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Aging-related changes of intracellular Ca2+ stores and contractile response of intestinal smooth muscle
Experimental Gerontology 41 (2006) 55–62 www.elsevier.com/locate/expgero
Aging-related changes of intracellular Ca2C stores and contractile response of intestinal smooth muscle Guiomar S. Lopes a, Alice T. Ferreira b, Maria Etsuko Oshiro b, Irina Vladimirova c, Neide H. Jurkiewicz a, Aron Jurkiewicz a, Soraya S. Smaili a,* a
Department of Pharmacology, Federal University of Sa˜o Paulo (UNIFESP), Rua Treˆs de Maio—100, Sa˜o Paulo 04044-020, Sa˜o Paulo, Brazil b Department of Biophysics, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Brazil c Bogomoletz Institute of Physiology, Kiev, Ukraine Received 10 June 2005; received in revised form 31 August 2005; accepted 4 October 2005
Abstract In this study, we investigated the effect of aging on intracellular Ca2C stores, as sarcoendoplasmic reticulum (SR) and mitochondria, and the influence of these compartments on contraction of rat colon smooth muscle [Bitar, K.N., 2003. Aging and neural control of the GI tract V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins. Am. J. Physiol. Gastrointest. Liver. Physiol. 284(1), G1–G7; Marijic, J., Li, Q.X., Song, M., Nishimaru, K., Stefani, E., Toro, L., 2001. Decreased expression of voltage-and Ca2C-activated KC channels in ´ con, P., Vila, E., 2002. coronary smooth muscle during aging. Circ. Res. 88, 210–234; Rubio, C., Moreno, A., Briones, A. Ivorra, M.D., D’O Alterations by age of calcium handling in rat resistance arteries. J. Cardiovasc. Pharmacol. 40(6), 832–840]. Calcium stores and contraction were evaluated by simultaneous measurements of fluorescence and tension in smooth muscle strips loaded with fura-2. Results showed that activation of muscarinic receptors by methylcholine (MCh, 10 mM), induced a greater contraction in aged rats than in adult animals. The inhibition of Ca2C ATPase by thapsigargin (TG, 1 mM) did not prevent the refilling of SR either in adult or aged rats. MCh, in the presence of TG, induced an increase in transient fluorescence, indicating a release of Ca2C from TG-insensitive compartment. The mitochondrial uncoupler, FCCP (5 mM), caused a greater increase in intracellular Ca2C and tension in aged rats, indicating that mitochondria may accumulate more Ca2C during aging. The present results show that changes in intracellular Ca2C stores, such as mitochondria and SR, affect contraction and may cause dysfunctions during aging that could culminate in severe alterations of Ca2C homeostasis and cell damage. q 2005 Elsevier Inc. All rights reserved. Keywords: Calcium store; Contraction; Smooth muscle; Aging
1. Introduction One of the current hypothesis of the aging process is related to changes in Ca2C homeostasis and cytotoxicity, which are associated with the degenerative diseases (Choi, 1995; Verhkratsky and Toescu, 1998; Barja, 1998). Thus, Ca2C accumulation in the cytoplasm and alterations in Ca2C buffering by the sarcoendoplasmic reticulum (SR) and mitochondria could lead to oxidative stress and dysfunctions during aging (Barja, 1998). Excitation and contraction of the intestinal smooth muscle depend on the increase in the cytosolic Ca2C concentration ([Ca2C]i). The activation of metabotropic receptors, as * Corresponding author. Tel.: C55 11 5576 4449; fax: C55 11 5571 1776 E-mail address: [email protected] (S.S. Smaili).
0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2005.10.004
muscarinic receptors, may result in IP3 formation and release of SR Ca2C. This event may evoke a transient increase in [Ca2C]i which seems to be involved in the phasic component of the smooth muscle contraction (Berridge, 1996; Smaili et al., 1998). On the other hand, the influx of Ca2C from the extracellular medium through voltage-operated (VOCCs) and receptoroperated (ROCCs) Ca2C channels can be involved in the tonic contraction (Berridge, 1996; Bolton, 1979; Smaili et al., 1998). Under certain conditions, Ca2C entry can occur through storeoperated channels (SOCCs) activated by the emptying of the SR (Gibson et al., 1998). The activation of SOCCs appears to be of greater importance in tonic smooth muscles, as observed in the gastric fundus and in the vascular smooth muscle (Smaili et al., 1998; Gibson et al., 1998). A family of Ca2C pumps, the sarcoendoplasmic reticulum 2C Ca -ATPases (SERCA), mediates the Ca2C-uptake mechanisms in the SR (Pozzan et al., 1994). Thapsigargin (TG) and
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cyclopiazonic acid (CPA) have been shown to inhibit some types of SERCA pump which leads to a depletion of the Ca2C in TG-sensitive stores present in different smooth muscle cells (Pozzan et al., 1994; Go´mez-Viquez et al., 2003). In addition, under certain circumstances, TG-insensitive stores were also described (Neusser et al., 1999; Vanoevelen et al., 2004) as the Golgi complex of secretory cells, where a different Ca2CATPase is present. This TG-insensitive pump (SPCA1) was shown to play a role in modulating cytosolic and intra-Golgi Ca2C homeostasis (Vanoevelen et al., 2004). Another important TG-insensitive Ca2C store are mitochondria which modulate and shape cytosolic Ca2C signaling (Smaili et al., 2003). Mitochondrial Ca2C (Ca2Cm) uptake and release, either via the uniporter or NaC-dependent and NaCindependent exchange mechanisms, may certainly regulate [Ca2C]i (Budd, 1998; Szado et al., 2003). In addition, Ca2Cm efflux also occurs by the opening of the permeability transition pore (PTP) which operates in high or low conductance modes (Szado et al., 2003). The transient pore opening, associated with the low conductance state, is expected to result in a redistribution of Ca2C between the intra- and extramitochondrial spaces. It has been also proposed that this mechanism may provide a rapid Ca2C efflux which could protect the organelles against Ca2C overload and also amplify cytosolic Ca2C signals (Ichas et al., 1997; Smaili and Russel, 1999; Lopes et al., 2004). Changes in Ca2C homeostasis are among the most important causes of age-related alterations in the nervous system. It is unknown whether similar changes occur in the gastrointestinal system and what would be the correlation with the colon motor diseases that occur during aging processes (Verhkratsky and Toescu, 1998). These alterations have been associated with a decrease in acetylcholine release, a decrease