Calcium- and superoxide anion-mediated mitogenic action of substance P on cardiac fibroblasts

Division of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India 695 011

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Substance P is released from nerve endings in the heart under pathological conditions like ischemia, but its action on cardiac cells has not been investigated. This study tested the hypothesis that substance P is mitogenic to adult cardiac fibroblasts and delineated the underlying mechanism(s). Substance P, acting via neurokinin-1 (NK-1) receptors, stimulated cellular hyperplasia over a range of 1-10 µmol/l. It elicited no change in net collagen production, total protein synthesis, or cell protein content but increased ⁴⁵Ca uptake and superoxide generation. EGTA, N-acetyl-cysteine, and superoxide dismutase attenuated the hyperplastic response to substance P. A combination of substance P and EGTA enhanced superoxide generation without an increase in DNA synthesis, showing that an increase in superoxide production does not result in hyperplasia when extracellular Ca²⁺ is chelated. Together, the data suggest that substance P may activate, via NK-1 receptors, a hyperplastic but not hypertrophic response in adult cardiac fibroblasts and that alterations in redox state and Ca²⁺ homeostasis may act in concert to mediate its mitogenic action.

peptidergic; neural pathway; calcium signaling; oxidant signaling; hyperplasia

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE HEART IS COMPOSED OF MYOCYTES and nonmyocytes, most of which are fibroblasts. For a long time, defective myocyte function was believed to be solely responsible for cardiac failure and very little attention was focused on the contribution of fibroblasts to the structural and functional integrity of the myocardium (36). However, whereas myocytes are terminally differentiated and lose their replicative ability soon after birth, fibroblasts retain their ability to proliferate in response to humoral and mechanical stimuli even in the adult heart. Consequently, cardiac fibroblasts play a central role in wound healing and myocardial remodeling under various pathological conditions. Modulation of cardiac fibroblast function has therefore attracted increasing attention in the past decade (36). Extensive studies (3) have been carried out on the effects of angiotensin II on fibroblasts following identification of a renin-angiotensin system in these cells. The emphasis on the renin-angiotensin system and its role in fibrogenesis has, however, nearly precluded investigations on the possible involvement of other growth factors in regulating myocardial fibroblasts.

The discovery that the myocardium is innervated not only by cholinergic and adrenergic nerves but also by peptidergic nerves that synthesize and secrete neuromodulatory peptides, such as substance P (SP) (37), raises the possibility that these neuropeptides may modulate myocardial metabolism under normal and/or pathological conditions. SP, a potent peripheral and coronary vasodilator (39), is believed to be involved in inflammation (18), tissue repair, and fibrosis (8). More recently, it has been shown that SP acts as a trophic agent in certain cell types, such as arterial endothelial cells (33) and skin fibroblasts (15). Although SP is released from nerve endings in the heart under pathological conditions such as hypoxia and ischemia (13, 32), no attempt has been made to understand its action on cardiac cells. Moreover, because SP immunoreactivity is marked in the connective tissue of the heart (35), myocardial fibroblasts may be particularly susceptible to its action. It is surprising therefore that regulation of fibroblast function by SP has not hitherto been investigated.

The present investigation had two objectives: 1) to test the hypothesis that SP is a stimulus for cardiac fibroblast proliferation, and 2) to delineate the mechanism(s) underlying the mitogenic action of SP on cardiac fibroblasts, with focus on the possible involvement of superoxide anion and Ca²⁺. The results of this study suggest for the first time that SP, acting via the neurokinin-1 (NK-1) receptor, is a mitogenic stimulus for cardiac fibroblasts and that concerted increases in superoxide production and Ca²⁺ influx may contribute to its mitogenic action.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All chemicals were purchased from Sigma and were of the best available grade. [³H]thymidine (specific activity, 18 Ci/mmol), [³H]phenylalanine (specific activity, 6.4 Ci/mmol), and ⁴⁵Ca (specific activity, 12.35 mCi/g) were obtained from the Bhabha Atomic Research Center, India.

Isolation of Adult Rat Myocardial Fibroblasts

Ventricular fibroblasts from adult Sprague-Dawley rats were isolated following the method of Eghbali et al. (4) with minor modifications. Briefly, the ventricular tissue was minced and subjected to repeated enzymatic digestions with a mixture of collagenase type IA, trypsin, pancreatin, and deoxyribonuclease. Isolated cells were preplated for 150 min and the unattached cells were discarded. The adherent cells were grown in Dulbecco's modified Eagle's medium (DMEM)-medium 199 (M199)-fetal bovine serum (7:2:1). Cells from passages 3 and 4 were used for the experiments, and their fibroblastic nature was confirmed by immunocytochemistry using antibodies against vimentin, factor VIII-related antigen, and desmin. The cultures were negative for factor VIII-related antigen and desmin, ruling out endothelial and smooth muscle cells, respectively, but positive for vimentin, indicating that they were fibroblasts (99% purity). Serum deprivation lasted 24 h in DMEM-M199 (8:2) supplemented with insulin and transferrin at 5 µg/ml each. The same medium was also used for the assays except where indicated otherwise. SP was used at a concentration of 10 µmol/l in all the experiments except for the dose response.

