C-type natriuretic peptide induces human colonic myofibroblast relaxation

Taned Chitapanarux, Stephen L. Chen, Helen Lee, Andrew C. Melton, and Hal F. Yee, Jr.

Departments of Medicine and Physiology, CURE Digestive Diseases Research Center, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095

Submitted 29 July 2003 ; accepted in final form 2 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intestinal response to injury requires coordinated regulation of the tension exerted by subepithelial myofibroblasts (SEM). However, the signals governing relaxation of intestinal SEM have not been investigated. Our aim was to test the hypothesis that signal transduction pathways initiated by C-type natriuretic peptide (CNP) induce intestinal SEM relaxation. We directly quantified the effects of CNP on isometric tension exerted by cultured human colonic SEM. We also measured the effects of CNP on cGMP content, myosin regulatory light chain (MLC) phosphorylation, and cytosolic Ca2+ concentration. CNP induced relaxation of SEM within 10 s. By 10 min, relaxation reached a plateau that was sustained for 2 h. CNP-induced relaxation was saturable, with a maximal decrease in tension (51.7 ± 3.8 dyn) observed at 250 nM. SEM relaxation in response to CNP constituted ~23% of total basal tension. CNP increased intracellular cGMP content and reduced MLC phosphorylation. Effects of CNP on cGMP and MLC exhibited the same dose dependence as CNP-induced relaxation. MLC phosphorylation decreased within 2 min of CNP exposure and was sustained for at least 45 min. CNP also stimulated a large transient increase in cytosolic Ca2+ concentration that occurred within 30 s and was nearly complete by 1 min. We also observed that calyculin-A, a potent inhibitor of MLC phosphatase, completely abolished the reduction in MLC phosphorylation induced by CNP. These results suggest that CNP induces intestinal SEM relaxation through cGMP-associated reductions in MLC phosphorylation. Moreover, these findings raise the possibility that CNP plays a role in intestinal wound healing.

guanosine 3',5'-cyclic monophosphate; contraction; myosin; subepithelial myofibroblast; intestine


SUBEPITHELIAL MYOFIBROBLASTS (SEM) mediate the injury response of the large and small intestine (41). These cells lie tightly apposed to the basal surface of the epithelium. When the epithelium is damaged, SEM respond by contracting and rapidly reducing the surface area of the wound (11, 34, 35). Subsequently, SEM migrate to and proliferate at sites of injury (18, 42, 47, 55). The expanded population of SEM secretes inflammatory mediators (32, 55), which direct epithelial cell chemotaxis and proliferation. These SEM also produce extracellular matrix proteins and matrix metalloproteases (1, 33, 42, 55), which participate in tissue restitution and scar formation. In addition to initially diminishing wound surface area, alterations in the tension (i.e., tensile force) exerted by cells of this type are believed to contract granulation tissue, orient extracellular matrix proteins, and remodel scars (9, 10, 13, 15, 31, 45). Thus coordinated regulation of contraction (i.e., increase in tension) and relaxation (i.e., reduction in tension) is essential for mucosal repair in the intestine. We recently showed (20) that the inflammatory mediator endothelin-1 stimulates colonic SEM contraction through pathways that signal through rhoassociated kinase and myosin. The extra- and intracellular signals governing the relaxation of this cell type have, however, not been investigated.

C-type natriuretic peptide (CNP) is a potent cardiac and smooth muscle relaxing factor (3, 36, 38, 56). In smooth muscle, CNP activates its cognate natriuretic peptide receptor, NPR-B (6, 40). NPR-B, a member of the membrane-bound guanalyl cyclase family, catalyzes the synthesis of cGMP within the cytosol (6, 40). CNP has been measured in the intestine by radioimmunoassay (25). Moreover, transcripts for CNP and NPR-B have been detected in the intestinal mucosa (22). Finally, this natriuretic peptide stimulates intestinal cGMP production (19, 22, 44) and increases cGMP levels in cultured colonic SEM (54). Together these findings suggest the possibility that CNP might act as a relaxing factor for intestinal SEM.