in Ca2C influx via plasma membrane Ca2C channels or a decline in the Ca2C channel current (Roberts et al., 1994; Xiong et al., 1993). On the other hand, recent studies have shown that aging is associated with an increase in [Ca2C]i resulted from a decline in the Ca2C buffering capacity and a Ca2C store overload (Pottorf et al., 2000; Rubio et al., 2002). These results are in agreement with the study in forebrain neurons which showed that aging caused an increase in the Ca2Cm buffering capacity as a compensatory response to the increase in Ca2C influx (Murchison and Griffith, 1998). In fact, aging might alter mitochondrial function, as the uniporter, the membrane potential or the PTP activity (Satru´stegui et al., 1996; Lopes et al., 2004). Altogether indicate that during aging several aspects of Ca2C homeostasis might be affected, as Ca2C influx, release of Ca2C from intracellular stores and Ca2C uptake processes by the SR and mitochondria. The present study aimed to characterize the changes in the SR and mitochondrial intracellular Ca2C stores in colon smooth muscle from adult and aged rats. We investigated the influence of these changes on [Ca 2C ] i signaling and contraction. The relationship between [Ca2C]I and tension was assessed by recording both parameters simultaneously. The results showed that aging may alter Ca2C stores and contraction in colon smooth muscle and these changes might
affect smooth muscle contractility after metabotropic receptor stimulation. 2. Materials and methods 2.1. Isolation and preparation of rat colon Female Wistar 2BWA adult rats (4–6 mo) and aged rats (24–30 mo) were killed with an overdose of ether, and a segment of their terminal colon was excised. The smooth muscle layer was freed of the adjacent mucosa and washed in Tyrode solution with the following composition: 145 mM/l NaCl; 5.5 mM/l KCl; 2.5 mM/l CaCl2; 1 mM/l MgCl2, 10 mM/l HEPES; and 10 mM/l glucose. 2.2. Incubation and preparation of colon smooth muscle Samples of colon smooth muscle were mounted vertically in a cuvette and a designed holder fixed on one end of the muscle to the bottom of the cuvette while the other at the top was attached to an isometric force transducer. The resting tension was adjusted to 1 g (1 V). The tissue was perfused with 2.5 ml Tyrode solution at 37 8C, bubbled continuously with 95% O2 and 5% CO2. The muscle strip was incubated with 4 mM fura2/AM plus 0.02% pluronic acid F-127 for 3.5 h at room temperature. 2.3. Simultaneous measurements of fluorescence and contraction A system containing a transducer and spectrofluorimeter coupled to a computer system allowed simultaneous measurements of dual wavelength fluorimetry and tension. Experiments were performed using a fluorimeter (PTI, Photon Technology International, Inc, New Jersey, USA). After fura2 loading, tissues were subjected to excitation wavelengths of 340 and 380 nm. Emitted fluorescence was collected at 510 nm. The ratio of 340–380 nm excitation (R340/380) was reported as a relative measurement of [Ca2C]i (Himpens and Somlyo, 1988). Results were expressed in fluorescence ratio (340/380) values after background subtraction which was approximately 40% of the total fluorescence measured at the 340 or 380 nm. Fluorescence intensities were normalized in relation to the average of the baseline values. Briefly, fluorescence intensity in the non-zero pixels were averaged (F) and ploted as normalized fluorescence (DF/F0 X100) against time (Smaili and Russel, 1999). DF was calculated as the difference between the mean value of the first 20 data points prior to stimulation of the cell (Fo) and F. Passive tension of 1 g was initially applied and the tissue was allowed to equilibrate for 30 min. Isometrically recordings were obtained with a force–displacement transducer and tension values were expressed in grams. Fluorescence and tension were analyzed in order to obtain four parameters for comparison between groups. These included the maximum increase in fluorescence and tension expressed in percentage over the baseline values (all Figs. C and D) and the rate of
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decline of the calcium and tonic contractile response (All Figs. E and F). The decline expresses the percentage of decrease in the respective measurement from the peak to the stabilization of the response. MCh (10 mM) was applied after 3 min of steady level under a resting tension and tested in normal and nominally (without EGTA) Ca2C-free solution. Drugs were added by medium replacement and the samples were washed five times with Tyrode between different drugs. The amplitude of the agonistevoked increase in the transient fluorescence ratio (R340/380) was taken as intracellular Ca2C increase. In some experiments TG was incubated for 10 min followed by MCh (10 mM) addition. We tested whether the increase in fluorescence was due to the Ca2C influx through VOCC. Therefore, some experiments were performed in the presence of verapamil (10 mM), a VOCC antagonist that was added to the incubation medium 30 min before the beginning of the experiments. In other experiments, SKF96365 (4 mM), an antagonist of the ROCC was also added, to avoid interference and allow observation of transport from Ca2C stores during intracellular Ca2C measurements.
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In another experiment, the mitochondrial uncoupler FCCP, which causes a rapid and reversible collapse of the proton gradient across the mitochondrial membrane (DJm), was also used (Budd and Nicholls, 1996). This drug inhibits mitochondrial Ca2C uptake and may be used to evaluate the effect of this mechanism in Ca2Cm accumulation (Budd, 1998; Ichas et al., 1997). Under certain conditions and depending on the system, FCCP may also cause a release of Ca2Cm (Smaili and Russel, 1999). Because FCCP can cause a severe ATP consumption by reversal of the F0–F1 ATP synthase, FCCP was always added together with oligomycin (1 mg/m), which inhibits mitochondrial ATP synthase and prevents ATP consumption in the presence of FCCP (Budd and Nicholls, 1996; Bernardi and Petronilli, 1996). Oligomycin alone had no significant effect on basal levels of Ca2C and tension (data not shown). At the end of all experiments, calibration was performed with the addition of digitonin (100 mM) followed by MnCl2 (2 mM) and EGTA (4 mM). All fluorescence and tension values were normalized to the basal fluorescence and basal tension.