Measurement of DNA Synthesis

DNA synthesis was measured in terms of [³H]thymidine incorporation into trichloroacetic acid-insoluble material, as described earlier (23). Briefly, serum-deprived, subconfluent cultures were exposed to SP for 24 h. During the last 4 h, the cells were pulsed with 2.5 µCi/ml [³H]thymidine and processed for determination of acid-precipitable radioactivity.

Determination of Cell Number

Serum-deprived, subconfluent cultures were treated with SP for 24 h. The cells were then washed, detached with trypsin-EDTA solution, and resuspended in growth medium. Cell counts were performed with the use of a Neubauer counting chamber.

Measurement of Total Protein Synthesis and Cell Protein Content

Protein synthesis was measured in terms of [³H]phenylalanine incorporation into trichloroacetic acid-insoluble material, as described by Schorb et al. (21). After serum deprivation, confluent cultures were exposed to SP and 2.5 µCi/ml [³H]phenylalanine for 20 h. The cells were then processed for determination of acid-precipitable counts. Protein content per dish was measured by a modified Lowry assay (38).

Measurement of Net Collagen Production

Confluent cultures were serum deprived and exposed to SP for 24 h. Net collagen production (present in cell monolayer and medium) was determined by a hydroxyproline-based assay, as described earlier (34).

Measurement of Superoxide Production

Superoxide generation was measured in terms of nitroblue tetrazolium (NBT) reduction into formazan, as described by Siwik et al. (25) with some modifications. The cells were incubated for 4 h in Krebs-Ringer phosphate buffer, pH 7.4, containing 1.5 mmol/l NBT, with or without SP. After the assay, the cells were washed and color was extracted for spectrophotometric quantification at 490 nm.

Measurement of Ca²+ Influx

⁴⁵Ca uptake by cardiac fibroblasts was assayed as described earlier (7). Cells were preincubated for 10 min in 1.0 ml of HEPES-buffered physiological salt solution (HBPSS) composed of (in mmol/l) 145 NaCl, 5 KCl, 1 MgCl₂, 1.2 CaCl₂, 5 HEPES, and 10 glucose (pH 7.4 at 37°C). ⁴⁵Ca influx measurements were initiated by adding 5 µCi/ml ⁴⁵CaCl₂. After incubation of the cells for 0, 15, 30, and 60 min, 1.0 ml of ice-cold HBPSS containing 2 mmol/l lanthanum chloride was added and the medium was discarded within 10-15 s. The cells were washed three times in HBPSS containing 1.0 mmol/l lanthanum chloride for a total of 15 s and solubilized in 0.5 ml of 0.1 N NaOH. Radioactivity was measured by liquid scintillation spectroscopy. Kinetics of ⁴⁵Ca uptake in the presence of SP was determined by the inclusion of SP in the incubation buffer. The time 0 value was subtracted from other time points.

Statistical Analysis

Statistical evaluation of data was by one-way ANOVA, followed by a Bonferroni test. A value of P < 0.05 was considered significant. The difference in ⁴⁵Ca uptake between the control and SP-treated groups at each of the time points was evaluated by Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SP Exerts Mitogenic Action on Adult Cardiac Fibroblasts

As shown in Fig. 1, SP, at 10 µmol/l, increased incorporation of [³H]thymidine into DNA by ~46% (n = 20). Moreover, it was observed that even in the absence of insulin and transferrin in the culture medium, SP had a stimulatory effect of comparable magnitude on [³H]thymidine incorporation, indicating that SP exerts the effect even in the absence of other growth factors (data not shown). To ascertain whether the increase in DNA synthesis with SP reflects a proliferative response, cell count was performed after treatment with SP. Furthermore, the dose-response was studied to determine the concentration range over which SP exerts mitogenic effect on adult cardiac fibroblasts in vitro. As shown in Fig. 2, even a dose of 1 nmol/l SP produced a small but significant increase in cell number. The effect was more marked at higher concentrations of SP. Although not statistically significant, the effect with 10 µmol/l SP was higher than with 100 nmol/l SP and was therefore chosen for further experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Effect of substance P (SP) on [3H]thymidine incorporation into DNA. Serum-deprived, subconfluent cultures of fibroblasts were treated with SP at 10 µmol/l for 24 h. [3H]thymidine was included in the medium at 2.5 µCi/ml during the final 4 h of incubation with SP. To assess the effect of EGTA on SP-induced fibroplasia, cells were incubated with 1 mmol/l EGTA in presence of SP after pretreatment of the cells with EGTA for 15 min. Values were computed on a per-dish basis and are expressed as means ± SD of 20 separate determinations. a Significantly different from control; b significantly different from SP-treated cells.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of SP on cell number. Serum-deprived, subconfluent cultures of fibroblasts were treated with SP at the indicated concentrations for 24 h. Cell count was performed using a Neubauer counting chamber. Values are expressed as means ± SD. Control, n = 16. a Significantly different from control; b significantly different from 1 nmol/l SP; 10 µmol/l SP vs. 100 nmol/l SP, not significant.

