and 8.9 ± 1.6%, respectively; P < 0.05), suggesting that PKC activation is necessary for its translocation to the membrane. D: 500 µg of cytosolic fractions were immunoprecipitated with anti-PKC-alpha antibody and were subjected to SDS-PAGE and Western blotted with anti-RhoA antibody (1:200). Representative of 3 blots showing there was a barely detectable background association of PKC-alpha with RhoA in the cytosolic fraction of resting unstimulated smooth muscle cells or with stimulation of the cells with ACh. E: 500 µg of cytosolic fractions were immunoprecipitated with anti-RhoA antibody and were subjected to SDS-PAGE and Western blotted with anti-PKC-alpha antibody (1:200). Representative of 3 blots showing there was a barely detectable background association of PKC-alpha with RhoA in the cytosolic fraction of resting unstimulated smooth muscle cells or with stimulation of the cells with ACh.

Translocation of HSP27, RhoA, and PKC-alpha to the Particulate Fraction in Response to Contraction by Acetylcholine

To confirm that the increase in association is due to agonist-induced translocation of each of the proteins per se to the particulate fraction, cells were stimulated with acetylcholine (0.1 µM) for 30 s or 4 min, and immunoprecipitates of RhoA (anti-RhoA antibody) from 500 µg of particulate fractions were subjected to SDS-PAGE and Western blotted with anti-RhoA antibody (1:200). Sustained increase in the amount of RhoA translocated to the membrane and immunoprecipitated with RhoA antibody was observed (Fig. 4A). Similarly, particulate fractions from cells stimulated with acetylcholine (0.1 µM) for 30 s or 4 min were subjected to immunoprecipitation followed by Western blot with anti-PKC-alpha antibody. Significant increase in the amount of PKC translocated to the particulate fraction was evident (Fig. 4B). Translocation was inhibited when the cells were preincubated with calphostin C (Fig. 4B).


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4.   Translocation of HSP27, RhoA, and PKC-alpha to the particulate fraction in response to contraction by ACh. A: sustained increase in the amount of RhoA translocated to the membrane and immunoprecipitated with RhoA antibody was observed. B: particulate fractions from cells stimulated with ACh (0.1 µM) for 30 s or 4 min were subjected to immunoprecipitation followed by Western blot with anti-PKC-alpha antibody. Significant increase in the amount of PKC translocated to the particulate fraction. Translocation was inhibited when the cells were preincubated with calphostin C. C: particulate fractions from cells treated with ACh (0.1 µM) subjected to immunoprecipitation followed by Western blot with anti-HSP27 antibody (IP: anti-HSP27 antibody, IB: HSP27 antibody). Significant and sustained increase in the amount of HSP27 translocated to the membrane was observed. Lower-molecular-weight proteins did not seem to translocate with stimulation with ACh.

Furthermore, particulate fractions from cells treated with acetylcholine (0.1 µM) subjected to immunoprecipitation followed by Western blot with anti-HSP27 antibody (anti HSP27 antibody, HSP27 antibody). Significant and sustained increase in the amount of HSP27 translocated to the membrane was observed. Lower-molecular-weight proteins did not seem to translocate on stimulation with acetylcholine (Fig. 4C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory has previously reported that preincubation of smooth muscle cells from the rabbit rectosigmoid with a monoclonal antibody to HSP27 inhibits PKC-mediated contraction (5). Our laboratory has also previously shown that RhoA may regulate smooth muscle contraction through cytoskeletal reorganization of HSP27 (40). The following physiological paradigm arises: "Does agonist-induced contraction result in phosphorylation of HSP27 and in an increase in association between HSP27 and PKC, which is paralleled by an increase in the association of PKC with RhoA?" Here, we have attempted to study the association of RhoA with PKC in agonist-induced sustained contraction of rabbit rectosigmoid smooth muscle cells. The data suggest that HSP27 may serve as a link between the two signal transduction pathways leading to contraction on stimulation by contractile agonists.

Ca2+ and MLC phosphorylation are key regulators of dynamic actin filament reorganization. Because the contraction-to-Ca2+ ratio is not always proportional, the Ca2+/calmodulin-dependent MLC kinase pathway cannot solely account for the Ca2+-induced contraction (8, 26, 35). In past years, evidence accumulated that the ras-related, small, GTP-binding protein Rho is another important signaling element that mediates various actin-dependent cytoskeletal functions, including smooth muscle contraction (38). However, its roles in different signal transduction cascades may vary depending on cell type. RhoA has been shown to play pivotal roles in Ca2+ sensitization (24, 26). Several Rho targets have been identified, including protein kinase N, Rho kinase, and the MBS of myosin phosphatase (23). It has been proposed that Rho activates Rho kinase, which inhibits myosin phosphatase and results in increase in phosphorylated MLC (23, 24, 26, 36).