Fig. 1. Effect of MCh (10 mM) on fluorescence and tension measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A) MCh induced a transient followed by a sustained elevation in fluorescence ratio. (B) Contraction induced by MCh also showed a phasic and a tonic component. The phasic response was greater in the aged rats. (C) Histogram shows the maximum fluorescence observed with MCh and compared the effects in adult and aged animals. (D) Histogram compares the maximal tension obtained with MCh which was significantly higher in aged rats. (E) Shows the rate of decline of the sustained phase of fluorescence. (F) the rate of decline in tension, which were not different between adults and aged animals. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
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Fig. 2. Effect of MCh (10 mM) on fluorescence and tension in Ca2C-free solution measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A) MCh induced similar increase in transient fluorescence both in adult and aged rats. (B) MCh-induced tension was higher in aged than in adult animals. (C,D) Histograms show that there is no significant difference between adult and aged rats either in the maximum fluorescence ratio or tension induced by MCh. (E,F) In Ca2C-free solution the rate of decline in fluorescence and tension were significantly faster in aged rats. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
Drugs used were methylcholine (MCh), FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), oligomycin, verapamil and thapsigargin (Sigma Chemical Co., St. Louis, MO, USA); SKF96365 (Calbiochem, La Jolla, CA, USA); fura-2/AM and pluronic acid F127 (Molecular Probes Inc., Eugene, OR, USA). 2.4. Statistics The normalized fluorescence and tension were expressed as means GSEM plotted against time. Data express the means of at least six experiments and statistical analyses were performed by Student t-test and Welch correction. 3. Results 3.1. Effects of MCh Simultaneous measurements in fura-2-loaded strips of rat colon were tested with MCh (10 mM), which caused an increase in fluorescence ratio and contraction. MCh induced a transient increase that was followed by a sustained
elevation in fluorescence. This response was similar to the contractile pattern where the phasic and tonic contraction was recorded (Fig. 1A,B). We measured the amplitude of the transient increase in fluorescence and the decline of sustained phase as well as the increase of phasic contraction and the decline of tonic phase contraction and compared them between adult and aged rats. Results show that the increase in Ca2C and the fluorescence rate decline were very similar in both groups of animals (Fig. 1A,C,E). However, the maximum amplitude of tension (phasic component) in aged animals was significantly higher when compared with adult rats (Fig. 1B–D), and the decline in contraction in aged animals was not significantly different from the one observed in adult animals (Fig. 1F). To investigate the influence of extracellular Ca2C influx during muscarinic receptor stimulation, experiments were performed in the absence of external Ca2C. Fig. 2(A,B) shows that even when the muscles were incubated in nominally Ca2C-free solution, MCh still induced a transient Ca2C increase with similar magnitude in both adult and aged rats (Fig. 2A,D). It is interesting to point out that under this condition the contraction in aged rats was similar to that
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Fig. 3. Effect of MCh (10 mM) in the presence of TG (1 mM) on fluorescence and tension measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A,B) TG did not induce a sustained elevation in fluorescence but increased [Ca2C]i oscillations in both groups, with higher amplitude in aged rats. In the presence of TG, the effect of MCh was higher in aged rats. (C,D) Maximum fluorescence and tension induced by MCh in the presence of TG were significantly increased in aged rats. (E,F) There is no difference in the rate of decline of the MCh responses when TG was present. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
observed when the normal solution was used (Figs. 1D and 2D). Although the contraction in aged rats in Ca2C-free solution was slightly increased in relation to the normal solution, this was not significantly different (Figs. 1B and 2B). As shown in Fig. 2, the decrease in Ca2C was faster in aged rats which might be related to the release of Ca2C from intracellular compartments. In fact, ER and other compartments can be depleted after incubation with Ca2C-free solution. On the other hand, the declines in fluorescence and contraction were significantly faster in aged rats (Fig. 2A–F), which allowed the restoration to the basal Ca2C levels. These results indicate that the phasic components of fluorescence and contraction are dependent on intracellular Ca2C in both groups of animals. However, the sustained increase in intracellular Ca2C and the tonic phase of contraction seem to be more dependent on extracellular Ca2C in aged rats, or the kinetics of the [Ca2C]i buffering system is faster in these animals. It is interesting to point out that the oscillations observed in Fig. 1 were not present when tissues were incubated in Ca2C-free solution. Thus, the nature and the source of the intracellular Ca2C mobilization were then investigated in both groups of animals.
3.2. Effects of MCh (10 mM) in the presence of TG (1 mM) In these experiments, the Ca2C-ATPase blocker, TG was applied for 10 min before the addition of MCh. The presence of TG resulted in transient increases in fluorescence and tension with an oscillatory pattern both in adult and aged animals. These oscillations were more marked in aged rats (Fig. 3A,B). MCh caused elevation of the intracellular Ca2C that was significantly higher in aged rats (25.2G5.7% in adult and 89.1G17.1% in aged) (Fig. 3C) and more elevated than in the control in the absence of TG (56.4G6.1% in the absence and 89.1G17.1% in the presence of TG) (Fig. 1C and 3 C). However, in adult animals the TG treatment had no significant effect on the MCh-induced transient increase in Ca2C or on the amplitude of the sustained contraction. However, MCh contraction was significantly higher in aged rats (Fig. 3B,D). The decline of fluorescence and tonic contraction was not significantly different between the two groups (Fig. 3E,F). These results indicate that in adult animals, MCh response might be dependent on TG-insensitive Ca2C stores. On the other hand, in aged animals other TG-insensitive compartments may play an important role during MCh response.
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Fig. 4. Effect of MCh (10 mM) in the presence of FCCP (5 mM) plus oligomycin (1 mg/ml) on fluorescence and tension measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A,B) FCCP caused an increase in fluorescence and tension. In the presence of FCCP, MCh still causes an increase in fluorescence and tension that were higher in aged rats. (C,D) Elevations in fluorescence and tension caused by FCCP were significantly increased in aged animals. (E,F) In the presence of FCCP, MCh evoked increases in fluorescence and tension that were significantly greater in aged rats than in the adult group. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
Therefore, studies were conducted to investigate the influence of mitochondria on these effects. All the experiments were performed in the presence of verapamil and SKF96365, to avoid extracellular Ca2C influx and interference during activation of SR stores by TG and MCh (see above). These Ca2C channel blockers did not affect responses to TG and MCh. 3.3. Effects of MCh in the presence of FCCP (5 mM) In order to test the influence of the Ca2C store at the mitochondrial level, FCCP plus oligomycin (see Section 2) were added to the colon strips. FCCP was used to collapse DJ and reduce the electrochemical driving force for mitochondrial Ca2C import and allow mitochondria to release accumulated Ca2C (Smaili and Russel, 1999; Bernardi and Petronilli, 1996). Fig. 4A,C shows that FCCP caused an increase of transient fluorescence in both preparations and this was significantly higher in aged rats. It was also observed that FCCP induced a further increase in tension in aged rats (Fig. 4B,D). In the presence of FCCP, the effects of MCh on Ca2C transients and
contraction were significantly higher in aged rats (Fig. 4E–F). The peak of Ca2C in aged rats in response to MCh and in the presence of FCCP, was significantly greater when compared to the effect in the absence of this drug (108.1G13.6% in the presence and 56.4G6.1% in the absence of FCCP) (Figs. 4E and 1C). It is important to point out that independently of the absence or presence of FCCP or TG, contraction in aged rats was increased in relation to their controls (Figs. 1, and 4). These results indicate that Ca2C may accumulate in the intracellular stores, especially into the mitochondria, during aging. 4. Discussion The aim of the present study was to investigate the agerelated changes in the intracellular Ca2C stores and contractile response in the colon smooth muscle. Our results showed that smooth muscle contraction induced by MCh involves an increase in the [Ca2C]i as a result of Ca2C release from intracellular stores. The simultaneous measurement of Ca2C and tension showed that the contraction was accompanied by