SP Does Not Influence Total Protein Synthesis and Net Collagen Production

To determine whether SP has any effect on total protein synthesis, the incorporation of [³H]phenylalanine into proteins was measured in confluent cultures of adult cardiac fibroblasts. Figure 3 shows that SP had no effect on protein synthesis at 20 h, suggesting that SP does not exert a hypertrophic action on these cells. This was confirmed by a lack of effect of SP on cell protein content per dish.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effect of SP on total protein synthesis and protein content. After serum deprivation, confluent cultures of fibroblasts were exposed to SP at 10 µmol/l and [3H]phenylalanine at 2.5 µCi/ml for 20 h. Acid-precipitable radioactivity was measured. Protein content was determined by a modified Lowry assay. Values were computed on per dish basis and expressed using arbitrary units as means ± SD of 12 separate determinations.

Because cardiac fibroblasts are the main source of collagens, the effect of SP on net collagen production (collagen deposition) was determined. The cell monolayer and medium were pooled and used for determination of collagen-associated hydroxyproline content. Results presented in Fig. 4 show that treatment of adult cardiac fibroblasts with SP did not alter net collagen production by these cells (n = 6).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effect of SP on net collagen production. Confluent cultures were serum deprived for 24 h and then exposed to SP at 10 µmol/l for 24 h. Net collagen production was measured in terms of hydroxyproline content of the monolayer and the medium. Values are means ± SD for 6 separate measurements.

SP Enhances Superoxide Production in Cardiac Fibroblasts

Consistent with its postulated role in tissue injury and repair, SP has been shown to promote superoxide production in certain cell types (28, 29). Therefore, we designed experiments to ascertain whether SP increases endogenous superoxide production in cardiac fibroblasts and to examine whether superoxide mediates the mitogenic action of SP on cardiac fibroblasts. SP was found to enhance superoxide production consistently. In confluent cultures (Fig. 5A), after exposure to SP for 4 h, the increase was found to be ~18% (n = 20). However, in subconfluent cultures (Fig. 5B), treatment with SP for 24 h resulted in a 35% (about twofold higher; Fig. 5A) increase in superoxide generation (n = 4). The observed effect of superoxide dismutase (SOD) was consistent with the stimulatory action of SP on superoxide formation (Fig. 5B). The stimulatory effect remained unaffected when cells were exposed to SP under Ca²⁺-free conditions (Fig. 5A).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of SP on superoxide production. A: confluent cultures of cardiac fibroblasts were exposed to SP at 10 µmol/l (n = 20) in Krebs-Ringer phosphate buffer spantide (KRPBS) for 4 h in presence of nitroblue tetrazolium (NBT). To assess the effect of spantide (n = 20) and EGTA (n = 20), cells were incubated with spantide at 10 µmol/l and EGTA at 1 mmol/l in presence of SP after pretreatment of the cells with spantide-EGTA for 15 min. Ca2+-free incubation was in KRPBS without Ca2+ (n = 4). Values are means ± SD. To determine significant differences between each of these treatments, statistical comparisons by one-way ANOVA, followed by Bonferroni test, were made among three groups at a time: control, SP treated, and SP treated in presence of spantide, EGTA, or Ca2+-free medium. Control, n = 20. B: subconfluent cultures were serum-deprived for 24 h and then exposed to SP at 10 µmol/l (n = 4) with or without superoxide dismutase (SOD) at 200 U/ml (n = 4) for 24 h. During the last 4 h, the cells were transferred to KRPBS containing NBT to measure superoxide generation. Control, n = 4. a Significantly different from control; b significantly different from SP-treated cells.

Superoxide Anion Mediates Mitogenic Action of SP on Cardiac Fibroblasts

Although SP increases superoxide generation in certain cell types, such an effect has not been linked to a mitogenic response. In our study, the antioxidant N-acetyl-cysteine abolished the increase in cell numbers in response to SP, suggesting that SP-induced cellular hyperplasia may be mediated by a redox-sensitive mechanism (Fig. 6). The inhibitory effect of SOD on SP-induced mitogenesis (Fig. 6) further confirmed the role of superoxide in the hyperplastic effect.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effect of spantide, SOD, and N-acetyl-cysteine (NAC) on SP-induced hyperplasia. To assess the effect of spantide on SP-induced fibroplasia, subconfluent serum-deprived cells were incubated with 10 µmol/l spantide in presence of SP for 24 h after pretreatment of the cells with spantide for 15 min (n = 8). For the effect of SOD and N-acetyl-cysteine, subconfluent serum-deprived cultures were treated with 10 µmol/l SP in presence of SOD at 200 U/ml (n = 4) or 5 mmol/l N-acetyl-cysteine (n = 8) for 24 h. Values are means ± SD. To determine significant differences between each of these treatments, statistical comparisons by one-way ANOVA, followed by Bonferroni test, were made among three groups at a time: control, SP treated, and SP treated in presence of any of these agents. Control, n = 16; SP treated, n = 16. a Significantly different from control; b significantly different from SP-treated cells.

