The stretching of smooth muscle tissue modulates contraction through augmentation of Ca2+ transients, but the mechanism underlying stretch-induced Ca2+ transients is still unknown. We found that mechanical stretching and maintenance of mouse urinary bladder smooth muscle strips and single myocytes at 30% and 18% beyond the initial length, respectively, resulted in Ca2+ oscillations. Experiments indicated that mechanical stretching remarkably increased the production of nitric oxide (NO) as well as the amplitude and duration of muscle contraction. Stretch-induced Ca2+ oscillations and contractility increases were completely abolished by the NO inhibitor L-NAME or eNOS (also known as NOS3) gene inactivation. Moreover, exposure of eNOS-knockout myocytes to exogenous NO donor induced Ca2+ oscillations. The stretch-induced Ca2+ oscillations were greatly inhibited by the selective inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor xestospongin C and partially inhibited by ryanodine. Moreover, the stretch-induced Ca2+ oscillations were also suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, but not by the soluble guanylyl cyclase (sGC) inhibitor ODQ. These results suggest that stretching myocyte and maintenance at a certain length results in Ca2+ oscillations that are NO dependent , and sGC and cGMP independent, and results from the activation of PI3K in smooth muscle.

Hollow smooth muscle organs and even non-smooth muscle organs undergo substantial changes in wall tension and attendant changes in muscle tone associated with filling and distension. Our previous studies have indicated that rapid increases in cell length, by triggering the gating of ryanodine receptors (RYRs), results in Ca2+ release from sarcoplasmic reticulum (SR) in the form of sparks or propagated Ca2+ waves. (Ji et al., 2002; Wei et al., 2008). Evidence indicates that stretch induces tissue nitric oxide (NO) production in cardiac muscle (Petroff et al., 2001; Prosser and Ward, 2014; Umar et al., 2009; van der Wees, 2006), which might act to regulate force production through altering Ca2+ release by SR RYRs (Jian et al., 2014; Kakizawa et al., 2013; Petroff et al., 2001; Sun et al., 2001; Xu et al., 2012). NO, a cellular second messenger, can mediate numerous biological functions, such as anti-apoptosis activities (Hruby et al., 2008), heart rate, heart development (Liu and Feng, 2012; Ramchandra et al., 2014), vasodilation and muscle contractility (Denniff et al., 2014; Smiljic et al., 2014). There are three known isoforms of nitric oxide synthase (NOS), with eNOS (also known as NOS3) accounting for most of the NO production in smooth muscle (Grider and Murthy, 2008; Han et al., 2013; Yanai et al., 2008). Previous research has indicated that the pathway through which NO regulates muscle contraction is controversial. Some reports suggest that NO regulates muscle contraction through the soluble guanylyl cyclase (sGC)–cGMP– protein kinase G (PKG) pathway (Yu et al., 2005), whereas other reports suggest that NO regulates muscle contraction independently of the sGC–cGMP–PKG pathway (Petroff et al., 2001; Yanai et al., 2008).

Ca2+ oscillations play a fundamental role in various cell signaling processes, and Ca2+-dependent molecules are regulated by the amplitude and frequency of these Ca2+ oscillations (Dolmetsch et al., 1998; Smedler and Uhlen, 2014). Studies have indicated that inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the generation of Ca2+ oscillations in many cell types (Abou-Saleh et al., 2013; Berridge, 2007; Martin-Cano et al., 2009; Tamarina et al., 2005; Tamashiro and Yoshino, 2014), although this idea has been challenged by some authors (Johnston et al., 2005; Kannan et al., 1997; Nakayama et al., 2005). Moreover, some authors have recently claimed that Ca2+ oscillations occur through the activation of both IP3Rs and RYRs in PC12 and interstitial cells of the rabbit urethra (Johnston et al., 2005; Koizumi et al., 1999).

In the present study, we demonstrate for the first time that stretching mouse bladder smooth muscle and maintaining it at certain lengths induces Ca2+ oscillations, and that these stretch-induced Ca2+ oscillations are mediated by NO production. NO production is both necessary and sufficient to trigger Ca2+ oscillations, as stretch-induced Ca2+ oscillations were abrogated by NOS inhibition or eNOS gene inactivation, and the stretch-induced Ca2+ release was restored by exogenous NO in eNOS-null cells. These results indicate that stretch-induced Ca2+ oscillation is dependent on NO in smooth muscle.