Although the signals underlying smooth muscle relaxation are well characterized (49, 50), little is known about how relaxation is regulated in nonmuscle cell types and nothing is known about how SEM relaxation is controlled. In smooth muscle, cellular tension is induced by the myosin II motor protein complex (myosin), which is activated when its myosin regulatory light chain (MLC) is phosphorylated. MLC kinase phosphorylates MLC, whereas MLC phosphatase dephosphorylates MLC. Therefore, the net effects of MLC kinase and MLC phosphatase regulate the activity of myosin. MLC kinase is activated by increases in cytosolic Ca2+ concentration ([Ca2+]), whereas MLC phosphatase activity is stimulated by cGMP-dependent serine/threonine protein kinase (PKG) (52) and inhibited by rho-associated kinase (50). Consequently, reductions in MLC kinase activity or increases in MLC phosphatase activity can cause relaxation in smooth muscle cells.

The aims of this study were to determine whether CNP can induce intestinal SEM relaxation and, if so, to characterize the contributing signal transduction pathways. Therefore, we directly and quantitatively measured the effects of CNP on the isometric tension exerted by colonic SEM. We also determined the effects of this peptide on cGMP, MLC phosphorylation, and cytosolic [Ca2+]. Our results suggest that CNP induces intestinal SEM relaxation through a cGMP-associated reduction in MLC phosphorylation.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials and cellular model system. CNP was obtained from Calbiochem (La Jolla, CA). Cytochalasin D and calyculin-A were obtained from Sigma (St. Louis, MO). Biotrak cGMP competitive enzyme immunoassay (EIA) system, enhanced chemiluminescence (ECL) reagent, and anti-rabbit secondary antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).

Human colonic SEM (CCD-18Co) were obtained at passage 5 from the American Type Culture Collection (no. CRL-1459; Rockville, MD). SEM were used between passages 7 and 12 and grown in culture as described (20). All experiments were performed at 37°C.

Cellular tension measurements. Cellular tension was directly and quantitatively measured in SEM as described (20). Briefly, cells (1 x 106) were cultured for 3 days within a solid but elastic gel made of type I collagen. Each gel containing SEM was attached to an isometric force transducer, stretched to its original length, and submerged in HEPES-buffered physiological salt solution for 1 h to establish a basal level of cellular tension. Reagents were added directly to the bath. Cellular tension was recorded digitally and analyzed with commercial software (Virtual Bench Logger; National Instruments).

cGMP measurements. SEM, cultured on tissue culture plates (Microtest III 96-well, Falcon; Becton Dickinson, Franklin Lakes, NJ), were incubated in serum-free media for 1 h before each experiment. Reagents were added directly to each well. Intracellular cGMP levels were assayed with a commercial cGMP EIA system. cGMP levels in the experimental samples were calculated by using a standard curve that was generated from samples of known cGMP concentration.

MLC phosphorylation measurements. SEM cultures were incubated in serum-free media for 1 h before each experiment. Reagents were added directly to the cultures. MLC phosphorylation was determined as described previously (20, 58, 59). Briefly, SEM proteins were precipitated with ice-cold 10% trichloroacetic acid and 2 mM dithiothreitol, then solubilized in a 9 M urea buffer. Unphosphorylated and phosphorylated forms of MLC were separated electrophoretically (Mini-protean II; Bio-Rad, Hercules, CA) on a glycerol/acrylamide gel and then transferred to nitrocellulose (Mini Trans-blot system; Bio-Rad). Nitrocellulose blots were incubated first with an affinitypurified rabbit anti-MLC that was generated in this laboratory (59) and then with peroxidase-conjugated secondary antibody. The ECL system was used to detect phosphorylated and unphosphorylated forms of MLC. MLC phosphorylation is presented as the sum of phosphorylated MLC as a percentage of total MLC, as quantified by densitometry.