SP Enhances Ca²+ Uptake by Cardiac Fibroblasts

Figure 7 shows the kinetics of ⁴⁵Ca uptake by cardiac fibroblasts. SP enhanced ⁴⁵Ca influx into cardiac fibroblasts by 21% at 30 min (n = 8) and 170% at 60 min (n = 8). In the untreated cells, there was a linear increase in ⁴⁵Ca uptake for 30 min, followed by a dip, whereas in the SP-treated cells, the dip was not observed so that the difference between control and SP-treated cells was much higher at 60 min.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   Effect of SP on 45Ca uptake. Fibroblasts were exposed to SP at 10 µmol/l for different periods of time as shown. 45Ca uptake was measured in HEPES-buffered physiological salt solution after the addition of 45Ca at 5 µCi/ml (see MATERIALS AND METHODS). Values are expressed as means ± SD of 8 separate measurements. Difference in 45Ca uptake between the control and SP-treated groups at each time point was evaluated by Student's t-test. cpm, Counts per minute. * P < 0.001, control vs. SP.

Ca²+ is Involved in Mitogenic Action of SP on Cardiac Fibroblasts

SP is reported to stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate with resultant changes in Ca²⁺ homeostasis in some cell types (14). We hypothesized that Ca²⁺ may be involved in the expression of SP effects on cardiac fibroblasts. When extracellular Ca²⁺ was blocked by EGTA, the SP-induced increase in [³H]thymidine incorporation was abolished (Fig. 1). EGTA per se did not decrease cell number significantly (data not shown). This, in conjunction with the increase in ⁴⁵Ca uptake in response to SP (Fig. 7), suggested a role for Ca²⁺ influx in mediating the mitogenic action of SP.

SP Acts via NK-1 Receptors on Cardiac Fibroblasts

Spantide, an NK-1 receptor antagonist, attenuated the increase in cell number in response to SP, showing that SP acts via NK-1 receptors on cardiac fibroblasts (Fig. 6). Spantide was also found to inhibit the stimulatory effect of SP on superoxide generation (Fig. 5A), confirming the involvement of NK-1 receptors in mediating this response.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SP, a member of the tachykinin family of neuropeptides, is present in capsaicin-sensitive unmyelinated primary afferent nerve fibers in peripheral organs and envelopes the vasculature, with the highest density of SP-containing network in the aorta and vena cavae close to the heart (5). It is also localized in the adventitia and the medial-adventitial border of the vessel wall and is synthesized and released from arterial endothelial cells (13, 10). In the heart, SP is released from sensory nerve endings in response to ischemia (13, 32). Light-microscopic autoradiograms reveal that the SP binding sites occur within clusters of connective tissue cells (35), suggesting that these cells may be susceptible to SP activation. Therefore, it is possible that SP released from neurons in connection with tissue injury might, besides exerting vasodilatory effects, stimulate surrounding connective tissue cells to proliferate, and initiate a healing response in the heart. To test such a possibility, the present study examined whether SP exerts direct mitogenic action on cardiac fibroblasts. Although an in vitro model has obvious limitations, the use of isolated cultured cardiac fibroblasts permitted evaluation of the ability of SP to exert direct effects on cardiac fibroblasts in the absence of any complicating systemic effects such as increase in cardiac workload, inotropic mechanisms, or tissue metabolism.

The findings of the present study showed that SP elicits a hyperplastic response in vitro in adult cardiac fibroblasts, as evidenced by an increase in the incorporation of thymidine into DNA and cell number. Whereas the mitogenic action of SP on other cell types, including dermal fibroblasts (15), is mediated by the NK-1 receptor, the effect of spantide in the present study (Fig. 6) is the first demonstration of NK-1 receptor-mediated mitogenesis in cardiac fibroblasts. The magnitude of the mitogenic effect of SP was comparable to that reported with skin fibroblasts, in which ~18 and 38% of [³H]thymidine-labeled nuclei were observed in response to SP at 1 and 10 µmol/l, respectively (15). The hyperplastic effect of SP on cardiac fibroblasts, although modest, was very consistent and was validated by different observations, such as increase in cell number and thymidine incorporation, dose response, and the abolition or attenuation of its action by the receptor antagonist, EGTA, N-acetyl-cysteine, and SOD.