Mechanic stretch induces Ca2+ oscillations in smooth muscle

Our previous studies have indicated that rapid stretching causes Ca2+ sparks or propagating Ca2+ waves in both mouse and rabbit bladder myocytes (Ji et al., 2002; Wei et al., 2008). Here, we first examined the effect of a maintained mechanical stretching of mouse bladder tissue strips on Ca2+ release in smooth muscle. The tissue strips were incubated with Fluo-4 AM (10 µM) plus 0.02% pluronic acid for 50–60 min at room temperature (23°C) and transferred into an optical recording chamber fixed with a Kevlar retaining clip (Warner Instruments). The strips were elongated and maintained at 30% beyond their initial length. As shown in Fig. 1, in control experiments (without stretch), there were only sporadic and small Ca2+ transients. In contrast to the control, a maintained stretch resulted in a number of repeated Ca2+ release events in some cells (Fig. 1B). The frequency of the stretch-induced Ca2+ oscillations were significantly higher (7.86±1.48 per 10 min; means±s.e.m.) in stretched tissue compared with the control (1.34±0.86 per 10 min) (Fig. 1C). Analysis of the amplitude (measured as F/F0) of Ca2+ oscillations indicated that the amplitude of stretch-induced Ca2+ oscillations were also significantly higher than that of the control (1.76±0.31 versus 0.52±0.22, respectively, Fig. 1D, P<0.01). These results suggest that maintained stretching of the tissues to a certain length induces significant Ca2+ oscillations in smooth muscle.

Fig. 1.

Stretch-induced Ca2+ oscillations in mouse bladder tissue segments. Stretching of Fluo-4-AM-loaded mouse bladder tissue strips to 30% over initial length leads to Ca2+ oscillations. (A) A control (without stretch) confocal microscopy x-y image indicates that there is no fluorescence change. The lower panels are profiles taken from above image as indicated by number. (B) Tissue strip stretched and maintained at 30% over their initial their length results in multiple cells producing discrete Fluo-4 fluorescence transients (upper images). The lower panels show the profiles taken as indicated from the image. (C) Number of Ca2+ transients per 10 min and (D) fluorescence intensity for experiments such as those shown in A and B. Results are mean±s.e.m. **P<0.01 compared with control (Student's t-test).

Fig. 1.

Stretch-induced Ca2+ oscillations in mouse bladder tissue segments. Stretching of Fluo-4-AM-loaded mouse bladder tissue strips to 30% over initial length leads to Ca2+ oscillations. (A) A control (without stretch) confocal microscopy x-y image indicates that there is no fluorescence change. The lower panels are profiles taken from above image as indicated by number. (B) Tissue strip stretched and maintained at 30% over their initial their length results in multiple cells producing discrete Fluo-4 fluorescence transients (upper images). The lower panels show the profiles taken as indicated from the image. (C) Number of Ca2+ transients per 10 min and (D) fluorescence intensity for experiments such as those shown in A and B. Results are mean±s.e.m. **P<0.01 compared with control (Student's t-test).

Nitric oxide mediates stretch-induced Ca2+ oscillations in urinary bladder smooth muscle

NO synthesis has previously been implicated in stretch-induced Ca2+ release in cardiac (Petroff et al., 2001) and smooth (Wei et al., 2008) muscle. In the present study, we demonstrated, as described above, that stretching smooth myocytes causes Ca2+ oscillations, which were not only dependent on the stretch length but also on the time over which the stretch was maintained. Thus, we hypothesized that the stretch-induced Ca2+ oscillations might be related to the production of NO. Therefore, a series of experiments were performed to examine the effects of a NOS inhibitor and an NO donor on Ca2+ oscillations induced by stretching in mouse bladder smooth myocytes. We first tested the effect of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), on isolated mouse bladder smooth myocytes. Cells were incubated with Fluo-4 AM at room temperature and were imaged with an Olympus inverted confocal microscope (IX81; Olympus). As shown in Fig. 2 and Fig. S1, the stretch-induced Ca2+ oscillations were completely abolished by application of L-NAME (1 mM) or eNOS gene inactivation. The abolishment of the Ca2+ oscillations by the application of NOS inhibitor and eNOS gene inactivation suggests that NO plays a role in stretch-induced Ca2+ oscillations in smooth muscle. To further test the effect of NO on stretch-induced Ca2+ oscillations in mouse bladder smooth myocytes, the spontaneous NO-generating compound S-nitroso-N-acetylpenicillamine (SNAP) was used. As shown in Fig. 2B and Fig. S1B, application of SNAP (10 µM) could enhance or restore the effect of the stretch-induced Ca2+ oscillations. The amplitude of the oscillations was increased by 35.3±0.25% (means±s.e.m.) compared to the amplitude in the positive control (stretched, but in the absence of SNAP). However, the frequency of the Ca2+ oscillations was not significantly altered by the application of SNAP (Fig. 2B,D). Thus, our results indicate that NO is involved in the stretch-induced Ca2+ oscillations in smooth muscle. Next, we examined the production of NO by stretching smooth muscle.