The possible interrelationship between RhoA and other serine/threonine kinases or tyrosine kinases is more complex. Our laboratory's previous data (40) have shown that herbimycin A (pp60src inhibitor) and genistein (tyrosine kinase inhibitor) did not inhibit RhoA translocation from the cytosol to the membrane. The suggestion was that RhoA activation by endothelin and ceramide is either independent or upstream of these tyrosine kinases. It has been suggested that RhoA is regulating Ca2+ sensitivity in smooth muscle via the PKC and mitogen-activated protein kinase pathway or through a PKC-mediated effect on MLC phosphatase (18, 19). Thus activation of RhoA could be upstream of PKC-alpha , or the translocation of RhoA could be is independent and parallel to the translocation and activation of PKC-alpha . Others have reported that in endothelial and epithelial cells, Rho inhibitors block PKC translocation and activation, suggesting RhoA requirement for PKC activation and/or translocation (17).

The accepted model of activation of PKC by lipids is that on binding of diacylglycerol in the presence of the phospholipid cofactor, a conformational change in PKC results in the removal of the pseudosubstrate from its binding site and in the activation of the enzyme. The cysteine-rich domain, the C2 domain, and the pseudosubstrate domains are involved in phospholipid binding. The pseudosubstrate domain contributes to membrane binding. Membrane association is reflected in a shift in subcellular localization or translocation of cytosolic PKC to membrane compartments. This process is controlled by protein-protein interactions. In addition to binding to lipids, PKC can also bind with proteins via protein-protein interactions. These interactions play an important role in the localization and function of PKC isozymes. PKC-binding proteins bind PKC directly via a non-substrate-binding site, which may or may not be PKC substrates and may require cofactors for binding to PKC. PKC isozymes associate with cytoskeletal proteins (22). The interaction between PKCs and cytoskeletal proteins is in part isozyme selective. It has been shown that PKC-zeta binds to tubulin via the pseudosubstrate region (13). PKC-epsilon binds to actin via an actin-binding site on the C1 region (31, 32). PKC-beta II interacts with F actin, and this interaction protects PKC-beta II from degradation and downregulation. (6).

Actin-binding proteins play a key role in shaping the actin cytoskeleton. To further understand how RhoA protein affects actin filament dynamics induced by agonists in smooth muscle contraction, we assessed the correlation between RhoA and the low-molecular-weight heat shock protein HSP27 identified as an actin-binding protein. Our laboratory has previously shown that HSP27 plays an integral role in the orientation or activation of the contractile machinery necessary to maintain a sustained contraction in rabbit gastrointestinal smooth muscle (43). HSP27 distribution during contraction of smooth muscle is not well understood. Recent data have shown that, in vascular smooth muscle, HSP27 redistributes from a cytosolic to a particulate fraction (9). It has been reported that inhibition of RhoA by C3 exoenzyme could block endothelin-induced cytoskeletal actin reorganization in cultured astrocytes (25). In cells transiently transfected with the dominant negative RhoA, our laboratory has observed (40) that ceramide- or endothelin-induced redistribution of HSP27 disappeared, which suggested that RhoA may exert its effect on cytoskeletal reorganization via HSP27. Our data (Fig. 4C) confirm an agonist-induced translocation of HSP27 to the particulate fraction, which could be a step in mediating the association of HSP27 with RhoA and with PKC-alpha .

There are several indications in other cell systems that suggest that RhoA- and PKC-mediated pathways interact. This was suggested to occur through the binding of RhoA to PKC-alpha (11). Evidence that a cellular colocalization of PKC isozymes and RhoA and an apparent synergistic effect on cellular functions are very sketchy. Slater and co-workers (34) report that PKC-alpha and RhoA could be coimmunoprecipitated from PC-12 cell lysates. The potentiating effect of RhoA was found to be specific for PKC-alpha . Slater et al. also reported that the membrane- or filamentous-actin associated PKC-alpha activity, induced by phorbol ester, is further potentiated by RhoA. This may result from a distinct activating conformational change, suggesting that PKC- and RhoA-mediated signaling pathways may converge on a direct PKC-alpha -RhoA interaction (33). Chang and co-workers (11) have suggested that Rho associates with PKC-alpha in vivo and that membrane association and residues within the effector domain of Rho are required for maximal enhancement of activator protein-1 transcriptional activity, further underscoring the importance of the association of these proteins. Hippenstiel and co-workers (17) suggest that the interaction between these proteins require the cooperative interaction with other molecules. Similar results were reported in human epithelial cells.