SP did not have any effect on total protein synthesis or protein content per dish in confluent cultures (Fig. 3). SP may thus elicit a hyperplastic but not a hypertrophic response in adult cardiac fibroblasts. The neuropeptide did not influence net collagen production (Fig. 4). The lack of an effect of SP on net collagen production may reflect an incomplete response of the cells under in vitro conditions. In vivo, other factors may act on fibroblasts, after activation by SP, to increase collagen production. Consistent with our finding, SP was shown to enhance proliferation of pulp cells without an increase in the production of matrix proteins, and it was suggested that it might enhance proliferative activity without influencing functional activity (30).

A concentration of SP, higher than its plasma concentration, was required to elicit a proliferative response in cardiac fibroblasts. The dose response was reported to display a bell-shaped distribution for DNA synthesis in endothelial cells, requiring an SP concentration of 10-100 µmol/l for a growth-stimulating response (33). In rheumatoid synoviocytes, however, SP induced mitogenesis at 10-1 µmol/l (12). Thus the cell type in question and the experimental conditions employed may be determinants of the effective concentration of SP. In the present study, SP produced a small but significant stimulatory effect on cell number at a concentration as low as 1 nmol/l but the effect was more pronounced at 100 and 10 µmol/l (Fig. 2). It may be noted that the cells were exposed to SP in the absence of other serum-derived growth factors. In vivo, a lower concentration of SP may act synergistically with other growth factors to exert more pronounced effects. It is also possible that fibroblasts are exposed to higher local concentrations of SP in vivo, especially under pathological conditions.

Mechanism of Action of SP

Ca²+ and superoxide are both involved in mitogenic action of SP on cardiac fibroblasts. SP has been shown to modulate Ca²⁺ homeostasis in many cell types (20, 28, 29). For example, in Chinese hamster ovary (CHO) cells expressing the SP receptor clone (CHO-SPR), SP induces Ca²⁺ entry through activation of cation channels. Furthermore, it has been suggested that D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] may regulate both Ca²⁺ entry and Ca²⁺ mobilization from intracellular stores in CHO-SPR cells (14). In neutrophils, SP stimulates the hydrolysis of PIP₂ into diacylglycerol and Ins(1,4,5)P₃ with a rise in intracellular Ca²⁺ (28). SP and related tachykinins have been shown to evoke Ca²⁺ signaling in cultured myenteric neurons by the influx of extracellular Ca²⁺ through L- and N-type Ca²⁺ channels. These signals are abolished on removal of extracellular Ca²⁺ or by the addition of Ca²⁺ channel blockers, lanthanum chloride, and nickel chloride (20).

In contrast, very little is known about regulation of Ca²⁺ homeostasis in fibroblasts and the role of Ca²⁺ in mediating the effects of growth factors on fibroblasts. The results of this study show that SP has a stimulatory effect on ⁴⁵Ca uptake by the cells (Fig. 7). To our knowledge, this is the first demonstration of agonist-induced enhancement of ⁴⁵Ca uptake by cardiac fibroblasts. Furthermore, it was found that chelation of extracellular Ca²⁺ using EGTA completely abolishes the stimulation of fibroblast proliferation by SP (Fig. 1), suggesting that the mitogenic effect of SP is dependent on Ca²⁺ influx from the extracellular space. Thus our data show that changes in Ca²⁺ homeostasis may play a role in the stimulation of cardiac fibroblast proliferation by SP.

SP has been shown to stimulate superoxide production in synovial cells (29) and human neutrophils (28). Tanabe et al. (29) have reported that NK-1 receptor/phospholipase C-linked diacylgycerol formation and activation of protein kinase C is the signal transduction pathway for SP-stimulated oxyradical production in synovial cells. Liberation of free radicals in gastrointestinal tissue as part of an inflammatory reaction in response to tachykinins has also been demonstrated (11). In the present study, SP consistently increased superoxide production in cardiac fibroblasts (Fig. 5). Exposure of subconfluent cultures to SP for 24 h elicited a more pronounced (about twofold higher) effect than exposure of confluent cultures to SP for 4 h (Fig. 5, A and B). The effect of SOD (Fig. 5B) confirmed the stimulatory action of SP on superoxide generation. Probing a role for extracellular Ca²⁺ in the enhancement of superoxide production by SP, fibroblasts were exposed to SP under Ca²⁺-free conditions. It was observed that whereas the effect of SP on superoxide production remained unaffected in a Ca²⁺-free medium, a combination of SP and EGTA had a more pronounced effect on superoxide generation than SP alone (Fig. 5A). However, it was subsequently found that EGTA and Ca²⁺-free incubation per se enhanced superoxide production in adult cardiac fibroblasts (22). Thus it is not clear from these experiments whether the effect of SP on superoxide production is dependent on extracellular Ca²⁺. It is intriguing that both SP (Fig. 5) and EGTA (22) stimulate superoxide generation, although the former enhances Ca²⁺ uptake and the latter chelates extracellular Ca²⁺. The mechanisms underlying the effects of SP and extracellular Ca²⁺ on superoxide production may be different and need to be addressed.