Fig. 2.

Effect of NOS inhibitor and NO stimulator on stretch-induced Ca2+ oscillations in smooth muscle. (A) Application of L-NAME, a NOS inhibitor, completely blocks the stretch-induced Ca2+ oscillations in smooth muscle strips. (B) In the presence of SNAP, an NO stimulator, the Ca2+ oscillations of smooth muscle strips are enhanced. (C) Summary of fluorescence intensity of the oscillations: the peak Ca2+ oscillations that were increased by elongation of the mouse bladder tissue segments were significantly inhibited by L-NAME (n=7) and enhanced by SNAP (n=5). (D) Frequency of Ca2+ oscillations (number of oscillations per 10 min): note that L-NAME, but not SNAP altered, the frequency of the Ca2+ oscillations induced by stretch. Results are mean±s.e.m. *P<0.05, **P<0.01 as indicated (ANOVA).

Fig. 2.

Effect of NOS inhibitor and NO stimulator on stretch-induced Ca2+ oscillations in smooth muscle. (A) Application of L-NAME, a NOS inhibitor, completely blocks the stretch-induced Ca2+ oscillations in smooth muscle strips. (B) In the presence of SNAP, an NO stimulator, the Ca2+ oscillations of smooth muscle strips are enhanced. (C) Summary of fluorescence intensity of the oscillations: the peak Ca2+ oscillations that were increased by elongation of the mouse bladder tissue segments were significantly inhibited by L-NAME (n=7) and enhanced by SNAP (n=5). (D) Frequency of Ca2+ oscillations (number of oscillations per 10 min): note that L-NAME, but not SNAP altered, the frequency of the Ca2+ oscillations induced by stretch. Results are mean±s.e.m. *P<0.05, **P<0.01 as indicated (ANOVA).

Direct detection of NO production by stretching smooth myocytes

Electron spin resonance (ESR) spin-trapping-based detection was used to measure the NO production in bladder tissue undergoing stretch as previously described (Dikalov and Fink, 2005; Wei et al., 2008). As shown in Fig. 3, NO production was significantly increased when the strips were elongated to 30% beyond their initial length, which was measured by trapping of NO in the diethyldithiocarbamate (DETC)-iron (II) complex, relative to that in the control (557.3±44.6 versus 354.5±17.8 in arbitrary units, respectively, P<0.05) in six experiments. These results were further supported by demonstrating the effect in individual myocytes loaded with the NO-sensitive indicator 4,5-diaminofluorescein (DAF-2). Milliseconds of acute cell stretch could increase NO production, as discrete DAF-2 fluorescence transients were recorded in multiple cells that were elongated by an average of 30% beyond their initial length (Fig. 4). Similarly, elongation of a single cell by ∼18% beyond its initial length and maintaining this stretch led to a rapid rise in fluorescence in five out of the six trials (Fig. 4B). In stretched unloaded cells, tissue segments, L-NAME-loaded myocytes or tissue strips, fluorescence alteration was not found (data not shown). Thus, eNOS activity and NO production are regulated by relevant mechanical forces on smooth muscle.

Fig. 3.

Measurement of NO in mouse urinary bladder smooth muscle tissue. Upper panel, ESR spectra of NO trapped by the DETC-iron (II) complex in mouse bladder smooth muscle strips. Compared to control (unstretched tissue strips, gray line), NO was substantially increased in stretched tissue strips (black line). Lower panel, summary of NO production. NO production was significantly different between the control group and the stretch group. Results are mean±s.e.m., n=7 *P<0.05 (Student's t-test).

Fig. 3.

