Long-term regulation of contractility and calcium current in smooth muscle

Maria Gomez and Karl Swärd

Department of Physiology and Neuroscience, Lund University, S-223 62 Lund, Sweden

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 10-3 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.

Phasic spontaneous contractile activity was evaluated by inspection of the force traces during the 15-min equilibration period. Individual strips were considered to display activity if spontaneous contractions were observed over a period of 3 min.

Solutions used in tissue bath experiments were of the following composition (in mM): 135.5 NaCl, 5.9 KCl, 1.2 MgCl2, 11.6 glucose, and 11.6 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.35, 37°C. High-K+ solutions were obtained by isosmolar substitution of NaCl for KCl. The solution with the highest extracellular Ca2+ concentration (32 mM) in the fura 2 experiments was constructed to have the same osmolarity as the solution with 16 mM Ca2+ by exchange of 24 mM NaCl for 16 mM CaCl2. Solutions for evaluation of the sensitivity of force to extracellular Ca2+ contained 1 µM atropin, 1 µM phentolamine, and 10 µM propranolol to exclude effects of autonomic transmitters.

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 MOmega ) 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).

Steady-state activation curves were estimated from the peak inward current, corrected for the change in driving force at each of the test potentials. Driving force was calculated from the difference between test potential and the observed reversal potential (+55 mV). Steady-state inactivation curves were obtained using a two-pulse protocol in which the membrane was clamped from -90 mV to different voltages for 15 s (prepulse) and then stepped to +10 mV for 280 ms (test pulse). The difference between peak current and late current measured at the end of the test pulse was plotted against prepulse potential.

The control extracellular solution used during whole cell recordings contained (in mM) 105 NaCl, 5.4 KCl, 1 MgCl2, 10 BaCl2, 30 tetraethylammonium chloride, 10 HEPES, and 5 glucose. To decrease the Ca2+-mediated inactivation of the Ca2+ channels, Ba2+ was used as the charge carrier. Pipette solution was of the following composition (in mM): 144 CsCl, 2 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 HEPES, 5 ATP, and 5 phosphocreatine. The solution was titrated to pH 7.2 with CsOH.

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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Fig. 1.   Relationships between extracellular Ca2+ concentration ([Ca2+]EC) and force at 60 mM K+ in longitudinal smooth muscle strips from guinea pig ileum, maintained for 5 days under different conditions. Concentration of Ca2+ giving 50% of asymptotic maximum force (EC50, in mM) is indicated. A: force of strips maintained in 0% fetal calf serum (0% FCS, open circle , n = 50) and 10% FCS (bullet , n = 35). P < 0.05 at all [Ca2+]EC values. B: effect of 1 µM verapamil added to medium with 10% FCS (black-square, n = 6). P < 0.05 at all [Ca2+]EC values except at 16 mM. C: lack of effect of 1 µM verapamil in the absence of FCS (square , n = 9). D: effects of 0.5 and 2 µM ionomycin (down-triangle and triangle , n = 8 and 5, respectively). P < 0.05 except at 0.5 µM ionomycin in combination with Ca2+ concentrations >1 mM.

FCS is widely used as a stimulator of growth and has been shown to stimulate an increase of intracellular Ca2+ in vascular smooth muscle (18). The rightward shift in the sensitivity of force to extracellular Ca2+ might be considered to depend on this effect. To test if influx of Ca2+ is necessary for the effect, 1 µM verapamil was added to medium with 10% FCS to block voltage-gated Ca2+ channels during the culture period. Treatment with verapamil inhibited the effect of FCS on Ca2+ sensitivity, as is shown in Fig. 1B, but had no effect at 0% FCS (Fig. 1C).

If an increase in intracellular Ca2+ during culture is not only necessary but also sufficient to decrease sensitivity to extracellular Ca2+, it should be possible to induce a similar effect with a Ca2+ ionophore without the presence of FCS. To test this, strips were cultured with 0.5 and 2 µM ionomycin, which has been shown to increase intracellular Ca2+ in several cell types, including smooth muscle (1, 27). Figure 1D illustrates that culture with ionomycin decreased sensitivity of force to extracellular Ca2+ in a concentration-dependent manner.

