Long depolarizations cause a steady tonic contraction and induce sarcoplasmic reticulum (SR) Ca2+-uptake in trout atrial myocytes. Simultaneous measurements of cytosolic [Ca2+]([Ca2+]i) and whole membrane current showed an elevated[Ca2+]i throughout the depolarization. Rapid caffeine(Caf) applications at –80 mV before and after a long depolarization were used to determine SR Ca2+ loading and its dependency on membrane potential and [Ca2+]i during depolarization. Following a 10 s depolarization, the maximal SR Ca2+ load was 597 μmol l–1 and loading was half-maximal at –12 mV. Theβ-adrenergic agonist isoproterenol (ISO) did not affect the maximal SR Ca2+ loading but shifted the potential for half-maximal loading by–26 mV. Following a 3 s depolarization, the maximal SR Ca2+uptake rate (V̇max) was 418μmol l–1 s–1 in control conditions. ISO did not affect V̇max, but significantly lowered the average free Ca2+ transient during the depolarization and shifted the K0.5 for the relationship between SR Ca2+ uptake and [Ca2+]i from 1.27 in control to 0.8 μmol l–1 with ISO. Following repetitive 200 ms depolarizations, ISO increased the l-type Ca2+current (ICa) amplitude by 91±29% and the peak Ca2+ transient by 41±10%, and decreased the half life of the Ca2+ transient from 151±12 to 111±6 ms. Using the relationship between [Ca2+]i and SR Ca2+uptake to calculate the total SR Ca2+ uptake during a Ca2+ transient elicited by a 200 ms depolarization, a significant increase in the SR Ca2+ uptake from 37±6 μmol l–1 in control to 68±4 μmol l–1with ISO was seen. When normalized to the total Ca2+ transport the contribution of the SR was not significantly different in the absence(35±6%) or presence of ISO (41±4%). Exposure of cells to ISO and low extracellular [Ca2+] increased ICa by 67±40%(N=5) but significantly reduced SR Ca2+ uptake at membrane potentials above –30 mV. Together, these results suggest that (i) ISO has a stimulatory effect on the SR Ca2+ pump that may contribute to the faster decay of the Ca2+ transient, and (ii) the relative contribution of the SR to the Ca2+ removal during relaxation is not altered by ISO in trout atrial myocytes.
In the mammalian heart, β-adrenergic stimulation has both inotropic and lusitropic effects. The inotropic effect is due to phosphokinase A(PKA)-dependent effects on both l-type Ca2+ current(Hartzell et al., 1991; Hove-Madsen et al., 1996; Hove-Madsen and Tort, 1998; Jurevicius and Fischmeister,1996) and the SR function, while stimulation of SR Ca2+uptake through a PKA-dependent phosphorylation of phospholamban(Chu et al., 2000; Mattiazzi et al., 1994) has been reported to account for the lusitropic effect(Li et al., 2000). Indeed, the effect of β-adrenergic agonists and PKA-dependent phosphorylation on SR Ca2+ uptake has been examined in both intact and permeabilized myocytes, measuring the effects on Ca2+ sparks(Gomez et al., 1996; Zhou et al., 1999; Ziolo et al., 2001), caffeine(Caf) induced Ca2+ transients(Li et al., 1998) or oxalate stimulated Ca2+ uptake in the SR(Mattiazzi et al., 1994).
In the teleost heart, the effect of β-adrenergic stimulation on ICa has recently been characterized(Hove-Madsen and Tort, 1998; Vornanen, 1998), with a two-to threefold stimulation of ICa. This is similar to values obtained in mammals, but much smaller that the seven- to tenfold stimulation reported in frog (Fischmeister and Shrier,1989). Based on the stimulatory effect of β-adrenergic stimulation of ICa, it has been suggested that sarcolemmal Ca2+ influx is sufficient to activate contraction under these conditions (Vornanen, 1998).β-adrenergic stimulation does, however, also affect other important factors such as myofilament sensitivity and SR function, which critically determine the amplitude and kinetics of cell shortening(Freestone et al., 2000; Li et al., 2000).