Measurement of NO in mouse urinary bladder smooth muscle tissue. Upper panel, ESR spectra of NO trapped by the DETC-iron (II) complex in mouse bladder smooth muscle strips. Compared to control (unstretched tissue strips, gray line), NO was substantially increased in stretched tissue strips (black line). Lower panel, summary of NO production. NO production was significantly different between the control group and the stretch group. Results are mean±s.e.m., n=7 *P<0.05 (Student's t-test).

Fig. 4.

Production of NO in elongated mouse bladder tissue strips. The tissue segments were incubated with the NO indicator DAF-2 for 60 min (or 10 min for single cells) at room temperature before the experiment. (A) Stretching and maintenance of the tissue strip at 30% over their initial length resulted in multiple cells producing discrete DAF-2 fluorescence transients (right panel). The lower panels show the profiles taken as indicated from the above images. (B) Single-cell experiments demonstrated a similar result to that observed in the tissue strips. (C) DAF-2 fluorescence transients intensity and probability (percentage of tissue segments or cells with DAF-2 fluorescence transients). Results are mean±s.e.m., n=6.**P<0.01 compared with control (without stretching) (Student's t-test).

Fig. 4.

Production of NO in elongated mouse bladder tissue strips. The tissue segments were incubated with the NO indicator DAF-2 for 60 min (or 10 min for single cells) at room temperature before the experiment. (A) Stretching and maintenance of the tissue strip at 30% over their initial length resulted in multiple cells producing discrete DAF-2 fluorescence transients (right panel). The lower panels show the profiles taken as indicated from the above images. (B) Single-cell experiments demonstrated a similar result to that observed in the tissue strips. (C) DAF-2 fluorescence transients intensity and probability (percentage of tissue segments or cells with DAF-2 fluorescence transients). Results are mean±s.e.m., n=6.**P<0.01 compared with control (without stretching) (Student's t-test).

NO mediates stretch-induced contraction of smooth muscle

To explore the potential functional link between NO formation and contractile activity, eNOS-null bladders were used in the force-production experiments. As shown in Fig. 5A, in stretched tissue strips, the contraction amplitude of eNOS-null bladder segments was increased by 2.3-fold compared to that in control tissues (P<0.01, n=7). Moreover, the stretch-induced increases in contraction amplitude were reversed by L-NAME (1 mM). In contrast to wild-type tissues, in eNOS-knockout tissue strips, the amplitude or frequency of contractions were not affected by neither stretching nor the application of L-NAME (Fig. 5B). Finally, after exposure to the NO donor SNAP, the amplitude of contractions were significantly increased by 2.1-fold in wild-type and 2.0-fold in eNOS-knockout myocytes (n=6, P<0.01) (Fig. 5C). Taken together, these experiments suggest that NO production mediates stretch-induced Ca2+ oscillations and contractions in mouse bladder smooth myocytes.

Fig. 5.

NO mediates stretch-induced contraction of smooth muscle. (A,B) Samples of spontaneous contractions recorded in wild-type (A) and eNOS-deleted (B) mouse urinary bladder smooth muscle strips. After being stretched from the control state (0.2 g) to 0.5 g (gram), the amplitude of the contraction was significantly increased in wild-type myocytes but not in eNOS-knockout myocytes. The stretch-induced increase in amplitude of the contraction could be inhibited by the application of L-NAME. In contrast to wild-type strips, eNOS deficient strips did not respond to L-NAME but did respond to SNAP. The frequency of contraction was significantly decreased by stretching and by the application of the exogenous NO donor SNAP. (C,D) Quantitative summary of the amplitude and frequency of the stretch-induced contraction. Results are mean±s.e.m., n=5 or 7, as indicated.*P<0.05, **P<0.01 (ANOVA).

Fig. 5.

NO mediates stretch-induced contraction of smooth muscle. (A,B) Samples of spontaneous contractions recorded in wild-type (A) and eNOS-deleted (B) mouse urinary bladder smooth muscle strips. After being stretched from the control state (0.2 g) to 0.5 g (gram), the amplitude of the contraction was significantly increased in wild-type myocytes but not in eNOS-knockout myocytes. The stretch-induced increase in amplitude of the contraction could be inhibited by the application of L-NAME. In contrast to wild-type strips, eNOS deficient strips did not respond to L-NAME but did respond to SNAP. The frequency of contraction was significantly decreased by stretching and by the application of the exogenous NO donor SNAP. (C,D) Quantitative summary of the amplitude and frequency of the stretch-induced contraction. Results are mean±s.e.m., n=5 or 7, as indicated.*P<0.05, **P<0.01 (ANOVA).