The inhibitory effect of N-acetyl-cysteine and SOD on the mitogenic action of SP showed that SP-induced fibroplasia is mediated, at least in part, by reactive oxygen species (ROS). This is the first demonstration of the involvement of ROS in mediating the mitogenic action of SP and is in agreement with the current thinking that ROS exert critical signaling functions to regulate cellular growth in response to growth factors (9, 6). Platelet-derived growth factor (26) and thrombin (17) have been shown to stimulate ROS production in smooth muscle cells and suppression of ROS inhibited platelet-derived growth factor- and thrombin-induced mitogenesis in these cells. Furthermore, angiotensin II elicits a hypertrophic response in smooth muscle cells via the production of both superoxide and H₂O₂ (40, 31).

It has been reported that free-radical generation and alterations in Ca²⁺ homeostasis may be related in some cell types. Oxidants have been shown to stimulate Ca²⁺ signaling by increasing cytosolic Ca²⁺ concentration (27), suggesting a physiological role of ROS and oxidative stress in the regulation of Ca-induced signaling. Increase in intracellular Ca²⁺ was detected in response to H₂O₂ treatment of vascular smooth muscle cells (19) and, in endothelial cells treated with H₂O₂ (2), there was a transient release of Ca²⁺ from intracellular stores. Furthermore, a link between SP-induced superoxide production and Ca²⁺ flux has been suggested. Tanabe et al. (28) have reported that NK-1 receptor/G protein-coupled D-myo-inositol 3-phosphate formation with resulting Ins(1,4,5)P₃-induced transient increase in intracellular Ca²⁺ concentration is the main signal transduction pathway for SP-stimulated O production in human neutrophils, although in synovial cells superoxide production was not found to be linked to Ins(1,4,5)P₃-induced Ca²⁺ mobilization (29). In the present study, whereas the mitogenic action of SP was dependent on superoxide production (Fig. 6), a combination of SP and EGTA enhanced superoxide generation (Fig. 5A) but without an increase in [³H]thymidine incorporation (Fig. 1). The observations show that an increase in superoxide production in response to SP does not result in increased cell proliferation when extracellular Ca²⁺ is chelated. It appears, therefore, that SP-induced fibroplasia may depend on both Ca²⁺- and superoxide-sensitive pathways and that blocking either of these would abolish the proliferative response. Thus, whereas the literature points to the link between ROS and Ca²⁺ homeostasis in certain cell types, our results suggest for the first time that SP-triggered Ca²⁺ and oxidant signaling pathways may, in addition, be functionally coupled to a hyperplastic response in cardiac fibroblasts.

Model of SP-induced cardiac fibroblast proliferation. SP is reported to exert its mitogenic action via its G protein-coupled NK-1 receptor and activation of extracellular signal-regulated kinases (ERKs) (1). The SP antagonist (D-Arg1, D-Trp5,7,9, and Leu11) inhibits both G protein-coupled receptor-mediated signal transduction and cellular DNA synthesis in Swiss 3T3 cells and the activation of ERK-1 and ERK-2, induced by G protein-coupled receptor agonists (24). Moreover, activation of ERK-1 and ERK-2 is required for DNA synthesis in cardiac fibroblasts (16). ERKs represent an important intracellular target of an altered redox environment and activation of ERKs by ROS has been implicated in the growth response of smooth muscle cells stimulated with growth factors (6). A model of SP-induced mitogenesis in cardiac fibroblasts, mediated by Ca²⁺- and redox-sensitive pathways and activation of ERKs, is consistent with our data and recent literature and may provide a basis for future investigations.

In summary, a fibroproliferative response in the heart is important for recovery from injury, but factors that regulate fibroblast proliferation in the heart remain largely unidentified. Despite the exclusive reliance on an in vitro model, the present study suggests that SP may be an "activator" of a mitogenic response in cardiac fibroblasts and proposes that SP-induced alterations in redox state and Ca²⁺ homeostasis may act in concert to mediate its mitogenic action. The idea of a neural pathway of activation of cardiac fibroblasts is attractive and needs to be explored because it may be particularly relevant in conditions like myocardial ischemia that stimulate cardiac sensory nerve endings and promote increased local release of SP and other neuropeptides. Future investigations should delineate the Ca²⁺ and oxidant signaling pathways, and any cross-talk between them, that seem to mediate the mitogenic action of SP on cardiac fibroblasts.

	ACKNOWLEDGEMENTS

The authors thank Dr. P. Sankara Sarma of Achutha Menon Center for Health Science Studies, Sree Chitra Tirunal Institute for Medical Sciences and Technology, for assistance in statistical analyses of data.

	FOOTNOTES

This work was supported by a research grant to K. Shivakumar from the Department of Science and Technology, Government of India. C. Kumaran acknowledges the Department of Science and Technology for a junior research fellowship and the Council of Scientific and Industrial Research, Government of India, for a senior research fellowship.