With respect to the SR function, the short duration of the Ca2+transient in the mammalian cardiac myocyte and the simultaneous regulation of the intercellular [Ca2+] by several mechanisms have made it difficult to characterize the relationship between[Ca2+]i and the rate of Ca2+ uptake in the SR in intact mammalian cardiac myocytes. In contrast to this, long depolarizations in lower vertebrate myocytes lead to a tonic cell shortening,a maintained outward current (Hove-Madsen et al., 1998, 2000) and presumably an intracellular [Ca2+] that is maintained elevated. Furthermore, a voltage dependent Ca2+ uptake by the SR takes place during long depolarizations in these cells and can be used to estimate the average SR Ca2+ uptake rate (Hove-Madsen et al., 1998, 2000). Using simultaneous measurements of intracellular [Ca2+] and whole membrane current,the present study has taken advantage of these properties of trout cardiac myocytes in order to (1) characterize the relationship between membrane potential, [Ca2+]i and the SR Ca2+ uptake rate under basal and phosphorylating conditions, (2) use this relationship between cytosolic [Ca2+] and SR Ca2+ uptake to calculate the SR Ca2+ uptake during a Ca2+ transient induced by a 200 ms depolarization, and (3) determine the relative contribution of SR Ca2+ uptake to total Ca2+ transport in the absence and presence of β-adrenergic stimulation.
Materials and methods
Trouts Oncorhynchus mykiss Walbaum were purchased from a local fish farm and acclimated in tanks at the facilities at the Universitat Autònoma de Barcelona to the water temperature at the fish farm(16°C). Atrial myocytes were obtained by enzymatic digestion of the heart at 18–20°C as described previously(Hove-Madsen et al., 1999). The cell isolation procedure and the experimental protocols were approved by the local ethical committee (DARP no. 1017).
Whole membrane current was measured using a software driven patch-clamp amplifier (EPC-9, Heka, Germany) or an Axopatch 200, (Axon, California, USA). After seal formation, the cell was placed in front of one of eight capillaries containing the desired extracellular solution. Standard internal and external solutions were used to eliminate Na+ and K+ currents. The pipette solution contained (in mmol l–1): CsCl 100, MgATP 3.1; MgCl2 1; sodium phosphocreatine 5; Li2GTP 0.42;EGTA 0.025 (no EGTA was included when using fluo-3 loaded cells); Hepes 10;tetraethyl ammonium (TEA) 20. The pH was adjusted to 7.2 with CsOH. The external medium contained (in mmol l–1): NaCl 107; CsCl 20;MgCl2 1.8; NaHCO3 4; NaH2PO4 0.8;CaCl2 1.8; Hepes 10; glucose 5; pyruvate 5. The pH was adjusted to 7.4 with NaOH. The Na+ current was eliminated with 1 μmol l–1 tetrodotoxin (TTX). Whole membrane current was measured in the ruptured or the perforated patch configuration with 200–250 μg ml–1 amphotericin B (Figs 4, 5, 6, 7) at room temperature(20°C). The pipette resistance was 2–5 MΩ, seal resistance was 2–20 GΩ, and the access resistance was 6.5±1.2 MΩ for ruptured patches. When using amphotericin B, equilibration of the preparation by continuous stimulation at 0.2 Hz was started when access resistance had decreased to ∼20 MΩ, and the access resistance was 11.6±3.1 MΩ at the end of the experiments. Cells showing a sudden drop in access resistance were discarded.
The cells were stimulated continuously with a 200 ms depolarization from–80 mV to 0 mV every 5 s. This protocol allowed evaluation of ICa and Caf induced Na+–Ca2+ exchange(NCX) current (INCX) under steady state conditions in the absence and presence of β-adrenergic stimulation. β-adrenergic stimulation was produced by exposing cell to 1 μmol l–1 isoproterenol(ISO). The Caf induced INCX allows determination of the SR Ca2+ content in the absence and presence of ISO as described previously (Hove-Madsen et al.,1998; Negretti et al.,1995; Varro et al.,1993). The basic experimental protocol outlined in Fig. 1 was used to determine the effect of β-adrenergic stimulation on Ca2+ uptake in the SR. First the SR Ca2+ content was cleared with a brief exposure to 10 mmol l–1 caffeine (Caf). Then Ca2+ uptake was induced by depolarizing the cell to different membrane potentials for 3 or 10 s (labeled Load in Fig. 1). The amount of Ca2+ accumulated in the SR was determined from the INCX elicited by exposing the cell to 10 mmol l–1Caf after repolarizing the cell to –80 mV (labeled Caf in Fig. 1). The SR Ca2+uptake rate was calculated by dividing the Ca2+ accumulated by the SR with the duration of the depolarization. For conversion of the time integrals of Ca2+ carrying currents to Ca2+concentrations, a conversion factor of 15.4 pF pl–1 was used(Hove-Madsen and Tort, 1998)and the Ca2+ accessible cell volume was considered to equal the non-mitochondrial cell volume, which in trout corresponds to 55% of the total cell volume (Vornanen,1998).