Cytosolic [Ca2+] measurements. SEM, cultured for 2–3 days on glass coverslips at a density of 1.5 x 104 cells/22 mm2 coverslip, were equilibrated in a physiological salt solution for 30 min at 37°C. Cytosolic [Ca2+] measurements were performed as described previously (20) with the following modifications. The cells were exposed to 5 µM of the membrane-permeant acetoxymethyl ester form of the calcium-sensitive fluorescent dye fluo-3 (Molecular Probes, Eugene, OR) for 45 min at 25°C, washed twice, and placed in a 37°C imaging chamber. Following excitation at a light wavelength of 488 nm, microscopic images (x40, 0.75 numerical aperture objective) were collected with a charge-coupled device camera every 30 s at a light wavelength of 535 nm. Fluorescence intensity was measured (Metamorph; Universal Imaging, West Chester, PA) by creating a region of interest around the entire cytosol. Alterations in cytosolic [Ca2+] were determined as the change in fluorescence intensity divided by the basal fluorescence intensity. Transmitted light images were compared before and after treatment with CNP and fetal bovine serum (20%). No changes in cell shape were observed.

Statistical analysis. Data were presented as means ± SE. Statistical comparisons were made by Student's t-test. A statistical difference was defined as P < 0.05.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CNP induces relaxation of colonic SEM. To directly and quantitatively investigate SEM relaxation, collagen gels populated with colonic SEM were attached to sensitive isometric force transducers. The tension exerted by these cells was recorded digitally as depicted in Fig. 1. We tested the hypothesis that CNP induces relaxation of SEM by exposing these cells to varying concentrations (0–500 nM) of this natriuretic peptide. Exposure of SEM to 250 nM CNP resulted in a rapid and sustained reduction from the basal tension exerted by these cells (Fig. 1). Relaxation, defined as the decrement in tension, occurred within 10 s of the addition of CNP (Fig. 1, inset) and achieved a plateau by 10 min (Fig. 1). This plateau was sustained for up to 2 h, which was the longest experiment performed (data not shown). CNP-induced relaxation was dose dependent and saturable (Fig. 2), with a maximal decrease in cellular tension (51.7 ± 3.8 dyn) observed at 250 nM. These results suggest that CNP induces SEM relaxation.



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Fig. 1. Representative cellular tension tracing for colonic subepithelial myofibroblasts (SEM) exposed first to 250 nM C-type natriuretic peptide (CNP) and then to 2 nM endothelin-1 (ET-1) at the times indicated. Inset: expansion of the portion of the cellular tension tracing indicated by the rectangular box.

 


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Fig. 2. CNP dose-response relationship for the maximal change ({Delta}) in cellular tension. Values are means ± SE of 4 independent experiments.

 

To exclude the possibility that CNP-induced decreases in SEM tension resulted from cellular injury or damage to the contractile machinery, CNP-treated SEM were subsequently exposed to endothelin-1 or cytochalasin D. Twenty minutes after exposure to CNP, treatment with endothelin-1 (2 nM) stimulated an increase in cellular tension substantially greater than that observed at baseline (Fig. 1). In contrast, subsequent exposure to cytochalasin D (1 µM), which completely abolishes cellular tension, resulted in substantial additional relaxation. In these experiments (n = 4), CNP (250 nM) reduced cellular tension by 54 ± 2 dyn, whereas 1 µM cytochalasin D caused an additional 180 ± 18-dyn decrease in tension. Thus CNP-induced relaxation represented ~23% of the total basal tension exerted by the cells. These observations indicate that CNP did not act by damaging the contractile apparatus or causing generalized cellular toxicity and thus support the hypothesis that CNP induces SEM relaxation.