Address for reprint requests and other correspondence: K. Shivakumar, Div. of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India 695 011 (E-mail: shivak{at}sctimst.ker.nic.in).

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.

10.1152/ajpheart.00747.2001

Received 20 August 2001; accepted in final form 4 December 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.   Castagliuolo, I, Valenick L, Liu J, and Pothoulakis C. Epidermal growth factor receptor transactivation mediates substance P-induced mitogenic responses in U-373 MG cells. J Biol Chem 275: 26545-26550, 2000[Abstract/Free Full Text].

2.   Doan, TN, Gentry DL, Taylor AA, and Elliott SJ. Hydrogen peroxide activates agonist-sensitive Ca²⁺-flux pathways in canine venous endothelial cells. Biochem J 297: 209-215, 1994.

3.   Dostal, DE, and Baker KM. The cardiac renin-angiotensin system: conceptual, or a regulator of cardiac function? Circ Res 85: 643-650, 1999[Abstract/Free Full Text].

4.   Eghbali, M, Tomek R, Woods C, and Bhambi B. Cardiac fibroblasts are predisposed to convert into myocyte phenotype: specific effect of transforming growth factor . Proc Natl Acad Sci USA 88: 795-799, 1991[Abstract/Free Full Text].

5.   Furness, J, Papka RE, Della NG, Costa M, and Eskay RL. Substance P-like immunoreactivity in nerves associated with the vascular system of guinea-pigs. Neuroscience 7: 447-459, 1982[Web of Science][Medline].

6.   Irani, K. Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apop totic signaling. Circ Res 87: 179-183, 2000[Abstract/Free Full Text].

7.   Jiang, H, St. Ulme D, Dickens G, Chabuk A, Lavarreda M, Lazarovili P, and Guroff G. Both p140^trk and p75^NGFR nerve growth factor receptors mediate nerve growth factor-stimulated calcium uptake. J Biol Chem 272: 6835-6837, 1997[Abstract/Free Full Text].

8.   Katayama, I, and Nishioka K. Substance P augments fibrogenic cytokine-induced fibroblast proliferation: possible involvement of neuropeptide in tissue fibrosis. J Dermatol Sci 15: 201-206, 1997[Web of Science][Medline].

9.   Kunsch, C, and Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res 85: 753-766, 1999[Abstract/Free Full Text].

10.   Linnik, M, and Moskowitz M. Identification of immunoreactive substance P in human and other mammalian endothelial cells. Peptides 10: 957-962, 1989[Web of Science][Medline].

11.   Lordal, M, Soder O, and Hellstrom PM. Tachykinins stimulate lipid peroxidation mediated by free radicals in gastrointestinal tract of rat. Dig Dis Sci 42: 1524-1529, 1997[Web of Science][Medline].

12.   Lotz, M, Carson DA, and Vaughan JH. Substance P activation of rheumatoid synoviocytes: neural pathway in pathogenesis of arthritis. Science 235: 893-895, 1987[Abstract/Free Full Text].

13.   Milner, P, Ralevic V, Hopwood A, Feher E, Lincoln J, Kirkpatrick K, and Burnstock G. Ultrastructural localization of substance P and choline acetyltransferase in endothelial cells of rat coronary artery and release of substance P and acetylcholine during hypoxia. Experientia 45: 121-125, 1989[Web of Science][Medline].

14.   Mochizuki-Oda, N, Nakajima Y, Nakanishi S, and Ito S. Characterization of the substance P receptor-mediated calcium influx in cDNA transfected Chinese hamster ovary cells. A possible role of inositol 1,4,5-trisphosphate in calcium influx. J Biol Chem 269: 9651-9658, 1994[Abstract/Free Full Text].

15.   Nilsson, J, von Euler A, and Dalsgaard C. Stimulation of connective tissue growth by substance P and substance K. Nature 315: 61-63, 1985[Medline].

16.   Pages, G, Lenormand P, L'allemain G, Chambard J-C, Meloche S, and Pouyssegur J. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA 90: 8319-8323, 1993[Abstract/Free Full Text].

17.   Patterson, C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C, and Runge MS. Stimulation of vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47 (phox) may participate in forming this oxidase in vitro and in vivo. J Biol Chem 274: 19814-19822, 1999[Abstract/Free Full Text].

18.   Payan, D, Brewster D, and Goetzl E. Specific stimulation of human T lymphocytes by substance P. J Immunol 131: 1613-1615, 1983[Abstract].

19.   Roveri, A, Coassin M, Maiorino M, Zamburlini A, van Amsterdam FT, Ratti E, and Ursini F. Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells. Arch Biochem Biophys 297: 265-270, 1992[Web of Science][Medline].

20.   Sarosi, GAJ, Kimball BC, Barnhart DC, Zhang W, and Mulholland MW. Tachykinin neuropeptide-evoked intracellular calcium transients in cultured guinea pig myenteric neurons. Peptides 19: 75-84, 1998[Web of Science][Medline].