Measurement of intracellular [Ca2+]
The intracellular Ca2+ concentration[Ca2+]i was measured with the Ca2+ indicator fluo-3. Cells were incubated with 5 μmol l–1 fluo-3 AM for 10–15 min at room temperature. After incubation cells were washed and left for 30 min at room temperature before starting experiments. Fluo-3 was excited at 488±1 nm and fluorescence emission was measured using a 530 nm bandpass filter. The bandpass width was ±20 nm. A sampling interval of 10 ms was used to achieve a reasonable signal-to-noise ratio. This sampling interval was necessary as the cell volume of trout atrial myocytes is 2–4 pl, which is about ten times less than the cell volume of mammalian cardiac myocytes. Calibration of the fluo-3 signal in each cell was done by subtraction of background fluorescence and measurement of the maximal fluorescence at the end of each experiment. As long depolarizations lead to a tonic elevation of [Ca2+]i and contraction in trout cardiac myocytes (Hove-Madsen et al., 1998, 2000), maximal fluorescence was obtained by depolarizing the cell for 3 s to increasingly more positive potentials until an irreversible cell contracture was achieved. The fluo-3 signal measured at irreversible cell contracture was taken as the maximal fluorescence since it also coincided with a large and irreversible increase in leak, and it was similar to the fluorescence obtained when leak was induced by squeezing the cell with the patch pipette. [Ca2+]i was then calculated from[Ca2+]i=F×Kd/(Fmax–F)where F is the measured net fluorescence, Fmax is the maximal net fluorescence and Kd is the dissociation constant for Ca2+ for fluo-3(Trafford et al., 1999). A Kd for fluo-3 in cells of 1100 nmol l–1(Harkins et al., 1993) was used in the present study.
Data analysis and statistics
Unless otherwise stated, cells served as their own control when the effects of β-adrenergic stimulation were examined. For statistical analysis of the data, Student's t-test for paired samples was used to compare two experimental conditions. Values are given as means ± s.e.m. Analysis of variance (ANOVA) was used to evaluate the effect of β-adrenergic stimulation on the relationships between membrane potential, [Ca2+]i and SR Ca2+ uptake (Figs 3, 6 and 9). While SR Ca2+uptake does not depend directly on the membrane potential, it does depend on the [Ca2+]i concentration, and since the[Ca2+]i concentration depends on the membrane potential,the Boltzmann equation was used to empirically fit the relationship between[Ca2+]i or SR Ca2+ uptake and membrane potential (Figs 3 and 9). Similarly, the relationship between SR Ca2+ uptake and [Ca2+]i has been described by a Hill equation (Hove-Madsen and Bers, 1993) and this equation was therefore used to fit the data in Fig. 6C.
β-adrenergic stimulation of SR Ca2+ uptake
Fig. 2A shows typical NCX currents elicited by Caf after a 10 s depolarization to –70, –30 and –10 mV. Current traces obtained in the absence and the presence of 1μmol l–1 ISO are shown for each membrane potential. The amount of Ca2+ released from the SR was determined from the time integrals of the Caf induced NCX currents as shown in Fig. 2B. Fig. 3 summarizes the dependency of the SR Ca2+ accumulation on the membrane potential in the absence and the presence of ISO. The lines represent empirical fits of data from six cells with a Boltzmann equation. This fit gave slopes of 22.2 and 23.8 in the absence and the presence of ISO, respectively, and a basal SR Ca2+ loading of 14 μmol l–1 and 20 μmol l–1, respectively. K0.5 was –12 mV in control and –38 mV in the presence of 1 μmol l–1ISO while the maximal SR Ca2+ load was 38.8 and 39.2 amol pF–1 or μmol l–1 in control and ISO,respectively. This corresponds to a maximal SR Ca2+ content of 1086 and 1097 μmol l–1 non-mitochondrial volume in control and ISO, respectively. While ISO changed the relationship between voltage and SR Ca2+ uptake significantly (P<0.001, N=6),causing a shift in K0.5 of –26 mV, it had no significant effect on basal or maximal SR Ca2+ uptake. Fitting of data points from individual experiments showed a significant difference between the average K0.5 in the absence and the presence of ISO (P<0.01), while ISO did not significantly change the slope,the basal or the maximal SR Ca2+ loading.