Our data indicate that PKC-alpha and RhoA do not associate in the cytosolic fractions of resting/unstimulated smooth muscle cells or in response to contractile agonists. PKC-alpha and RhoA coimmunoprecipitate in the particulate fraction of colon smooth muscle cells in response to different contractile agonists. The literature does not provide evidence for a direct interrelationship between these two molecules. Data presented here show that both PKC-alpha and RhoA coimmunoprecipitate with HSP27 in the particulate fraction. A possible explanation could be due to an association between Rho proteins and a PKC-adaptor protein. Our data suggest that HSP27 is a good candidate for such interaction. We propose a model by which HSP27 binds to PKC-alpha and to RhoA on the membrane, as a result of activation of both PKC-alpha and RhoA in contracting smooth muscle. With phosphorylation, HSP27 may bind to activated PKCalpha and activated RhoA. Conversely, PKC-alpha , as it translocates to the membrane, would phosphorylate HSP27 and thus favors its association with RhoA. Thus HSP27 may function as a facilitator in thin-filament regulation of smooth muscle contraction.


    ACKNOWLEDGEMENTS

We thank Mercy Pawar for technical assistance.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-42876.

Address for reprint requests and other correspondence: K. N. Bitar, Univ. of Michigan Medical School, 1150 W. Medical Center Dr., MSRB I, Rm. A520, Ann Arbor, MI 48109-0658 (E-mail: bitar{at}umich.edu).

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.

Received 15 March 2001; accepted in final form 31 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amano, M, Chihara K, Nakamura N, Fukata Y, and Yano T. Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase. Genes Cells 3: 177-188, 1998[Abstract].

2.   Amano, M, Ito M, Kimura K, Fukata Y, and Chihara K. Phosphorylation and activation of myosin by Rho-associated kinase. J Biol Chem 271: 20246-20249, 1996[Abstract/Free Full Text].

3.   Benndorf, R, Hayess K, Ryazantsev S, Wieske M, Behlke J, and Lutsch G. Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity. J Biol Chem 269: 20780-20784, 1994[Abstract/Free Full Text].

4.   Bitar, KN, Hillemeier C, Biancani P, and Balazovich KJ. Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin. Life Sci 47: 2429-2434, 1990[ISI][Medline].

5.   Bitar, KN, Kaminski MS, Hailat N, Cease KB, and Strahler JR. Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin. Biochem Biophys Res Commun 181: 1192-1200, 1991[ISI][Medline].

6.   Blobe, GC, Stribling DS, Fabbro D, Stabel S, and Hannun YA. Protein kinase C beta II specifically binds to and is activated by F-actin. J Biol Chem 271: 15823-15830, 1996[Abstract/Free Full Text].

7.   Bourne, HR, Sanders DA, and McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature 349: 117-127, 1991[Medline].

8.   Bradley, AB, and Morgan KG. Alterations in cytoplasmic calcium sensitivity during porcine coronary artery contractions as detected by aequorin. J Physiol (Lond) 385: 437-448, 1987[Abstract/Free Full Text].

9.   Brophy, CM, Molinaro JR, and Dickinson M. The macromolecular associations of heat shock protein-27 in vascular smooth muscle. Surgery 128: 320-326, 2000[ISI][Medline].

10.   Brophy, CM, Woodrum D, Dickinson M, and Beall A. Thrombin activates MAPKAP2 kinase in vascular smooth muscle. J Vasc Surg 27: 963-969, 1998[ISI][Medline].

11.   Chang, J, Pratt JC, Sawasdikosol S, Kapeller R, and Burakoff SJ. The small GTP-binding protein Rho potentiates AP-1 transcription in T cells. Mol Cell Biol 18: 4986-4993, 1998[Abstract/Free Full Text].

12.   Dekker, LV, and Parker PJ. Protein kinase C---a question of specificity. Trends Biochem Sci 19: 73-77, 1994[ISI][Medline].