21.   Schorb, W, Booz GW, Dostal DE, Conrad KM, Chang KC, and Baker KM. Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ Res 72: 1245-1254, 1993[Abstract/Free Full Text].

22.   Shivakumar, K, and Kumaran C. L-type calcium channel blockers and EGTA enhance superoxide production in cardiac fibroblasts. J Mol Cell Cardiol 33: 373-377, 2001[Web of Science][Medline].

23.   Shivakumar, K, Renuka Nair R, and Valiathan MS. Paradoxical effect of cerium on collagen synthesis in cardiac fibroblasts. J Mol Cell Cardiol 24: 775-780, 1992[Web of Science][Medline].

24.   Sinnett-Smith, J, Santiskulvong C, Duque J, and Rozengurt E. [D-Arg1, D-Trp5,7,9, Leu11] substance P inhibits bombesin-induced mitogenic signal transduction mediated by both Gq and G12 in Swiss 3T3 cells. J Biol Chem 275: 30644-30652, 2000[Abstract/Free Full Text].

25.   Siwik, DA, Tzortzis JD, Pimental DR, Chang-F, Pagano PJ, Singh K, Sawyer DB, and Colucci WS. Inhibition of copper-zinc superoxide dismutase induces cell growth, hypertrophic phenotype, and apop to sis in neonatal rat cardiac myocytes in vitro. Circ Res 85: 147-153, 1999[Abstract/Free Full Text].

26.   Sundaresan, M, Yu ZX, Ferrans VJ, Irani K, and Finkel T. Requirement for generation of H₂O₂ for platelet-derived growth factor signal transduction. Science 270: 296-299, 1995[Abstract/Free Full Text].

27.   Suzuki, YJ, Forman HJ, and Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 22: 269-285, 1997[Web of Science][Medline].

28.   Tanabe, T, Otani H, Bao L, Mikami Y, Yasukura T, Ninomiya T, Ogawa R, and Inagaki C. Intracellular signaling pathway of substance P-induced superoxide production in human neutrophils. Eur J Pharmacol 299: 187-195, 1996[Web of Science][Medline].

29.   Tanabe, T, Otani H, Mishima K, Ogawa R, and Inagaki C. Mechanisms of oxyradical production in substance P-stimulated rheumatoid synovial cells. Rheumatol Int 16: 159-167, 1996[Web of Science][Medline].

30.   Trantor, IR, Messer HH, and Birner R. The effects of neuropeptides (calcitonin gene-related peptide and substance P) on cultured human pulp cells. J Dent Res 74: 1066-1071, 1995[Abstract/Free Full Text].

31.   Ushio-Fukai, M, Alexander RW, Akers M, and Griendling KK. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem 273: 15022-15029, 1998[Abstract/Free Full Text].

32.   Ustinova, EE, Bergren D, and Schultz HD. Neuropeptide depletion impairs postischemic recovery of the isolated rat heart: role of substance P. Cardiovasc Res 30: 55-63, 1995[Web of Science][Medline].

33.   Villablanca, AC, Murphy CJ, and Reid TW. Growth-promoting effects of substance P on endothelial cells in vitro: synergism with calcitonin gene-related peptide, insulin, and plasma factors. Circ Res 75: 1113-1120, 1994[Abstract/Free Full Text].

34.   Villarreal, FJ, Kim NN, Ungab GD, Printz MP, and Dillmann WH. Identification of functional angiotensin II receptors on rat cardiac fibroblasts. Circulation 88: 2849-2861, 1993[Abstract/Free Full Text].

35.   Walsh, RJ, Weglicki WB, and Corres-de-Araujo R. Distribution of specific substance P binding sites in the heart and adjacent great vessels of the Wistar white rat. Cell Tissue Res 284: 495-500, 1996[Web of Science][Medline].

36.   Weber, KT, Sun Y, Katwa LC, and Cleutjens JPM Connective tissue: a metabolic entity. J Mol Cell Cardiol 27: 107-120, 1995[Web of Science][Medline].

37.   Weihe, E, Reinecke M, Opherk D, and Forssmann WG. Peptidergic innervation (substance P) in the human heart. J Mol Cell Cardiol 13: 331-333, 1981[Web of Science][Medline].

38.   Winterbourne, DJ. Chemical assays for protein. In: Methods in Molecular Biology, edited by Graham J, and Higgins J.. Totowa, NJ: Humana, 1993, vol. 19, p. 197-202.

39.   Yatani, A, Yokoyama M, Akita H, and Fukuzaki H. Endothelium-dependent vasodilating effect of substance P during flow-reducing coronary stenosis in the dog. J Am Coll Cardiol 15: 1374-1384, 1990[Abstract].

40.   Zafari, AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, and Griendling KK. Role of NADH/NADPH oxidase-derived H₂O₂ in angiotensin II-induced vascular hypertrophy. Hypertension 32: 488-495, 1998[Abstract/Free Full Text].