IP3R gates stretch-induced Ca2+ oscillations in smooth muscle

Previous studies have demonstrated that RYR gating underlies Ca2+ release in smooth muscle (Herrera and Nelson, 2002; Nelson et al., 1995; Yuan et al., 2014; Zheng et al., 2010) and stretch-induced Ca2+ sparks (Ibrahim et al., 2012; Iribe et al., 2009; Ji et al., 2002; Wei et al., 2008), as these events are eliminated by pre-exposure of myocytes to ryanodine. In the present study, we used a similar approach to determine whether stretch-induced Ca2+ oscillations were inhibited by the application of ryanodine and/or the selective IP3R inhibitor xestospongin C in mouse bladder smooth muscle. We first examined the effect of ryanodine on the stretch-induced Ca2+ oscillations. As shown in Fig. 6A, neither the amplitude nor the frequency of stretch-induced Ca2+ oscillations was significantly affected by the application of ryanodine (30 µM) (P>0.05 compared with stretching alone), suggesting that the stretch-induced Ca2+ oscillations are not RYR gated. We then investigated the role of IP3Rs in mediating stretch-induced Ca2+ oscillations in smooth muscle. As indicated in Fig. 6B, in the presence of xestospongin C (10 µM), the amplitude of the stretch-induced Ca2+ oscillations was significantly reduced to 1.37±0.06 relative to the control (absence of xestospongin C) value of 1.76±0.14 (n=7, mean±s.e.m.; P<0.05). It should be noted that xestospongin C alone could not completely abolish the stretch-induced Ca2+ oscillations, even when increasing the concentration of xestospongin C to 30 µM (data not shown), which suggests that IP3R gating is not the only mechanism of mediating stretch-induced Ca2+ oscillations in smooth muscle. It has previously been reported that rapid stretching muscle causes Ca2+ sparks through RYR2 in both cardiac and smooth myocytes (Ibrahim et al., 2012; Iribe et al., 2009; Ji et al., 2002; Wei et al., 2008). In the current experiment, the results indicated that ryanodine could, to a limited extent, inhibit the stretch-induced Ca2+ oscillations. Therefore, it is possible that RYRs are partially involved in regulating stretch-induced Ca2+ oscillations. Thus, we examined the effect of the co-application of xestospongin C and ryanodine on the stretch-induced Ca2+ oscillations in smooth muscle. In the co-presence of xestospongin C and ryanodine, the amplitude and frequency of the stretch-induced Ca2+ oscillations were almost completely abolished (Fig. 6B–D).

Fig. 6.

Effect of Ca2+ release channel inhibitors on stretch-induced Ca2+ oscillations. (A) Application of ryanodine did not exhibit inhibitory effects on stretch-induced Ca2+ oscillations. (B) Xestospongin C, a selective IP3R inhibitor, significantly reduced the stretch-induced Ca2+ oscillations. (C,D) Quantitative summary of peak Ca2+ oscillations amplitude and frequency: xestospongin C, but not ryanodine, decreased the peak amplitude and frequency (number of oscillations per 10 min) of stretch-induced Ca2+ oscillations in mouse bladder smooth muscle. Results are mean±s.e.m., n=6. *P<0.05 compared with stretched tissue but without NO inhibitors (ANOVA).

Fig. 6.

Effect of Ca2+ release channel inhibitors on stretch-induced Ca2+ oscillations. (A) Application of ryanodine did not exhibit inhibitory effects on stretch-induced Ca2+ oscillations. (B) Xestospongin C, a selective IP3R inhibitor, significantly reduced the stretch-induced Ca2+ oscillations. (C,D) Quantitative summary of peak Ca2+ oscillations amplitude and frequency: xestospongin C, but not ryanodine, decreased the peak amplitude and frequency (number of oscillations per 10 min) of stretch-induced Ca2+ oscillations in mouse bladder smooth muscle. Results are mean±s.e.m., n=6. *P<0.05 compared with stretched tissue but without NO inhibitors (ANOVA).

Taken together, these results indicate that the stretch-induced Ca2+ oscillations are gated primarily by IP3 Ca2+ release channels in mouse bladder smooth muscle.

