Department of Physiology and Neuroscience, Lund University, S-223 62 Lund, Sweden
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ABSTRACT |
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Longitudinal smooth muscle strips from guinea pig ileum were cultured in vitro for 5 days, and the relationship between extracellular Ca2+ and force in high-K+ medium was evaluated. In strips cultured with 10% fetal calf serum (FCS), this relationship was shifted to the right (50% effective concentration changed by 2-3 mM) compared with strips cultured without FCS. The shift was prevented by inclusion of verapamil (1 µM) during culture and mimicked by ionomycin in the absence of FCS. The intracellular Ca2+ concentration ([Ca2+]i) during stimulation with high-K+ solution or carbachol was reduced after culture with FCS, whereas the [Ca2+]i-force relationship was unaffected. Cells were isolated from cultured strips, and whole cell voltage-clamp experiments were performed. Maximum inward Ca2+ current (10 mM Ba2+), normalized to cell capacitance, was almost three times smaller in cells isolated from strips cultured with FCS. Culture with 1 µM verapamil prevented this reduction. These results suggest that increased [Ca2+]i during culture downregulates Ca2+ current density, with associated effects on contractility.
tissue culture; fura 2; patch clamp; verapamil; ionomycin
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INTRODUCTION |
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SMOOTH MUSCLE CELLS in culture rapidly lose their contractile phenotype (3). However, the presence of extracellular matrix constituents has been shown to slow the phenotypic transition (17, 28). Culture of smooth muscle tissue, rather than isolated cells, might provide an environment that better preserves the contractile phenotype, since the surrounding matrix is still present. When smooth muscle tissue is cultured under serum-free conditions, contractility is well preserved for periods of ~5 days (16). However, the presence of fetal calf serum (FCS) during such short-term culture of intestinal smooth muscle decreases maximum force- generating capacity (23, 26). In rat tail artery, it was shown that this effect of serum can be largely eliminated by the presence of the L-type Ca2+ channel blocker verapamil during culture with FCS, suggesting that the decreased contractility is associated with Ca2+ influx (18).
Motility in the gastrointestinal tract involves spontaneous depolarization of the smooth muscle cell membrane, which activates voltage-dependent Ca2+ channels. A voltage-activated, dihydropyridine-sensitive L-type Ca2+ channel has been shown to be present in longitudinal smooth muscle cells of guinea pig ileum (5, 6). It was considered that the effects of FCS on contractility might be mediated by influence on this channel type. This should lead to altered intracellular Ca2+ levels during activation. Depolarization causes a rise in intracellular Ca2+ that stimulates phosphorylation of the myosin regulatory light chains, leading to contraction. Several of these activation steps downstream of membrane depolarization might be affected by culture, in which case the relationship between intracellular Ca2+ and contractile force would be affected. In the present work, we investigated if changes in activation properties can be a factor behind the changes in contractility induced by FCS.
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MATERIALS AND METHODS |
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Animals and tissue culture.
Female guinea pigs weighing 300-500 g were killed by cervical
fracture. A 20- to 30-cm segment of the ileum was detached from mesenterium. Strips (0.1 × 0.2 × 15 mm) were teased along
natural lines of cleavage from the outer longitudinal muscle layer and then suspended isometrically on holders made of stainless steel wire
(0.3 mm in diameter). The holders were transferred to 2.5-ml culture
dishes with medium, and additions were made as specified in the text.
The culture dishes were incubated at 37°C for 5 days, or
12-108 h in time-course experiments, in a water-jacketed incubator with an atmosphere of 5% CO2 in
air. The medium consisted of Dulbecco's modified Eagle's medium and
Ham's F-12 (1:1) with 50 U/ml penicillin and 50 µg/ml streptomycin,
and either 0 or 10% FCS (all from Biochrom KG). Verapamil (Sigma
Chemical, St. Louis, MO) was prepared to a
103 M stock solution in
water, and ionomycin (Calbiochem, La Jolla, CA) was prepared to stock
solutions of 5 × 10
3
and 5 × 10
4 M in
ethanol.