The difference in sensitivity to Ca2+ in strips cultured with 0 and 10% FCS was not due to a difference in the concentration dependence of the responsiveness to high-K+ solution. In Fig. 2, concentration-response curves are shown for strips cultured with and without FCS. Contractions (5 min), separated by 5-min periods in normal solution, were elicited with different concentrations of K+, and both the peak and mean force of the resultant contraction were evaluated. The initial peak had a minute influence on the mean value of the entire contraction. Figure 2A shows the dependence of the mean force amplitude on the concentration of K+. Note that for both culture conditions mean force is maximal at 60 mM K+. The concentration dependence of the peak of contraction (Fig. 2B) was shifted to the right after culture with 10% FCS. Peak force amplitudes in response to 144 mM K+ for strips cultured in different conditions are shown in Fig. 2C. If peak force in response to 144 mM K+ is taken to represent full activation of the contractile machinery, there is a decrease in force-generating ability both with 10% FCS and with 0.5 and 2 µM ionomycin compared with controls. Inclusion of 1 µM verapamil during culture with FCS caused a significant increase in the peak 144 mM K+ response compared with 10% FCS alone.


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Fig. 2.   A: mean force over 5-min contractions induced by different concentrations of K+ after culture with 0% FCS (open circle , n = 10) and 10% FCS (bullet , n = 10). B: force at peak of contraction as a function of the concentration of K+. C: force at the peak of contraction at 144 mM K+ as a function of culture conditions (n values are given in legend to Fig. 1). Conditions for C are as follows: 0% FCS (a), 10% FCS (b), 10% FCS plus 1 µM verapamil (c), 0% FCS plus 1 µM verapamil (d), 0% FCS plus 0.5 µM ionomycin (e), 0% FCS plus 2 µM ionomycin (f). * P < 0.05 compared with B. black-lozenge  P < 0.05 compared with a.

The time course of the changes in sensitivity to extracellular Ca2+, peak K+ force, and phasic contractile activity are depicted in Fig. 3. The sensitivity to extracellular Ca2+, determined in each strip, was defined as the Ca2+ concentration giving 50% of the force at 16 mM Ca2+ (EC50). The change in sensitivity of force to extracellular Ca2+ occurred between 12 and 36 h (Fig. 3, top), with no further change during the study period (108 h). On the other hand, a decrease in peak force in response to 140 mM K+ had occurred already after 12 h (Fig. 3, middle). The fraction of mounted strips displaying phasic spontaneous activity was decreased in strips cultured with 10% FCS compared with controls at times exceeding 36 h (Fig. 3, bottom).


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Fig. 3.   Time course of changes in sensitivity to extracellular Ca2+ (top), peak force in response to 140 mM K+ (middle), and phasic spontaneous activity (bottom). Fraction of mounted strips presenting activity is indicated above the symbols at bottom. * P < 0.05, ** P < 0.01, and *** P < 0.001.

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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Fig. 4.   Records of intracellular Ca2+ concentration ([Ca2+]i) and force in a control strip maintained for 5 days at 0% FCS (top traces) and a strip maintained at 10% FCS (bottom traces). Experimental protocol is indicated between the recordings. CCh, carbachol.

As seen in Fig. 4, the stimulated [Ca2+]i was lower in the strip that had been treated with serum (bottom traces) compared with the control strip (top traces). Mean [Ca2+]i during stimulation with the muscarinic agonist carbachol was 386 ± 46 nM for controls and 197 ± 52 nM for serum-treated strips (P < 0.05, n = 11 and 7) at the normal extracellular Ca2+ concentration of 2.5 mM. The corresponding values during stimulation with high K+ were 564 ± 78 and 277 ± 72 nM (P < 0.05, n = 11 and 7).

Values of [Ca2+]i as a function of the extracellular Ca2+ concentration are given in Fig. 5A. Although the means of [Ca2+]i for serum-treated and control strips were significantly different only at 0.5 and 2 mM extracellular Ca2+, the means were shifted in the same direction by 100-400 nM also at the higher extracellular Ca2+ concentrations. Figure 5B gives the relationship between [Ca2+]i and relative force (the maximum force of each individual strip was taken to be 100%). No effect on the sensitivity of force to [Ca2+]i was detected.


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Fig. 5.   A: compiled data of [Ca2+]i as a function of [Ca2+]EC at 60 mM K+ for longitudinal ileal muscle strips cultured with 0% FCS (open circle , n = 11) and 10% FCS (bullet , n = 7); *** P < 0.001, * P < 0.05. B: normalized force (maximum = 100%) as a function of [Ca2+]i under the same conditions. Continuous line is best fit to data of a 4-parameter logistic function (EC50 = 326 nM). Inset: [Ca2+]i-force relationship of the same data set with absolute force on the ordinate.