Signaling mechanisms underlying CNP-induced colonic SEM relaxation. To test whether the effects of CNP might be mediated through NPR-B, we measured intracellular cGMP levels in response to varying concentrations (0–1,000 nM) of this natriuretic peptide. Exposure of SEM to CNP stimulated a robust increase in the levels of cGMP (Fig. 3). The increase in cGMP induced by CNP was dose dependent and reached a plateau at 250 nM. These findings suggest that the effects of CNP on SEM are transduced by NPR-B.



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Fig. 3. CNP dose-response relationship for the intracellular cGMP content. Values are means ± SE of 4 independent experiments.

 

To explore the mechanisms through which CNP induces SEM relaxation, we also measured changes in MLC phosphorylation in response to varying concentrations of CNP (0–1,000 nM). Exposure of SEM to CNP caused a rapid and sustained decrease in the fraction of total MLC that was phosphorylated (Fig. 4A). Maximal reduction in MLC phosphorylation was observed at 5 min and was sustained for at least 45 min. The effect of CNP on MLC phosphorylation was dose dependent and reached a plateau at 250 nM CNP (Fig. 4B). These results suggest that CNP induces relaxation of SEM by reducing the portion of phosphorylated MLC and consequently diminishing the activity of myosin.



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Fig. 4. A: time course for myosin regulatory light chain (MLC) phosphorylation in response to 250 nM CNP. MLC phosphorylation is presented as the sum of phosphorylated MLC as a percentage of total MLC. Values are means ± SE of >3 independent experiments. B: CNP dose-response relationship for MLC phosphorylation, which is presented as the sum of phosphorylated MLC as a percentage of total MLC. MLC phosphorylation was measured 5 min after addition of CNP or carrier. Values are means ± SE of >3 independent experiments.

 

To examine if CNP reduced the level of MLC phosphorylation in SEM by inhibiting the activity of Ca2+-dependent MLC kinase, we measured the effects of CNP on cytosolic [Ca2+]. CNP (250 nM) stimulated a rapid and transient elevation in cytosolic [Ca2+] (Fig. 5). This transient increase occurred within 30 s and was nearly complete at 1 min. The magnitude of the increase in cytosolic [Ca2+] was similar to that stimulated by fetal bovine serum (20%). The observation that CNP increased cytosolic [Ca2+] strongly argues against reductions in MLC kinase activity as the mechanism underlying the decrease in MLC phosphorylation seen in response to CNP.



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Fig. 5. Time course for changes in cytosolic Ca2+ concentration as measured by the change in fluorescence intensity divided by initial fluorescence intensity ({Delta}F/F0). Colonic myofibroblasts were first exposed to 250 nM CNP and then to 20% fetal bovine serum (FBS) at the times indicated. Values are means ± SE of 9 cells from 3 independent preparations.

 

To test whether CNP reduced the level of MLC phosphorylation in SEM by stimulating the activity of MLC phosphatase, we employed calyculin-A, a cell-permeable inhibitor of type 1 and 2A protein phosphatases. We confirmed that calyculin-A was a potent inhibitor of MLC phosphatase by evaluating its effect on the basal level of MLC phosphorylation. At 2 nM, calyculin-A resulted in a 5% increase in MLC phosphorylation, whereas 10 nM calyculin-A increased MLC phosphorylation by 15%. If CNP-induced relaxation is mediated by MLC phosphatase, then we would expect calyculin-A to inhibit the decrease in MLC phosphorylation caused by CNP. After treatment with calyculin-A, CNP-induced reductions in MLC phosphorylation were completely abolished (Fig. 6). These data are consistent with the notion that CNP reduces MLC phosphorylation in SEM through its effects on MLC phosphatase.