13.   Garcia-Rocha, M, Avila J, and Lozano J. The zeta isozyme of protein kinase C binds to tubulin through the pseudosubstrate domain. Exp Cell Res 230: 1-8, 1997[ISI][Medline].

14.   Guay, J, Lambert H, Gingras-Breton G, Lavoie JN, Huot J, and Landry J. Regulation of actin filament dynamics by p38 MAP kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci 110: 357-368, 1997[Abstract].

15.   Hall, A. The cellular functions of small GTP-binding proteins. Science 249: 635-640, 1990[Abstract/Free Full Text].

16.   Hickey, E, Brandon SE, Potter R, Stein G, Stein J, and Weber LA. Sequence and organization of genes encoding the human 27 kDa heat shock protein. Nucleic Acids Res 14: 4127-4145, 1986[Abstract/Free Full Text].

17.   Hippenstiel, S, Kratz T, Krull M, Seybold J, von Eichel-Streiber C, and Suttorp N. Rho protein inhibition blocks protein kinase C translocation and activation. Biochem Biophys Res Commun 245: 830-834, 1998[ISI][Medline].

18.   Hirata, K, Kikuchi A, Sasaki T, Kuroda S, Kaibuchi K, Matsuura Y, Seki H, Saida K, and Takai Y. Involvement of Rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 267: 8719-8722, 1992[Abstract/Free Full Text].

19.   Horowitz, A, Menice CB, Laporte R, and Morgan KG. Mechanisms of smooth muscle contraction (Review). Physiol Rev 76: 967-1003, 1996[Abstract/Free Full Text].

20.   Ibitayo, AI, Groblewski G, and Bitar KN. Ceramide induced phosphorylation of Hsp27 and modulation of its distribution within smooth muscle cells (Abstract). Gastroenterology 114: A769, 1998.

21.   Ibitayo, AI, Sladick J, Tuteja S, Louis-Jacques O, Yamada H, Groblewski G, Welsh M, and Bitar KN. HSP27 in signal transduction and association with contractile proteins in smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 277: G445-G454, 1999[Abstract/Free Full Text].

22.   Keenan, C, and Kelleher D. Protein kinase C and the cytoskeleton. Cell Signal 10: 225-232, 1998[ISI][Medline].

23.   Kimura, K, Fukata Y, Matsuoka Y, Bennett V, Matsuura Y, Okawa K, Iwamatsu A, and Kaibuchi K. Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase) and myosin phosphatase. J Biol Chem 273: 5542-5548, 1998[Abstract/Free Full Text].

24.   Kimura, K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, and Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245-248, 1996[Abstract].

25.   Koyama, Y, and Baba A. Endothelin-induced cytoskeletal actin re-organization in cultured astrocytes: inhibition by C3 ADP-ribosyltransferase. Glia 16: 342-350, 1996[ISI][Medline].

26.   Kureishi, Y, Kobayashi S, Amano M, Kimura K, Kanaide H, Nakano T, Kaibuchi K, and Ito M. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 272: 12257-12260, 1997[Abstract/Free Full Text].

27.   Landry, J, Lambert H, Zhou M, Lavoie JN, Hickey E, Weber LA, and Anderson CW. Human Hsp27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II. J Biol Chem 267: 794-803, 1992[Abstract/Free Full Text].

28.   Lutsch, G, Vetter R, Offhauss U, Wieske M, Grone HJ, Klemenz R, Schimke I, Stahl J, and Benndorf R. Abundance and location of the small heat shock proteins Hsp25 and alpha B-crystallin in rat and human heart. Circulation 96: 3466-3476, 1997[Abstract/Free Full Text].

29.   Parker, PJ, Coussens L, Totty N, Rhee L, Young S, Chen E, Stabel S, Waterfield MD, and Ullrich A. The complex primary structure of protein kinase C---the major phorbol ester receptor. Science 233: 853-858, 1986[Abstract/Free Full Text].

30.   Paterson, HF, Self AJ, Garrett MD, Just I, Aktories K, and Hall A. Microinjection of recombinant p21Rho induces rapid changes in cell morphology. J Cell Biol 111: 1001-1007, 1990[Abstract/Free Full Text].

31.   Prekeris, R, Hernandez RM, Mayhew MW, White MK, and Terrian DM. Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin. J Biol Chem 273: 26790-26798, 1998[Abstract/Free Full Text].

32.   Prekeris, R, Mayhew MW, Cooper JB, and Terrian DM. Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function. J Cell Biol 132: 77-90, 1996[Abstract/Free Full Text].