[Ca2+]i depends both on influx of Ca2+ and its removal. When removal of intracellular Ca2+ during relaxation in Ca2+-free depolarizing solution was analyzed in records from Fig. 4, no apparent difference between FCS-treated strips and controls was observed, as shown in Fig. 6. This was taken as an indication that influx, but not efflux, of Ca2+ had been decreased after culture of strips with 10% FCS. As control, both higher and lower rates of Ca2+ removal could be detected during relaxation in normal, nondepolarizing solution and after treatment with 0.5 µM ionomycin, respectively.


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Fig. 6.   Rate of removal of Ca2+ after switching from activating (144 mM K+-2.5 mM Ca2+) to relaxing (60 mM K+-0 Ca2+) solution in guinea pig ileum strips cultured for 5 days with 0% FCS (bullet , 2 strips), 10% FCS (triangle ), and 0% FCS + 0.5 µM ionomycin (square ). Decay of [Ca2+]i on exchange from 10 µM carbachol to normal, nondepolarizing solution (open circle ). Records were normalized with respect to the starting [Ca2+]i.

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 -80 mV. After patch rupture and establishment of whole cell recording conditions, inward currents were monitored until a stable current amplitude was attained. As shown in Fig. 7, peak current normalized to cell capacitance was significantly reduced in cells from strips cultured with FCS; 1 µM verapamil, added to medium with FCS, protected against the downregulation of Ca2+ current (Fig. 7). Cell size, estimated from capacitance, was not significantly affected by serum (40.6 ± 5.1 and 28.2 ± 5.0 pF for FCS and controls, P > 0.1).


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Fig. 7.   Peak inward currents, normalized to cell capacitance, in cells isolated from strips cultured as indicated. Currents were evoked by a depolarizing pulse to +10 mV from a holding potential of -80 mV. Bars have n values, from left to right, of 10, 8, 6, and 7. * P < 0.05, *** P < 0.001.

Figure 8A shows original recordings from two different cells isolated from strips cultured in the absence (top trace) and in the presence (bottom trace) of serum. The current-voltage relationship did not differ between serum-stimulated cells and controls, as illustrated in Fig. 8B. Currents were elicited by a series of increasing depolarizing steps, at every 5 s, from a holding potential of -80 mV. Inward current begins to be activated at -40 mV, peaks between +10 and +20 mV, and reaches the reversal potential at about +55 mV for both groups. Steady-state activation-inactivation properties were also examined and found to be similar under both conditions (Fig. 8, C and D). Voltage at one-half activation (V d0.5) and its slope factor (kd) were identical in cells isolated from strips cultured with and without serum(V d0.5 -4.6 ± 0.8 vs. -4.5 ± 0.4 mV, kd = 7.4 ± 0.7 vs. 5.3 ± 0.3). There were no significant differences in the inactivation parameters (V f0.5 and kf, respectively) between the two groups (V f0.5 = -35.6 ± 0.5 vs. -35.2 ± 2.0 mV, kf = 8.5 ± 0.5 vs. 10.8 ± 1.9).


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Fig. 8.   A: representative voltage-clamp traces from 2 different cells isolated from strips cultured in the absence (top trace, cell capacitance = 52.5 pF) and in the presence (bottom trace, cell capacitance = 53.3 pF) of serum. B-D: superimposed peak current-voltage curves (B; holding potential = -80 mV), steady-state activation relationship (C), and steady-state inactivation curves (D) obtained from cells isolated from strips cultured with (black-square) and without (square ) serum. Data from C and D were fitted, respectively, by the following Boltzmann equations: dinfinity (V) = {1 + exp[(V - V d0.5)/kd]}-1, where dinfinity (V) is the activation parameter, V d0.5 is voltage of half-activation, and kd is the slope factor, and finfinity (V) = {1 + exp[(V - V f0.5)/kf]}-1, where finfinity (V) is the inactivation parameter, V f0.5 is voltage of half-inactivation, and kf is the slope factor.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

We thank Professor Per Hellstrand for useful discussions and suggestions.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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4.   DeLorme, E. M., C. S. Rabe, and R. McGee, Jr. Regulation of the number of functional voltage sensitive Ca2+ channels on PC12 cells by chronic changes in membrane potential. J. Pharmacol. Exp. Ther. 244: 838-843, 1988[Abstract].

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