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Fig. 6. Effects of different concentrations of calyculin-A on MLC phosphorylation in response to carrier or 250 nM CNP. MLC phosphorylation was measured 10 min after addition of calyculin-A or carrier and 5 min after addition of CNP or carrier. Values are means ± SE of >3 independent experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we demonstrated that CNP induces relaxation in a colonic SEM cell line that has previously been broadly characterized (20, 21, 4648, 54, 57, 60). This natriuretic peptide and its cognate receptor, NPR-B, have previously been detected in the small and large intestine (22, 25). CNP was shown to increase cGMP levels in the intestine and in cultured colonic SEM (19, 22, 44, 54). Although CNP is a powerful relaxing factor for the heart and vasculature (3, 36, 38, 56), the role that CNP plays in the gut has been obscure. Emerging evidence suggests that the tension exerted by SEM plays an important role in the intestinal response to injury. Early after injury to the epithelium, increases in the tension generated by these cells are thought to shrink the wound (11, 34, 35). Substantial literature in other wound-healing models (9, 10, 13, 15, 31, 45) proposes that changes in the tension produced by SEM later in the wound response mediate tissue remodeling and subsequent mucosal restitution or scar formation. Hence, our observation that CNP causes SEM relaxation has important pathophysiological relevance for understanding the mechanisms underlying the intestinal response to injury. We have recently reported (20) that the inflammatory mediator endothelin-1 stimulates the contraction of colonic SEM. In addition, a role for CNP in inflammation and injury has been suggested (14, 43). Taking these observations together, we propose that CNP and endothelin-1 contribute to mucosal repair through their reciprocal effects on the tension exerted by intestinal SEM.

In addition to tissue remodeling, the tension exerted by SEM and other nonmuscle cell types is essential for fundamental processes such as cytokinesis and chemotaxis (8, 17, 24, 30, 39, 51). Little is known, however, about the molecular signals that govern nonmuscle cell relaxation. Most studies of nonmuscle cell tension have used techniques that only allow measurement of contraction. Thus much of what is believed about the regulation of relaxation in nonmuscle cells is based on studying the inhibition of contraction, rather than relaxation per se. In the present study, we have employed a method that permits direct and quantitative measurement of SEM relaxation. We have shown that CNP causes the relaxation of SEM within seconds. Moreover, the reduction in tension exerted by SEM in response to CNP was nearly one-third as great as the total tension produced by these cells in the absence of serum or other exogenous agonists. These findings suggest that the rate and magnitude of CNP-induced relaxation are sufficient to contribute significantly to vital cellular processes carried out by SEM.

To eliminate the possibility that the reduction in cellular tension induced by CNP resulted from toxic injury to the SEM or damage to their contractile machinery, control experiments were performed. SEM were exposed to either endothelin-1 or cytochalasin D after they had been treated with CNP. Subsequent exposure to endothelin-1 stimulated an increase in cellular tension that was greater than that observed at baseline before CNP treatment. In fact, the magnitude of the contraction induced by endothelin-1 after CNP was similar in magnitude to the contraction induced by endothelin-1 in the absence of CNP (20). This indicated that the contractile apparatus remained intact after treatment with CNP and that CNP did not cause generalized cellular toxicity. This interpretation was further substantiated by using cytochalasin D, which poisons actin polymerization (2, 27), to completely abrogate cellular tension. If the relaxation induced by CNP was due to loss of cellular or contractile integrity, then cytochalasin D should not further reduce cellular tension. Subsequent exposure to cytochalasin D induced substantial further relaxation by colonic myofibroblasts. These data indicate that CNP-induced decreases in cellular tension resulted from true physiological relaxation, rather than cellular injury or impairment of the contractile apparatus.

We investigated the molecular signals that transduce CNP-induced relaxation in SEM by measuring intracellular cGMP and MLC phosphorylation. To test the possibility that NPR-B mediated the actions of CNP, we measured the intracellular content of cGMP in colonic SEM. CNP significantly increased the level of cGMP in SEM. Furthermore, CNP induced changes in cGMP with a dose dependence similar to its effects on relaxation. In smooth muscle, membrane-bound guanalyl cyclases cause relaxation by eliciting a PKG-mediated reduction in MLC phosphorylation (4, 28, 29). We found that CNP reduced the level of MLC phosphorylation in colonic SEM with a time course similar to that of CNP-induced relaxation. Moreover, CNP reduced the magnitude of MLC phosphorylation with a dose-response relationship similar to the effects of CNP on both cellular tension and intracellular cGMP content. These results suggest that CNP-induced SEM relaxation is mediated by cGMP-dependent reductions in MLC phosphorylation.