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an increase in the intracellular Ca 2C level, although the changes in these parameters were not always correlated (Abe et al., 1996). In addition, MCh induced a transient increase in fluorescence and contraction and the last was more elevated in the aged animals (Fig. 1C,D). Several hypothesis could be raised to explain the difference between Ca2C responses and contraction in aged rats. One is the presence of the superficial buffer barrier (SBB). In this case, the peripheral SR could interact with the plasmalemma to perform a specialized function and regulate [Ca2C]i. This barrier could accumulate more Ca2C before it could reach the myofilaments (van Breemen et al., 1995). Thus, it is possible to suggest that in aged rats the SBB is less efficient, which would cause more accumulation of Ca2C in the cytosol and the increase in contraction observed (Fig. 1). Another possible explanation is the fact that these animals might show different contents in their intracellular Ca2C stores with different kinetic of Ca2C release. In fact, MCh-induced Ca2C signaling and contraction showed to be strongly dependent on the intracellular Ca2C stores, since the phasic responses were not significantly altered when in Ca2C free solution (Fig. 2A–D). However, in aged rats, the tonic components, were more affected by the Ca2Cremoval, where the rate of decline was faster. Therefore, it is possible that either this sustained response is partially dependent on extracellular Ca2C or the intracellular Ca2C stores could be more rapidly emptied. To investigate the intracellular Ca2C stores in adult and aged animals, we performed experiments in the presence of the inhibitors of these compartments. We showed that TG itself did not induce a sustained elevation of [Ca2C]i or tension in adult and aged rats. However, it increased the amplitude of the spontaneous Ca2C oscillations. On this point, it is interesting to note that Ca2C oscillations were markedly present in aged animals. Since the SR is very important for these oscillatory changes in Ca2C signaling (Smaili et al., 2003), these results indicate that the SR can be affected with aging and may accumulate more Ca2C. This result is in agreement with the studies performed in vascular smooth muscle from aged and spontaneously hypertensive rats (Neusser et al., 1999; Matz et al., 2003). However, MCh response in the presence of TG was not affected in adult rats, indicating that MCh may evoke TGinsensitive stores to promote its effect. In addition, the contraction induced by MCh in this condition, was much higher in aged rats, suggesting that these TG-insensitive store might be more important and store more Ca2C in these animals. TG-insensitive stores include mitochondria, thus, we have investigated this compartment and its effect on the response during aging. We observed that FCCP caused a larger increase in [Ca2C]i and contraction in aged rats (Fig. 4A–D). This effect might be associated with the release of Ca2Cm accumulated in the organelle, since the experiments were performed in the presence of verapamil and SKF96365 to avoid interference of FCCP with the plasma membrane Ca2C channels. Thus, it appears that in aged rats mitochondria accumulate and store more Ca2C than in adult animals to protect cells from injury associated with higher levels of [Ca2C]i. This idea is reinforced
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by the fact that the contraction is usually more elevated in aged rats (Figs. 1, 3 and 4) and the mitochondrial buffering system may work to decrease Ca2C at the cytosolic level, as has been reported in basal forebrain neurons during aging (Murchison et al., 2004). On the other hand, the increase in Ca2Cm accumulation might be a key step in the damage process, since it causes an increase in ROS production and stimulate other mechanisms that lead to massive cell death (Ermak and Davies, 2001; Smaili et al., 2003). In fact, recent results from our laboratory have shown that aging causes a decrease in the transient openings of PTP, which might be an important mechanism to attenuate Ca2Cm accumulation and ROS enhancement (Lopes et al., 2004). These alterations might be related to the mitochondrial degeneration and apoptosis observed in colon smooth muscle (Lopes et al., 2004). Aging processes have shown to be closely related to the dysfunction of Ca2C homeostasis (Roberts et al., 1994; Xiong et al., 1993; Pottorf et al., 2000; Murchison and Griffith, 1998; Satru´stegui et al., 1996). Although some hypothesis has been proposed, the nature of these alterations are not well understood. Herein we present some evidences that agonist-induced [Ca2C]i increase and storage might be affected in colon smooth muscle of aged rats. These alterations were observed by simultaneous measurements of Ca2C and contraction, which allowed us to assess the correlations between the Ca2C stores, buffering systems and the contractile function. Based on these findings, we suggest that aging induces an increase in Ca2C stores (mitochondria, SR and other compartments), which may cause a Ca2C overload. The increase in intracellular Ca2C levels promotes alterations in the contractile responses to agonists. On the other hand, Ca2C accumulation in the organelles might be related to tissue injuries, which may contribute to cell death associated with aging. Acknowledgments This Project was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). References Abe, F., Karaki, H., Endoh, M., 1996. Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle. Br. J. Pharmacol. 118, 1711–1716. Barja, G., 1998. Mitochondral free radical production and aging in mammals and birds. Ann. N.Y. Acad. Sci. 854, 225–238. Bernardi, P., Petronilli, V., 1996. The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J. Bioenerg. Biomembr. 28, 131–138. Berridge, M.J, 1996. Elementary and global aspects of calcium signaling. J. Physiol. 499 (2), 291–306. Bolton, T.B., 1979. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Rev. 59, 606–718. Budd, S., 1998. Mechanisms of neuronal damage in brain hypoxia/ischemia: focus on the role of mitochondrial calcium accumulation. Pharmacol. Ther. 80 (2), 203–229.
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Pozzan, T., Rizzuto, R., Volpe, P., Meldolesi, J., 1994. Molecular and cellular physiology of intracellular calcium stores. Physiol. Rev. 74, 595–636. Roberts, D., Gelperin, D., Wiley, W., 1994. Evidence for age-associated reduction in acetycholine release and smooth muscle response in the rat colon. Am. J. Physiol. 267, G515–G522. ´ con, P., Vila, E., 2002. Rubio, C., Moreno, A., Briones, A., Ivorra, M.D., D’O Alterations by age of calcium handling in rat resistance arteries. J. Cardiovasc. Pharmacol. 40 (6), 832–840. Satru´stegui, J., Villalba, M., Pereira, R., Bogo´nez, E., Martinez, A., 1996. Cytosolic and mitochondrial calcium in synaptosomes during aging. Life Sci. 59 (5–6), 429–434. Smaili, S.S., Russel, J.T., 1999. The permeability transition pore regulates both mitochondrial membrane potential and agonist-evoked Ca2C signals in oligodendrocyte progenitors. Cell Calcium 26, 1–10. Smaili, S.S., Cavalcanti, P.M.S., Oshiro, M.E., Ferreira, A.T., Jurkiewicz, A., 1998. Ca2C release-activated channels in rat stomach smooth muscle cells. Eur. J. Pharmacol. 342, 119–122. Smaili, S.S., Hsu, Y.T., Carvalho, A.C., Rosenstock, T.R., Sharpe, J.C., Youle, R.J., 2003. Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling. Braz. J. Med. Biol. Res. 36 (2), 183–190. Szado, T., Kuo, K.H., Helary, K.B., Pburko, D., Lee, C.H., Seow, C., Ruegg, U.T., Van Breemen, C., 2003. Agonist-induced mitochondrial Ca2C transients in smooth muscle. FASEB J. 17, 28–37. van Breemen, C., Chen, Q., Laher, I., 1995. Superficial buffer barrier function of smooth muscle sarcoplasmic reticulim. Trends Pharmacol. Sci. 16, 98–104. Vanoevelen, J., Raeymaekers, L., Parys, J.B., De Smedt, H., Baelen, V., Callewaert, G., Wuytack, F., Missiaen, L., 2004. Inositol trisphosphate producing agonists do not mobilize the thapsigargin-insensitive part of the endoplasmic-reticulum and Golgi Ca2C store. Cell Calcium 35 (2), 115–121. Verhkratsky, A., Toescu, E.C., 1998. Calcium and neuronal ageing. Trends Neurosci. 21, 2–7. Xiong, Z., Sperelakis, N., Noffsinger, A., Fenoglio, P.C., 1993. Changes in calcium channel current densities in rat colonic smooth muscle cells during development and aging. Am. J. Physiol. 265, C617–C625.