Effect of GC and PI(3)K inhibitors on stretch-induced Ca2+ release in smooth myocytes

We also designed and performed experiments to prove that endogenous NO acts directly on IP3R receptors rather than through the secondary effects of sGC activation, and cGMP and PKG. Mouse bladder smooth myocytes were co-incubated with Fluo-4 AM and the selective sGC inhibitor 1-H-[1,2,4,]-oxadiazole [4,3-α]quinoxaline-1-one (ODQ, 10 µM). As shown in Fig. 7, the presence of ODQ did not alter the amplitude or frequency of Ca2+ oscillations (Fig. 7A, upper panel, four experiments). It is known that the production of NO can also lead to Ca2+ release from intracellular Ca2+ stores through the second messenger cyclase ADP-ribose (cADPR) after the activation of ADP-ribosyl cyclase by NO (Willmott et al., 1996). To exclude this pathway, we tested the effects of the PKG inhibitor KT5823. As predicted, KT5823 exhibited no effects on Ca2+ oscillations in smooth muscle (Fig. 7A, middle panel), suggesting that the stretch-induced Ca2+ oscillations resulted from the direct effect of NO on IP3Rs rather than on the sGC–PKG and cADPR pathway.

Fig. 7.

Ca2+ oscillations were not altered by sGC or PKG inhibitors but were abolished by a PI3K inhibitor. (A) Representative experiments obtained from stretched smooth myocytes in the presence of ODQ, KT5823 or LY294002. Neither ODQ nor KT5823 altered the amplitude or frequency of the stretch-induced Ca2+ oscillations (upper and middle panels). The PI3K inhibitor LY294002 abolished the Ca2+ oscillations completely (lower panel). B,C. Quantitative summary data of data as shown in A. Results are mean±s.e.m., n=7. **P<0.01 compared to control (no LY294002) (ANOVA).

Fig. 7.

Ca2+ oscillations were not altered by sGC or PKG inhibitors but were abolished by a PI3K inhibitor. (A) Representative experiments obtained from stretched smooth myocytes in the presence of ODQ, KT5823 or LY294002. Neither ODQ nor KT5823 altered the amplitude or frequency of the stretch-induced Ca2+ oscillations (upper and middle panels). The PI3K inhibitor LY294002 abolished the Ca2+ oscillations completely (lower panel). B,C. Quantitative summary data of data as shown in A. Results are mean±s.e.m., n=7. **P<0.01 compared to control (no LY294002) (ANOVA).

It has been reported that the stretch dependence of Ca2+ release is mediated by a phosphoinositide 3-kinase (PI3K)-dependent signaling pathway in cardiac myocytes (Petroff et al., 2001). However, it is not known whether this is also the case in smooth myocytes. Therefore, the effect of the PI3K inhibitor LY294002 was tested. The addition of LY294002 (30 µM) entirely eliminated the stretch-induced Ca2+ oscillations in seven experiments (Fig. 7A, lower panel), suggesting that the stretch-induced Ca2+ oscillations in smooth muscle might be mediated by a PI3K-dependent signaling pathway, as has been reported in rat cardiac myocytes (Petroff et al., 2001).

Muscle stretching is a common physiological and pathological phenomenon in hollow smooth muscle organs. Previously, we found that rapid stretching of mouse and rabbit bladder smooth myocytes causes intracellular Ca2+ sparks and Ca2+ waves (Ji et al., 2002; Wei et al., 2008); here, we report that a maintained mechanical stretching (18% increase from the initial length for single cells, and 30% increase from the initial length for tissue strips) of mouse bladder smooth myocytes results in Ca2+ oscillations (Fig. 1). This is the first demonstration that increases in the length of myocytes from hollow smooth muscle organs induces Ca2+ oscillations. Our main finding is that stretch-induced Ca2+ oscillations are mediated by NO production and relate to mechanical forces, and that the action of NO is not dependent on the sGC-cGMP pathway. Single-cell and tissue elongation resulted in the increases in Ca2+ oscillations amplitude and frequency, and the increased Ca2+ oscillations were abrogated by NOS inhibitor L-NAME and promoted by the NO donor SNAP (Fig. 2). Moreover, elongation of smooth muscle resulted in an increase in NO production in tissue segments and in single cells (Figs 3 and 4), and eNOS gene inactivation resulted in the loss of stretch-induced Ca2+ oscillations in tissue strips (Fig. S1). For the first time, we demonstrated a functional role for the link between NO and stretch-induced contractions, as stretch-induced contractions were inhibited by L-NAME and enhanced by SNAP (Fig. 5). The enhancement of stretch-induced smooth myocyte contraction by NO is supported by previous studies. For example, Yanai et al. (2008) have reported that NO enhances the spontaneous contraction of guinea pig detrusor smooth muscle through a cGMP-independent mechanisms. The production of NO occurred before the induction of Ca2+ oscillations, but its action was sGC and PKG independent, as stretch-induced Ca2+ oscillations were not altered by sGC and PKG inhibitors (Fig. 7). By contrast, the stretch-induced Ca2+ oscillations were inhibited by the PI3K inhibitor LY294002 (Fig. 7A, lower panel). In the present study, we have also shown that the stretch-induced Ca2+ oscillations in smooth muscle are significantly inhibited by the IP3R inhibitor xestospongin C (Fig. 6), but not by high concentrations of ryanodine (30 µM) alone, suggesting the stretch-induced Ca2+ oscillations occur primarily through the activation of IP3R Ca2+ release channels. However, RYRs might be partially involved in the stretch-induced Ca2+ oscillations because only the co-application of xestospongin C and ryanodine were able to entirely eliminate the stretch-induced Ca2+ oscillations (Fig. 6B).