33.   Slater, S, Milano S, Stagliano B, Gergich K, Curry J, Taddeo F, and Stubbs C. Interaction of protein kinase C with filamentous actin: isozyme specificity resulting from divergent phorbol ester and calcium dependencies. J Am Chem Soc 39: 271-280, 2000.

34.  Slater SJ, Stagliano BA, Sietz J, and Stubbs CD. Interation of PKC with members of the rho family of small GTpases (Abstract). Keystone Symposium Feb. 2000, p. 109.

35.   Somlyo, AP, and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[Medline].

36.   Somlyo, AP, and Somlyo AV. Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol (Lond) 522: 177-185, 2000[Abstract/Free Full Text].

37.   Stasia, MJ, Jouan A, Bourmeyster N, Boquet P, and Vignais PV. ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility. Biochem Biophys Res Commun 180: 615-622, 1991[ISI][Medline].

38.   Takai, Y, Sasaki T, Tanaka K, and Nakanishi H. Rho as a regulator of the cytoskeleton. Trends Biochem Sci 20: 227-231, 1995[ISI][Medline].

39.   Tang, S, Morgan KG, Parker C, and Ware JA. Requirement for protein kinase C theta for cell cycle progression and formation of actin stress fibers and filopodia in vascular endothelial cells. J Biol Chem 272: 28704-28711, 1997[Abstract/Free Full Text].

40.   Wang, P, and Bitar KN. Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27. Am J Physiol Gastrointest Liver Physiol 275: G1454-G1462, 1998[Abstract/Free Full Text].

41.   Welsh, MJ, and Gaestel M. Small heat shock protein family; function in health and disease. In: Stress on Life from Molecules to Man, edited by Csermely P.. New York: New York Acad. Sci., 1998, p. 28-35.

42.   Wilkinson, JM, and Pollard I. Immunohistochemical localisation of the 25 kDa heat shock protein in unstressed rats: possible functional implications. Anat Rec 237: 453-457, 1993[Medline].

43.   Yamada, H, Strahler J, Welsh MJ, and Bitar KN. Activation of MAP kinase and translocation with HSP27 in bombesin-induced contraction of rectosigmoid smooth muscle. Am J Physiol Gastrointest Liver Physiol 269: G683-G691, 1995[Abstract/Free Full Text].

44.   Yamboliev, I, Wiesmann K, Singer C, Hedges J, and Gerthoffer W. Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle. Am J Physiol Cell Physiol 279: C352-C360, 2000[Abstract/Free Full Text].

J APPL PHYSIOL 92(1):41-49
8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. J. Sandoval, E. R. Injeti, W. T. Gerthoffer, and W. J. Pearce
Postnatal maturation modulates relationships among cytosolic Ca2+, myosin light chain phosphorylation, and contractile tone in ovine cerebral arteries
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2183 - H2192.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
D. P. Poole and J. B. Furness
PKC {delta}-isoform translocation and enhancement of tonic contractions of gastrointestinal smooth muscle
Am J Physiol Gastrointest Liver Physiol, March 1, 2007; 292(3): G887 - G898.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. B. Patil and K. N. Bitar
RhoA- and PKC-{alpha}-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells.
Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G83 - G95.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
H. Pang and K. N. Bitar
Direct association of RhoA with specific domains of PKC-{alpha}
Am J Physiol Cell Physiol, October 1, 2005; 289(4): C982 - C993.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Somara, H. Pang, and K. N. Bitar
Agonist-induced association of tropomyosin with protein kinase C{alpha} in colonic smooth muscle
Am J Physiol Gastrointest Liver Physiol, February 1, 2005; 288(2): G268 - G276.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. B. Patil, M. D. Pawar, and K. N. Bitar
Direct association and translocation of PKC-{alpha} with calponin
Am J Physiol Gastrointest Liver Physiol, June 1, 2004; 286(6): G954 - G963.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. B. Patil, M. D. Pawar, and K. N. Bitar
Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKC{alpha}
Am J Physiol Gastrointest Liver Physiol, April 1, 2004; 286(4): G635 - G644.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
K. N. Bitar
Aging and Neural Control of the GI Tract: V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins
Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G1 - G7.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
K. N. Bitar
HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction
Am J Physiol Gastrointest Liver Physiol, May 1, 2002; 282(5): G894 - G903.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (19)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bitar, K. N.
Right arrow Articles by Patil, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bitar, K. N.
Right arrow Articles by Patil, S. B.

<