Aging-related changes of intracellular Ca2C stores and contractile response of intestinal smooth muscle Guiomar S. Lopes a, Alice T. Ferreira b, Maria Etsuko Oshiro b, Irina Vladimirova c, Neide H. Jurkiewicz a, Aron Jurkiewicz a, Soraya S. Smaili a,* a
Department of Pharmacology, Federal University of Sa˜o Paulo (UNIFESP), Rua Treˆs de Maio—100, Sa˜o Paulo 04044-020, Sa˜o Paulo, Brazil b Department of Biophysics, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Brazil c Bogomoletz Institute of Physiology, Kiev, Ukraine Received 10 June 2005; received in revised form 31 August 2005; accepted 4 October 2005
Abstract In this study, we investigated the effect of aging on intracellular Ca2C stores, as sarcoendoplasmic reticulum (SR) and mitochondria, and the influence of these compartments on contraction of rat colon smooth muscle [Bitar, K.N., 2003. Aging and neural control of the GI tract V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins. Am. J. Physiol. Gastrointest. Liver. Physiol. 284(1), G1–G7; Marijic, J., Li, Q.X., Song, M., Nishimaru, K., Stefani, E., Toro, L., 2001. Decreased expression of voltage-and Ca2C-activated KC channels in ´ con, P., Vila, E., 2002. coronary smooth muscle during aging. Circ. Res. 88, 210–234; Rubio, C., Moreno, A., Briones, A. Ivorra, M.D., D’O Alterations by age of calcium handling in rat resistance arteries. J. Cardiovasc. Pharmacol. 40(6), 832–840]. Calcium stores and contraction were evaluated by simultaneous measurements of fluorescence and tension in smooth muscle strips loaded with fura-2. Results showed that activation of muscarinic receptors by methylcholine (MCh, 10 mM), induced a greater contraction in aged rats than in adult animals. The inhibition of Ca2C ATPase by thapsigargin (TG, 1 mM) did not prevent the refilling of SR either in adult or aged rats. MCh, in the presence of TG, induced an increase in transient fluorescence, indicating a release of Ca2C from TG-insensitive compartment. The mitochondrial uncoupler, FCCP (5 mM), caused a greater increase in intracellular Ca2C and tension in aged rats, indicating that mitochondria may accumulate more Ca2C during aging. The present results show that changes in intracellular Ca2C stores, such as mitochondria and SR, affect contraction and may cause dysfunctions during aging that could culminate in severe alterations of Ca2C homeostasis and cell damage. q 2005 Elsevier Inc. All rights reserved. Keywords: Calcium store; Contraction; Smooth muscle; Aging
1. Introduction One of the current hypothesis of the aging process is related to changes in Ca2C homeostasis and cytotoxicity, which are associated with the degenerative diseases (Choi, 1995; Verhkratsky and Toescu, 1998; Barja, 1998). Thus, Ca2C accumulation in the cytoplasm and alterations in Ca2C buffering by the sarcoendoplasmic reticulum (SR) and mitochondria could lead to oxidative stress and dysfunctions during aging (Barja, 1998). Excitation and contraction of the intestinal smooth muscle depend on the increase in the cytosolic Ca2C concentration ([Ca2C]i). The activation of metabotropic receptors, as * Corresponding author. Tel.: C55 11 5576 4449; fax: C55 11 5571 1776 E-mail address: [email protected] (S.S. Smaili).