Our findings demonstrate that stretching of smooth myocytes results in Ca2+ oscillations and establishes NO as a crucial coupling molecule linking membrane tension to SR Ca2+ release. Previous studies have demonstrated that the elongation of myocytes results in Ca2+ release from SR in smooth, skeletal and cardiac myocytes (Han et al., 2013; Iribe et al., 2009; Mutungi, 2003; Zou et al., 2002). This would agree with the results of the present study, which shows that stretching induces Ca2+ oscillations in mouse bladder smooth myocytes. NO has been shown to increase the activity of Ca2+ release receptors in cardiac (Petroff et al., 2001; van der Wees, 2006) and skeletal (Sun et al., 2001; Yamada et al., 2015) muscle. As the stimulation of guanylyl cyclase by NO is a prominent mechanism for smooth muscle relaxation (Rybalkin et al., 2003), the role of NO in stretch-induced Ca2+ oscillations seems counterintuitive. However, similar to the case in cardiac muscle, stretch-induced Ca2+ oscillations in smooth muscle are independent of the activation of guanylyl cyclase and PKG, indicating a compartmentalization of NO signaling.

Although RYRs might be involved in Ca2+ oscillations (Kannan et al., 1997; McHale et al., 2006; Nakayama et al., 2005; Plummer et al., 2011), studies suggest that Ca2+ oscillations are mainly generated by the activation of IP3R release channels in many cell types (Abou-Saleh et al., 2013; Berridge, 2007; Hruby et al., 2008; Manhas and Pardasani, 2014; Martin-Cano et al., 2009; Tamarina et al., 2005; Tamashiroand Yoshino, 2014). Our results suggest that the Ca2+ oscillations induced by the stretching of mouse bladder smooth myocytes was mainly mediated the activation of IP3Rs by NO production, although it was impossible to completely exclude the involvement of RYRs. Moreover, some studies have suggested that Ca2+ oscillation occurs through the activation of both IP3Rs and RYRs in PC12 and interstitial cells of the rabbit urethra (Johnston et al., 2005; Koizumi et al., 1999).

It remains unclear how NO affects Ca2+ release through IP3Rs. Future studies on the mechanical activation of muscle should focus on the mechanism tightly linking the action of NO to IP3R activation. This study might provide potentially attractive targets of therapeutic intervention for urinary continence, pregnancy and respiratory diseases.

Single-cell and tissue strep preparation

Mice, including eNOS-knockout mice, were obtained from the Jackson Laboratories (Bar Harbor, ME). All animal experiments were performed according to approved guidelines, and mice were anesthetized and killed in accordance with an approved laboratory animal protocol. Single-cells were prepared as described previously (Ji et al., 2002; Wei et al., 2008). Briefly, bladder myocytes were isolated by cutting the bladder into small pieces, which were incubated for 20 min in 1 mg/ml papain, 1 mg/ml dithioerythritol and 1 mg/ml bovine serum albumin (BSA) in Ca2+-free solution. The fragments were then transferred into a solution with 1 mg/ml collagenase type II (Worthington Biochemical) and 100 µM Ca2+, supplemented with 1 mg/ml BSA. The tissue was incubated for 10 min, triturated with a wide-bore Pasteur pipette, and passed through a 125-µm nylon mesh. Cells were concentrated by low-speed centrifugation, washed with fresh medium, resuspended and stored at 4°C.