Isometric force measurements.
Strips were removed from the holders, dissected to the approximate
dimensions of 0.1 × 0.1 × 10 mm, and mounted for force measurements as described (26). Strips were allowed to equilibrate for
15 min after mounting before further experimentation. Force was
recorded on a potentiometric recorder and on magnetic tape. Data were
later digitized for evaluation of mean force under different test
conditions. Strip dimensions were determined at four positions along
the length of the preparation using a microscope with an ocular scale,
and the mean diameter was used for calculation of cross-sectional area
assuming circular cross section. Strips cultured with serum, verapamil,
and ionomycin were always tested in parallel with controls. All
Ca2+-force data were fitted by the
following equation: F = (a d)/[1+ (x/c)b] + d, where F is force,
a and
d are asymptotic maximum and minimum values, respectively, x is Ca2+ concentration, c
is the concentration of Ca2+
giving 50% of maximum force
(EC50), and
b is a slope parameter.
Fura 2 measurements. Strips were mounted for surface fluorometry of fura 2 in an experimental setup described by Nilsson and Hellstrand (20). Preparations were loaded with 8 µM of the cell-permeant fura 2-acetoxymethyl ester (AM) at 22°C for 3-4 h. This solution was exchanged every hour. At these times, the preparations were flushed with prewarmed solution and contracted with 140 mM K+ before return to fresh loading solution. Force, in response to 140 mM K+ and after loading with fura 2, amounted to 106.1 ± 8.7% (0% FCS, n = 11) and 101.2 ± 14.9% (10% FCS, n = 7) of the response of the unloaded preparations, indicating that viability was not affected by the loading protocol. Strips were allowed to equilibrate for 15 min at 37°C after loading before the test protocol was started. At the end of the experiment, a calibration was performed in situ as described by Himpens et al. (9) and intracellular Ca2+ was calculated as described by Grynkiewicz et al. (8). The dissociation constant of fura 2 under the present intracellular conditions is not known but was taken to be 224 nM.
Preparation of cells. After removal of the cultured strips from their holders, the tissue was incubated for 10 min under continuous mechanical agitation at 35°C in 2 ml of the dispersion medium (DM, see below) containing 0.6 mg/ml collagenase (type I, Sigma), 0.5 mg/ml papain (type IV, Sigma), 2.5 mg/ml bovine serum albumin (type V, essentially fatty acid free), and 5 mM dithiothreitol. The DM solution contained (in mM) 110 NaCl, 5 KCl, 0.16 CaCl2, 2 MgCl2, 10 HEPES, 10 NaHCO3, 0.5 KH2PO4, 0.5 NaH2PO4, 10 glucose, 0.04 phenol red, 0.49 EDTA, and 10 taurine (pH 7.0 at room temperature). The enzyme mixture was then removed, and fresh DM was added. The tissue was gently agitated using a Pasteur pipette until a suspension of isolated cells was generated. After centrifugation for 5 min at 800 revolutions/min, the cells were resuspended, stored in DM solution at 4°C, and used within 4 h.
Whole cell recordings.
Membrane currents were recorded as described in previous work (6).
Whole cell voltage clamp was achieved with patch electrodes (2-5
M) using an Axopatch-200 amplifier (Axon Instruments, Foster City,
CA). Series resistance and capacitive currents were compensated, and
signals were filtered at 1 kHz (
3 dB) by the circuitry in the
Axopatch-200. Leakage was corrected by subtracting the summed response
to eight hyperpolarizing pulses with an amplitude equal to one-eighth
of the test pulse. All data were recorded and further analyzed by
pCLAMP software (Axon Instruments). All recordings were performed at
room temperature (21-24°C).