0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2005.10.004
muscarinic receptors, may result in IP3 formation and release of SR Ca2C. This event may evoke a transient increase in [Ca2C]i which seems to be involved in the phasic component of the smooth muscle contraction (Berridge, 1996; Smaili et al., 1998). On the other hand, the influx of Ca2C from the extracellular medium through voltage-operated (VOCCs) and receptoroperated (ROCCs) Ca2C channels can be involved in the tonic contraction (Berridge, 1996; Bolton, 1979; Smaili et al., 1998). Under certain conditions, Ca2C entry can occur through storeoperated channels (SOCCs) activated by the emptying of the SR (Gibson et al., 1998). The activation of SOCCs appears to be of greater importance in tonic smooth muscles, as observed in the gastric fundus and in the vascular smooth muscle (Smaili et al., 1998; Gibson et al., 1998). A family of Ca2C pumps, the sarcoendoplasmic reticulum 2C Ca -ATPases (SERCA), mediates the Ca2C-uptake mechanisms in the SR (Pozzan et al., 1994). Thapsigargin (TG) and
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cyclopiazonic acid (CPA) have been shown to inhibit some types of SERCA pump which leads to a depletion of the Ca2C in TG-sensitive stores present in different smooth muscle cells (Pozzan et al., 1994; Go´mez-Viquez et al., 2003). In addition, under certain circumstances, TG-insensitive stores were also described (Neusser et al., 1999; Vanoevelen et al., 2004) as the Golgi complex of secretory cells, where a different Ca2CATPase is present. This TG-insensitive pump (SPCA1) was shown to play a role in modulating cytosolic and intra-Golgi Ca2C homeostasis (Vanoevelen et al., 2004). Another important TG-insensitive Ca2C store are mitochondria which modulate and shape cytosolic Ca2C signaling (Smaili et al., 2003). Mitochondrial Ca2C (Ca2Cm) uptake and release, either via the uniporter or NaC-dependent and NaCindependent exchange mechanisms, may certainly regulate [Ca2C]i (Budd, 1998; Szado et al., 2003). In addition, Ca2Cm efflux also occurs by the opening of the permeability transition pore (PTP) which operates in high or low conductance modes (Szado et al., 2003). The transient pore opening, associated with the low conductance state, is expected to result in a redistribution of Ca2C between the intra- and extramitochondrial spaces. It has been also proposed that this mechanism may provide a rapid Ca2C efflux which could protect the organelles against Ca2C overload and also amplify cytosolic Ca2C signals (Ichas et al., 1997; Smaili and Russel, 1999; Lopes et al., 2004). Changes in Ca2C homeostasis are among the most important causes of age-related alterations in the nervous system. It is unknown whether similar changes occur in the gastrointestinal system and what would be the correlation with the colon motor diseases that occur during aging processes (Verhkratsky and Toescu, 1998). These alterations have been associated with a decrease in acetylcholine release, a decrease in Ca2C influx via plasma membrane Ca2C channels or a decline in the Ca2C channel current (Roberts et al., 1994; Xiong et al., 1993). On the other hand, recent studies have shown that aging is associated with an increase in [Ca2C]i resulted from a decline in the Ca2C buffering capacity and a Ca2C store overload (Pottorf et al., 2000; Rubio et al., 2002). These results are in agreement with the study in forebrain neurons which showed that aging caused an increase in the Ca2Cm buffering capacity as a compensatory response to the increase in Ca2C influx (Murchison and Griffith, 1998). In fact, aging might alter mitochondrial function, as the uniporter, the membrane potential or the PTP activity (Satru´stegui et al., 1996; Lopes et al., 2004). Altogether indicate that during aging several aspects of Ca2C homeostasis might be affected, as Ca2C influx, release of Ca2C from intracellular stores and Ca2C uptake processes by the SR and mitochondria. The present study aimed to characterize the changes in the SR and mitochondrial intracellular Ca2C stores in colon smooth muscle from adult and aged rats. We investigated the influence of these changes on [Ca 2C ] i signaling and contraction. The relationship between [Ca2C]I and tension was assessed by recording both parameters simultaneously. The results showed that aging may alter Ca2C stores and contraction in colon smooth muscle and these changes might
affect smooth muscle contractility after metabotropic receptor stimulation. 2. Materials and methods 2.1. Isolation and preparation of rat colon Female Wistar 2BWA adult rats (4–6 mo) and aged rats (24–30 mo) were killed with an overdose of ether, and a segment of their terminal colon was excised. The smooth muscle layer was freed of the adjacent mucosa and washed in Tyrode solution with the following composition: 145 mM/l NaCl; 5.5 mM/l KCl; 2.5 mM/l CaCl2; 1 mM/l MgCl2, 10 mM/l HEPES; and 10 mM/l glucose. 2.2. Incubation and preparation of colon smooth muscle Samples of colon smooth muscle were mounted vertically in a cuvette and a designed holder fixed on one end of the muscle to the bottom of the cuvette while the other at the top was attached to an isometric force transducer. The resting tension was adjusted to 1 g (1 V). The tissue was perfused with 2.5 ml Tyrode solution at 37 8C, bubbled continuously with 95% O2 and 5% CO2. The muscle strip was incubated with 4 mM fura2/AM plus 0.02% pluronic acid F-127 for 3.5 h at room temperature. 2.3. Simultaneous measurements of fluorescence and contraction A system containing a transducer and spectrofluorimeter coupled to a computer system allowed simultaneous measurements of dual wavelength fluorimetry and tension. Experiments were performed using a fluorimeter (PTI, Photon Technology International, Inc, New Jersey, USA). After fura2 loading, tissues were subjected to excitation wavelengths of 340 and 380 nm. Emitted fluorescence was collected at 510 nm. The ratio of 340–380 nm excitation (R340/380) was reported as a relative measurement of [Ca2C]i (Himpens and Somlyo, 1988). Results were expressed in fluorescence ratio (340/380) values after background subtraction which was approximately 40% of the total fluorescence measured at the 340 or 380 nm. Fluorescence intensities were normalized in relation to the average of the baseline values. Briefly, fluorescence intensity in the non-zero pixels were averaged (F) and ploted as normalized fluorescence (DF/F0 X100) against time (Smaili and Russel, 1999). DF was calculated as the difference between the mean value of the first 20 data points prior to stimulation of the cell (Fo) and F. Passive tension of 1 g was initially applied and the tissue was allowed to equilibrate for 30 min. Isometrically recordings were obtained with a force–displacement transducer and tension values were expressed in grams. Fluorescence and tension were analyzed in order to obtain four parameters for comparison between groups. These included the maximum increase in fluorescence and tension expressed in percentage over the baseline values (all Figs. C and D) and the rate of
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decline of the calcium and tonic contractile response (All Figs. E and F). The decline expresses the percentage of decrease in the respective measurement from the peak to the stabilization of the response. MCh (10 mM) was applied after 3 min of steady level under a resting tension and tested in normal and nominally (without EGTA) Ca2C-free solution. Drugs were added by medium replacement and the samples were washed five times with Tyrode between different drugs. The amplitude of the agonistevoked increase in the transient fluorescence ratio (R340/380) was taken as intracellular Ca2C increase. In some experiments TG was incubated for 10 min followed by MCh (10 mM) addition. We tested whether the increase in fluorescence was due to the Ca2C influx through VOCC. Therefore, some experiments were performed in the presence of verapamil (10 mM), a VOCC antagonist that was added to the incubation medium 30 min before the beginning of the experiments. In other experiments, SKF96365 (4 mM), an antagonist of the ROCC was also added, to avoid interference and allow observation of transport from Ca2C stores during intracellular Ca2C measurements.
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In another experiment, the mitochondrial uncoupler FCCP, which causes a rapid and reversible collapse of the proton gradient across the mitochondrial membrane (DJm), was also used (Budd and Nicholls, 1996). This drug inhibits mitochondrial Ca2C uptake and may be used to evaluate the effect of this mechanism in Ca2Cm accumulation (Budd, 1998; Ichas et al., 1997). Under certain conditions and depending on the system, FCCP may also cause a release of Ca2Cm (Smaili and Russel, 1999). Because FCCP can cause a severe ATP consumption by reversal of the F0–F1 ATP synthase, FCCP was always added together with oligomycin (1 mg/m), which inhibits mitochondrial ATP synthase and prevents ATP consumption in the presence of FCCP (Budd and Nicholls, 1996; Bernardi and Petronilli, 1996). Oligomycin alone had no significant effect on basal levels of Ca2C and tension (data not shown). At the end of all experiments, calibration was performed with the addition of digitonin (100 mM) followed by MnCl2 (2 mM) and EGTA (4 mM). All fluorescence and tension values were normalized to the basal fluorescence and basal tension.