Tissue segments were prepared by removing the fibrosal and mucosal layers from mouse bladders in an ice-cold Ca2+-free solution, and cutting segments of the remaining muscle layer into 2–3-mm-long strips, 100–200 µm in diameter, using a fine dissecting scissors.

Measurement of Ca2+ oscillations

Single myocytes were incubated with 10 µM Fluo-4 AM (Molecular Probes) for 10 min at room temperature in a recording chamber mounted on an inverted microscope (IX81, Olympus) and perfused with physiological salt solution for 40 min at room temperature. The extracellular solution was (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4, adjusted with NaOH). For the Ca2+-free extracellular solution, CaCl2 was omitted from the above solution or 3 mM EGTA and 1 mM CaCl2 was used to clamp free [Ca2+] at 100 nM, as indicated. Solutions were changed using a gravity perfusion system providing complete solution exchange within 30 s. Cell stretching was accomplished using two patch pipettes attached to separate manipulators. Pipettes were sealed to cells by suction to form a high-resistance seal, and cells were stretched by moving the pipettes along the longitudinal cell axis, stretching from slack length (L1) to a new length (L2), which was then maintained. For experiments on tissue segments, the preparations were incubated with Fluo-4 AM (10 µM) and 0.05% pluronic acid for 1 h. The strips were initially stretched by a few percent beyond their slack length to their approximate resting length, which was then maintained. All experiments were conducted at room temperature.

Fluorescence imaging was performed using a two-photon scanning laser microscope. An 800-nm beam from a Ti-sapphire laser (Tsunami, Spectra-Physics, Mountain View, CA) pumped with a 5 W, 525-nm diode laser (Millennia, Spectra-Physics) was scanned across the specimen with two oscillating mirrors (x- and y-scan) through an upright microscope (Olympus IX71FVSF-2). The emitted fluorescence was separated with a dichroic mirror (670uvdclp, Chroma Technology Corp, Rockingham, VT) and a long pass filter (E700SP or E600SP-2P, Chroma Technology Corp) positioned immediately below the objective. The emitted fluorescence was detected with a photomultiplier tube (PMT, R5929, Hamamatsu USA, Bridgewater, NJ). x-y images were recorded at an average frame rate of 30-ms intervals. Images were processed and analyzed using MATLAB 7.1 software (MathWorks).

Detection of NO by ESR in tissues

Mouse bladders were removed and dissected as described above and the muscle segments incubated in 30 mM diethyldithiocarbamate (DETC), 3 mM ferrous sulfate and 15 mM sodium dithionite for 25 min at room temperature. To test the stretch dependence of NO formation, tissue segments were elongated by ∼30% of their initial length, and the stretched and non-stretched control segments were homogenized. The adduct (DETC)2-Fe2+-NO was extracted with ethyl acetate and measured with an ESR spectrometer at room temperature. The process was carried out in a dark environment to avoid light-induced NO dissociation from the adduct.

Real-time detection of NO

Smooth myocytes were incubated with the NO-sensitive indicator 4,5-diaminofluorescein (DAF-2) for 10 min (single cells) or 60 min (tissues) at room temperature. NO fluorescence was detected using a laser scanning confocal head (FV1000; Olympus) attached to an inverted microscope (excitation at 488 nm, emission at 515−565 nm).

Data analysis

Image processing and data analysis were performed with custom software written in MATLAB. The results are expressed as the means±s.e.m. Significant differences were determined by Student's t-test. Data from three groups were compared by one-way repeated measures ANOVA, and significant differences between groups were determined by the Student–Newman–Keuls (SNK) test for paired comparisons.

We are grateful to Yanyun Wu and Qi Yuan for excellent technical assistance.

Author contributions

G.J. and B.W. designed the study. J.Z., K.Z., B.W. and Y.C. performed experiments. Y.C., X.Z. and L.M. analyzed data. J.Z. and G.J. wrote the manuscript.

Funding

This work was supported by grants from the National Basic Research Program of China [grant number 2011CB8091004]; by the National Natural Science Foundation of China [grant numbers 31300956 to K.Z., 81100539]; and by Natural Science Foundation Project of Chongqing [grant number CSTC2013jcyjA10140].

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Competing interests

The authors declare no competing interests.

Supplementary information