Statistics. The two-tailed unpaired Student's t-test, with the Bonferroni correction for multiple comparisons, was used for evaluation of statistical significance. The Fisher's exact test was used to calculate the probability that the difference in phasic spontaneous activity between strips cultured with 0 and 10% FCS for >60 h had arisen by chance (Fig. 3). P < 0.05 was considered statistically significant. Values given are means ± SE.
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RESULTS |
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Isometric force measurements. Strips of longitudinal ileum muscle were maintained in vitro under different conditions for 5 days and then used for isometric force measurements. After they were mounted, strips were twice depolarized with high (144 mM)-K+ solution. They were then transferred to Ca2+-free solution and, after relaxation, depolarized with 60 mM K+. In this state, extracellular Ca2+ was increased cumulatively. It was found that 60 mM K+ is optimal for maintained force responses, even though 144 mM K+ gives somewhat higher peak force (see Fig. 2B ). Each Ca2+ concentration was maintained for 5 min, and the mean force during this period was plotted as a function of the Ca2+ concentration. As illustrated in Fig. 1, the sensitivity of the strips to extracellular Ca2+ depended on the conditions during the incubation period. The force response after incubation with 10% FCS did not saturate even at 16 mM extracellular Ca2+, making an exact determination of sensitivity difficult. This does not, however, invalidate the conclusion that culture with 10% FCS decreases the sensitivity of force to extracellular Ca2+ by 2-3 mM (Fig. 1A).
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Fura 2 measurements. The altered sensitivity of the strips cultured with 10% FCS could in principle involve either the sensitivity of the contractile machinery to intracellular Ca2+ concentration ([Ca2+]i) or the level of [Ca2+]i in the depolarized cell at a given extracellular Ca2+ concentration. For evaluation of [Ca2+]i, strips were loaded with fura 2 and taken through the protocol shown in Fig. 4. After an initial period in HEPES, strips were contracted with 10 µM carbachol and 144 mM K+. This was followed by relaxation in Ca2+-free solution containing 60 mM K+ to depolarize the membrane and activate voltage-dependent channels. In this depolarized state, Ca2+ was introduced in a cumulative manner (0.5, 2, 16, 32 mM) after decline of [Ca2+]i and force to basal levels.
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Patch-clamp experiments. To test the hypothesis that the altered sensitivity to extracellular Ca2+ in depolarized tissue depends on altered Ca2+-influx mechanisms, whole cell inward currents were measured in cells isolated from the cultured strips. L-type Ca2+ channels have previously been demonstrated in freshly isolated cells from noncultured longitudinal smooth muscle of guinea pig ileum (5, 6).
Inward current responses were evoked by 300-ms depolarizing pulses to +10 mV from a holding potential of
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DISCUSSION |
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The main conclusion of the present study is that culture of intestinal smooth muscle in the presence of FCS decreases the sensitivity to extracellular Ca2+ and the density of Ca2+ current over the cell membrane. Intracellular relative to extracellular Ca2+ concentration was reduced in the depolarized muscle after culture, but the relationship between [Ca2+]i and force was unchanged. This indicates that the altered sensitivity to extracellular Ca2+ is due to altered Ca2+ handling at the membrane level. Because the rate of elimination of [Ca2+]i from the cytoplasm was unchanged, it can be concluded that inflow rather than outflow of Ca2+ is affected. These results make it likely that the decreased sensitivity of force to extracellular Ca2+ is a consequence of the decreased inward current.
Maintained force development in a depolarized muscle cell depends on the magnitude of the window current existing at the given membrane potential, which will depend on the channel density and on the activation and inactivation properties of the channels (15). Because the current-voltage relations, as well as activation and inactivation properties, were found to be unaltered after culture with FCS, it is likely that [Ca2+]i is reduced due to a functional decrease in channel density.
The present study also indicates that culture with FCS depresses the maximum force-generating ability (e.g., Fig. 5). The mechanism of this effect has not been addressed, but the effect could be considered to depend on a decrease in the amount of contractile proteins, such as myosin or actin. Further possibilities include disintegration of cells from the syncytium and cell death. It is, however, clear from the present results that Ca2+ inflow after serum treatment has become limiting for force generation at physiological concentrations of Ca2+.