Fig. 1. Effect of MCh (10 mM) on fluorescence and tension measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A) MCh induced a transient followed by a sustained elevation in fluorescence ratio. (B) Contraction induced by MCh also showed a phasic and a tonic component. The phasic response was greater in the aged rats. (C) Histogram shows the maximum fluorescence observed with MCh and compared the effects in adult and aged animals. (D) Histogram compares the maximal tension obtained with MCh which was significantly higher in aged rats. (E) Shows the rate of decline of the sustained phase of fluorescence. (F) the rate of decline in tension, which were not different between adults and aged animals. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
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Fig. 2. Effect of MCh (10 mM) on fluorescence and tension in Ca2C-free solution measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A) MCh induced similar increase in transient fluorescence both in adult and aged rats. (B) MCh-induced tension was higher in aged than in adult animals. (C,D) Histograms show that there is no significant difference between adult and aged rats either in the maximum fluorescence ratio or tension induced by MCh. (E,F) In Ca2C-free solution the rate of decline in fluorescence and tension were significantly faster in aged rats. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
Drugs used were methylcholine (MCh), FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), oligomycin, verapamil and thapsigargin (Sigma Chemical Co., St. Louis, MO, USA); SKF96365 (Calbiochem, La Jolla, CA, USA); fura-2/AM and pluronic acid F127 (Molecular Probes Inc., Eugene, OR, USA). 2.4. Statistics The normalized fluorescence and tension were expressed as means GSEM plotted against time. Data express the means of at least six experiments and statistical analyses were performed by Student t-test and Welch correction. 3. Results 3.1. Effects of MCh Simultaneous measurements in fura-2-loaded strips of rat colon were tested with MCh (10 mM), which caused an increase in fluorescence ratio and contraction. MCh induced a transient increase that was followed by a sustained
elevation in fluorescence. This response was similar to the contractile pattern where the phasic and tonic contraction was recorded (Fig. 1A,B). We measured the amplitude of the transient increase in fluorescence and the decline of sustained phase as well as the increase of phasic contraction and the decline of tonic phase contraction and compared them between adult and aged rats. Results show that the increase in Ca2C and the fluorescence rate decline were very similar in both groups of animals (Fig. 1A,C,E). However, the maximum amplitude of tension (phasic component) in aged animals was significantly higher when compared with adult rats (Fig. 1B–D), and the decline in contraction in aged animals was not significantly different from the one observed in adult animals (Fig. 1F). To investigate the influence of extracellular Ca2C influx during muscarinic receptor stimulation, experiments were performed in the absence of external Ca2C. Fig. 2(A,B) shows that even when the muscles were incubated in nominally Ca2C-free solution, MCh still induced a transient Ca2C increase with similar magnitude in both adult and aged rats (Fig. 2A,D). It is interesting to point out that under this condition the contraction in aged rats was similar to that
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Fig. 3. Effect of MCh (10 mM) in the presence of TG (1 mM) on fluorescence and tension measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A,B) TG did not induce a sustained elevation in fluorescence but increased [Ca2C]i oscillations in both groups, with higher amplitude in aged rats. In the presence of TG, the effect of MCh was higher in aged rats. (C,D) Maximum fluorescence and tension induced by MCh in the presence of TG were significantly increased in aged rats. (E,F) There is no difference in the rate of decline of the MCh responses when TG was present. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
observed when the normal solution was used (Figs. 1D and 2D). Although the contraction in aged rats in Ca2C-free solution was slightly increased in relation to the normal solution, this was not significantly different (Figs. 1B and 2B). As shown in Fig. 2, the decrease in Ca2C was faster in aged rats which might be related to the release of Ca2C from intracellular compartments. In fact, ER and other compartments can be depleted after incubation with Ca2C-free solution. On the other hand, the declines in fluorescence and contraction were significantly faster in aged rats (Fig. 2A–F), which allowed the restoration to the basal Ca2C levels. These results indicate that the phasic components of fluorescence and contraction are dependent on intracellular Ca2C in both groups of animals. However, the sustained increase in intracellular Ca2C and the tonic phase of contraction seem to be more dependent on extracellular Ca2C in aged rats, or the kinetics of the [Ca2C]i buffering system is faster in these animals. It is interesting to point out that the oscillations observed in Fig. 1 were not present when tissues were incubated in Ca2C-free solution. Thus, the nature and the source of the intracellular Ca2C mobilization were then investigated in both groups of animals.
3.2. Effects of MCh (10 mM) in the presence of TG (1 mM) In these experiments, the Ca2C-ATPase blocker, TG was applied for 10 min before the addition of MCh. The presence of TG resulted in transient increases in fluorescence and tension with an oscillatory pattern both in adult and aged animals. These oscillations were more marked in aged rats (Fig. 3A,B). MCh caused elevation of the intracellular Ca2C that was significantly higher in aged rats (25.2G5.7% in adult and 89.1G17.1% in aged) (Fig. 3C) and more elevated than in the control in the absence of TG (56.4G6.1% in the absence and 89.1G17.1% in the presence of TG) (Fig. 1C and 3 C). However, in adult animals the TG treatment had no significant effect on the MCh-induced transient increase in Ca2C or on the amplitude of the sustained contraction. However, MCh contraction was significantly higher in aged rats (Fig. 3B,D). The decline of fluorescence and tonic contraction was not significantly different between the two groups (Fig. 3E,F). These results indicate that in adult animals, MCh response might be dependent on TG-insensitive Ca2C stores. On the other hand, in aged animals other TG-insensitive compartments may play an important role during MCh response.
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Fig. 4. Effect of MCh (10 mM) in the presence of FCCP (5 mM) plus oligomycin (1 mg/ml) on fluorescence and tension measured simultaneously in colon smooth muscle from adult (dashed line) and aged (solid line) rats. (A,B) FCCP caused an increase in fluorescence and tension. In the presence of FCCP, MCh still causes an increase in fluorescence and tension that were higher in aged rats. (C,D) Elevations in fluorescence and tension caused by FCCP were significantly increased in aged animals. (E,F) In the presence of FCCP, MCh evoked increases in fluorescence and tension that were significantly greater in aged rats than in the adult group. Fluorescence and tension values were normalized as described in Section 2. Tension was expressed in grams. Data represent meansGSEM of at least six experiments.