Increasing the activity of MLC phosphatase or reducing the activity of MLC kinase can diminish MLC phosphorylation (49, 50). In smooth muscle, natriuretic peptides control cellular tension primarily by eliciting a PKG-mediated reduction in cytosolic [Ca2+], which decreases the activity of Ca2+-dependent MLC kinase (28, 29, 53). Thus an unanticipated finding of this study was that CNP induced a rapid and transient elevation in cytosolic [Ca2+] in SEM, which was similar in magnitude to that induced by serum. Indeed, one might expect that the observed increase in cytosolic [Ca2+] should have stimulated an increase in MLC kinase activity and a resultant increase in MLC phosphorylation and cellular tension. Emerging evidence from our laboratory and others (23, 37) can, in part, explain this apparent paradox. This evidence indicates that transient increases in cytosolic [Ca2+] are not sufficient to stimulate contraction in nonmuscle cell types, which contrasts with what we know about contractile regulation in smooth muscle cells. Prior reports that CNP increases cytosolic [Ca2+] in porcine kidney epithelial cells and a gonadotrope cell line (7, 12) support the validity of our finding that CNP induces an increase in cytosolic [Ca2+]. The physiological significance of the CNP-stimulated Ca2+ transients observed in these nonmuscle cell types remains to be explained.

If CNP increases cytosolic [Ca2+], then the reduction in MLC phosphorylation induced by this natriuretic peptide could not be explained by a decrease in MLC kinase activity. It has been reported that PKG can decrease MLC phosphorylation by activating MLC phosphatase (52). We used calyculin-A to test this alternate possibility. Calyculin-A is a membrane-permeant inhibitor of MLC phosphatase (5, 16, 26). We observed that exposing colonic SEM to calyculin-A before treatment with CNP completely blocked CNP-induced MLC phosphorylation. These data suggest that CNP-induced reductions in MLC phosphorylation and cellular tension in SEM are mediated through activation of MLC phosphatase. This stands in contrast to what we know about how CNP acts on smooth muscle.

Together, these novel results are consistent with a model for natriuretic peptide-induced relaxation of colonic SEM in which extracellular CNP directly stimulates synthesis of cGMP within the cytosol by engaging NPR-B. Increases in cGMP activate PKG, which in turn promotes the activity of myosin phosphatase. Enhanced myosin phosphatase activity leads to a net reduction in MLC phosphorylation and consequent relaxation. We hypothesize that by reducing the tension exerted by SEM, CNP acts in a reciprocal fashion with endothelin-1 to modulate the intestinal response to injury. Thus the findings of this study may have important implications for understanding the pathophysiology of intestinal restitution after acute epithelial injury, as well as scar and stricture formation during chronic mucosal injury. The signal transduction pathways described here present potential targets for therapy to facilitate normal wound healing and also prevent formation of strictures and other pathological manifestations of fibrosis.


    ACKNOWLEDGMENTS
 
We thank J. Han for valuable technical assistance and E. Arnold, K. Jiramongkolchai, P. Pham, and Drs. T. Chiu, S. Lidofsky, and E. Rozengurt for their thoughtful critiques of the manuscript.

GRANTS

This work was supported, in part, by awards to H. F. Yee, Jr., from the National Institute of Diabetes and Digestive and Kidney Diseases (K08-DK-02450, R03-DK-57532, and R01-DK-61532) and to T. Chitapanarux from the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. F. Yee, Jr., 675 Charles E. Young Dr. South, Rm. 1519, MacDonald Research Laboratories, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (E-mail: hyee{at}mednet.ucla.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.


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