FCS is a rich but unspecified source of growth factors that has been used in several studies to stimulate growth of smooth muscle cells (2, 21, 23, 24, 30). Application of FCS is found to acutely increase [Ca2+]i (18) and to stimulate progression of cells through the cell cycle (11), leading to increased DNA synthesis (18).
Decreased inflow of Ca2+ over the cell membrane, achieved by the presence of verapamil during culture, reversed the effect of FCS on Ca2+ sensitivity and current density, whereas increase of [Ca2+]i in the absence of FCS mimicked the effect on Ca2+ sensitivity. This indicates that chronically increased [Ca2+]i causes decreased functional expression of Ca2+ channels, implying that any constituent of serum with an ability to raise intracellular Ca2+, including, e.g., growth factors and serotonin, might cause the effects. The molecular mechanisms behind the effect on Ca2+ currents are, however, unknown. In PC-12 cells, it was shown that chronic exposure to high-K+ concentration or to ionomycin decreases [3H]nitrendipine binding and 45Ca2+ influx, which demonstrates a coupling between [Ca2+]i and functional channel expression also in this cell type (4).
In several types of smooth muscle, the density of Ca2+ (31) and Na+ currents (10, 14) has been shown to change during maturation, aging, and gestation. The events leading to these changes are, however, only partly understood. Support for cell cycle-dependent regulation of Ca2+ current density in aortic myocytes was recently obtained by Kuga et al. (13). These authors showed that L- and T-type currents were low in the G0 phase of the cell cycle, peaked in G1 and/or S, and then decreased. These data support and further extend earlier observations in myocytes of vascular origin (12, 19, 22). In the present study, information on how cells were distributed in different phases of the cell cycle was not obtained, and thus this factor cannot be ruled out.
We have earlier reported that the polyamines spermidine and spermine, ubiquitous cellular polycations associated with growth and differentiation, acutely inhibit L-type Ca2+ current and sensitize the contractile apparatus to Ca2+ in guinea pig ileum longitudinal smooth muscle (6, 25). It was also speculated that such influences could vary under different circumstances, such as during cellular growth when the concentrations of polyamines are known to increase (29). In the present study, no evidence of an altered sensitivity of force to [Ca2+]i was found after serum stimulation, which, under identical conditions, increased putrescine and spermidine but not spermine contents (26). On the other hand, inhibition of polyamine synthesis decreases Ca2+ sensitivity in cultured ileum while also causing maintained spontaneous activity, which is otherwise lost during culture with FCS (26). The electrophysiological basis of this latter effect remains to be established, but it is possible that polyamines, either directly or via their effects on cellular growth, may affect functional ion channel properties. It is, however, not likely that elevated intracellular polyamines could have directly influenced the present whole cell recordings, since the cell interior would be dialyzed by the pipette solution.
The connection between long-term changes in intracellular Ca2+ and functional Ca2+ channel expression as demonstrated by the present results may establish a link between growth stimulation, including growth in response to increased stretch or contractile activity, and alterations in pattern of myogenic tone and reactivity to neurotransmitters and hormones. However, much work remains to establish if these effects seen in cultured smooth muscle tissue are also present under in vivo conditions.
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ACKNOWLEDGEMENTS |
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We thank Professor Per Hellstrand for useful discussions and suggestions.
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FOOTNOTES |
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This study was supported by the Swedish Medical Research Council (Grant 04X-28), the Medical Faculty, University of Lund, and AB Astra-Hässle, Mölndal.
A preliminary report of this work has been published (7).
Address for reprint requests: K. Swärd, Dept. of Physiology and Neuroscience, Lund Univ., Sölvegatan 19, S-223 62 Lund, Sweden.
Received 5 May 1997; accepted in final form 23 July 1997.
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