Therefore, studies were conducted to investigate the influence of mitochondria on these effects. All the experiments were performed in the presence of verapamil and SKF96365, to avoid extracellular Ca2C influx and interference during activation of SR stores by TG and MCh (see above). These Ca2C channel blockers did not affect responses to TG and MCh. 3.3. Effects of MCh in the presence of FCCP (5 mM) In order to test the influence of the Ca2C store at the mitochondrial level, FCCP plus oligomycin (see Section 2) were added to the colon strips. FCCP was used to collapse DJ and reduce the electrochemical driving force for mitochondrial Ca2C import and allow mitochondria to release accumulated Ca2C (Smaili and Russel, 1999; Bernardi and Petronilli, 1996). Fig. 4A,C shows that FCCP caused an increase of transient fluorescence in both preparations and this was significantly higher in aged rats. It was also observed that FCCP induced a further increase in tension in aged rats (Fig. 4B,D). In the presence of FCCP, the effects of MCh on Ca2C transients and
contraction were significantly higher in aged rats (Fig. 4E–F). The peak of Ca2C in aged rats in response to MCh and in the presence of FCCP, was significantly greater when compared to the effect in the absence of this drug (108.1G13.6% in the presence and 56.4G6.1% in the absence of FCCP) (Figs. 4E and 1C). It is important to point out that independently of the absence or presence of FCCP or TG, contraction in aged rats was increased in relation to their controls (Figs. 1, and 4). These results indicate that Ca2C may accumulate in the intracellular stores, especially into the mitochondria, during aging. 4. Discussion The aim of the present study was to investigate the agerelated changes in the intracellular Ca2C stores and contractile response in the colon smooth muscle. Our results showed that smooth muscle contraction induced by MCh involves an increase in the [Ca2C]i as a result of Ca2C release from intracellular stores. The simultaneous measurement of Ca2C and tension showed that the contraction was accompanied by
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an increase in the intracellular Ca 2C level, although the changes in these parameters were not always correlated (Abe et al., 1996). In addition, MCh induced a transient increase in fluorescence and contraction and the last was more elevated in the aged animals (Fig. 1C,D). Several hypothesis could be raised to explain the difference between Ca2C responses and contraction in aged rats. One is the presence of the superficial buffer barrier (SBB). In this case, the peripheral SR could interact with the plasmalemma to perform a specialized function and regulate [Ca2C]i. This barrier could accumulate more Ca2C before it could reach the myofilaments (van Breemen et al., 1995). Thus, it is possible to suggest that in aged rats the SBB is less efficient, which would cause more accumulation of Ca2C in the cytosol and the increase in contraction observed (Fig. 1). Another possible explanation is the fact that these animals might show different contents in their intracellular Ca2C stores with different kinetic of Ca2C release. In fact, MCh-induced Ca2C signaling and contraction showed to be strongly dependent on the intracellular Ca2C stores, since the phasic responses were not significantly altered when in Ca2C free solution (Fig. 2A–D). However, in aged rats, the tonic components, were more affected by the Ca2Cremoval, where the rate of decline was faster. Therefore, it is possible that either this sustained response is partially dependent on extracellular Ca2C or the intracellular Ca2C stores could be more rapidly emptied. To investigate the intracellular Ca2C stores in adult and aged animals, we performed experiments in the presence of the inhibitors of these compartments. We showed that TG itself did not induce a sustained elevation of [Ca2C]i or tension in adult and aged rats. However, it increased the amplitude of the spontaneous Ca2C oscillations. On this point, it is interesting to note that Ca2C oscillations were markedly present in aged animals. Since the SR is very important for these oscillatory changes in Ca2C signaling (Smaili et al., 2003), these results indicate that the SR can be affected with aging and may accumulate more Ca2C. This result is in agreement with the studies performed in vascular smooth muscle from aged and spontaneously hypertensive rats (Neusser et al., 1999; Matz et al., 2003). However, MCh response in the presence of TG was not affected in adult rats, indicating that MCh may evoke TGinsensitive stores to promote its effect. In addition, the contraction induced by MCh in this condition, was much higher in aged rats, suggesting that these TG-insensitive store might be more important and store more Ca2C in these animals. TG-insensitive stores include mitochondria, thus, we have investigated this compartment and its effect on the response during aging. We observed that FCCP caused a larger increase in [Ca2C]i and contraction in aged rats (Fig. 4A–D). This effect might be associated with the release of Ca2Cm accumulated in the organelle, since the experiments were performed in the presence of verapamil and SKF96365 to avoid interference of FCCP with the plasma membrane Ca2C channels. Thus, it appears that in aged rats mitochondria accumulate and store more Ca2C than in adult animals to protect cells from injury associated with higher levels of [Ca2C]i. This idea is reinforced
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by the fact that the contraction is usually more elevated in aged rats (Figs. 1, 3 and 4) and the mitochondrial buffering system may work to decrease Ca2C at the cytosolic level, as has been reported in basal forebrain neurons during aging (Murchison et al., 2004). On the other hand, the increase in Ca2Cm accumulation might be a key step in the damage process, since it causes an increase in ROS production and stimulate other mechanisms that lead to massive cell death (Ermak and Davies, 2001; Smaili et al., 2003). In fact, recent results from our laboratory have shown that aging causes a decrease in the transient openings of PTP, which might be an important mechanism to attenuate Ca2Cm accumulation and ROS enhancement (Lopes et al., 2004). These alterations might be related to the mitochondrial degeneration and apoptosis observed in colon smooth muscle (Lopes et al., 2004). Aging processes have shown to be closely related to the dysfunction of Ca2C homeostasis (Roberts et al., 1994; Xiong et al., 1993; Pottorf et al., 2000; Murchison and Griffith, 1998; Satru´stegui et al., 1996). Although some hypothesis has been proposed, the nature of these alterations are not well understood. Herein we present some evidences that agonist-induced [Ca2C]i increase and storage might be affected in colon smooth muscle of aged rats. These alterations were observed by simultaneous measurements of Ca2C and contraction, which allowed us to assess the correlations between the Ca2C stores, buffering systems and the contractile function. Based on these findings, we suggest that aging induces an increase in Ca2C stores (mitochondria, SR and other compartments), which may cause a Ca2C overload. The increase in intracellular Ca2C levels promotes alterations in the contractile responses to agonists. On the other hand, Ca2C accumulation in the organelles might be related to tissue injuries, which may contribute to cell death associated with aging. Acknowledgments This Project was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). References Abe, F., Karaki, H., Endoh, M., 1996. Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle. Br. J. Pharmacol. 118, 1711–1716. Barja, G., 1998. Mitochondral free radical production and aging in mammals and birds. Ann. N.Y. Acad. Sci. 854, 225–238. Bernardi, P., Petronilli, V., 1996. The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J. Bioenerg. Biomembr. 28, 131–138. Berridge, M.J, 1996. Elementary and global aspects of calcium signaling. J. Physiol. 499 (2), 291–306. Bolton, T.B., 1979. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Rev. 59, 606–718. Budd, S., 1998. Mechanisms of neuronal damage in brain hypoxia/ischemia: focus on the role of mitochondrial calcium accumulation. Pharmacol. Ther. 80 (2), 203–229.
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