β-adrenergic stimulation of steady state ICa and Ca2+ transients
These results show that ISO stimulates Ca2+ accumulation, so it was important to determine whether the stimulation was a result of an increased sarcolemmal Ca2+ entry or a direct stimulation of SR Ca2+ uptake. Therefore, the free [Ca2+]i and the whole membrane current resulting from a long depolarization were measured simultaneously. First, the steady state effect of ISO on ionic current and[Ca2+]i was established using continuous stimulation with 200 ms depolarizations from –80 to 0 mV at a frequency of 0.2 Hz. As shown in Fig. 4, ISO increased both ICa (Fig. 4A) and Ca2+ transient(Fig. 4B) significantly by 91±29 (P<0,01, N=7) and 41±10%,respectively (P<0.01, N=7). Furthermore, when fitting the decaying phase of the Ca2+ transient with a single exponential, ISO decreased the half life of the Ca2+ transient significantly from 151±12 to 111±6 ms (P<0.001, N=7).
Effect of β-adrenergic stimulation on the relationship between[Ca2+]i and SR Ca2+ uptake
Fig. 5 shows superimposed fluo-3 traces (Fig. 5A) and ionic currents (Fig. 5B)elicited by exposure to 10 mmol l–1 Caf (right panels) after a 3 s depolarization to +10 mV (left panels), before (black traces) and after stimulation with 1 μmol l–1 ISO (gray traces). The membrane potential is indicated in the panel above the traces and the period of Caf application is shown. Notice that both the[Ca2+]i and the whole membrane currents are permanently elevated throughout the 3 s depolarization to +10 mV. Also notice that except from the first few hundred ms, ISO reduced the [Ca2+]iwhile it stimulated the outward current during depolarization. This observation was consistent in all seven cells examined. Fig. 5C shows that the effect of ISO on the Caf induced ionic current and Ca2+ transient was reversible. This was also true for the effects of ISO on the whole membrane current and the Ca2+ transient elicited with long depolarizations or with repetitive 200 ms depolarizations (data not shown). Fig. 6A shows recordings of the[Ca2+]i during depolarization to different membrane potentials and caffeine applications in the absence (left panels) and the presence of 1 μmol l–1 ISO (right panels). The membrane potential is given in the top of the two bars above the recordings, and the absence (–) or presence of 10 mmol l–1 Caf (+) in the bath solution in the bottom bar. Fig. 6B compares the average Ca2+ transient during depolarization (black bars) and the peak Caf induced Ca2+ transient(white bars) after depolarization in the absence (left panel) or the presence of 1 μmol l–1 ISO (right panel). The membrane potential during the depolarization is given below the bars. Notice that there was no significant difference between the average Ca2+ transient during depolarization and the peak Ca2+ transient elicited by Caf after depolarization in control conditions, while the Caf induced Ca2+transient was significantly bigger than the average Ca2+ transient during the preceding depolarization in the presence of ISO(P<0.001, N=7). This suggest that ISO directly stimulates SR Ca2+ uptake, and Fig. 6C shows the relationship between SR Ca2+ uptake(dCa/dt) and the average [Ca2+]i during depolarization without (filled circles) and with 1 μmol l–1 ISO (open circles). The lines represent fits of the data points with a Hill equation. This gave Hill slopes of 1.74 and 2.24 without and with ISO, respectively,while the V̇max values were unchanged at 418 and 435 μmol l–1 s–1,respectively. K0.5 was shifted from 1.27 μmol l–1 in the control to 0.80 μmol l–1 with 1 μmol l–1 ISO. Fitting of data points from individual experiments showed that there was a significant difference between the average K0.5 in the absence and the presence of ISO(P<0.05), while ISO did not significantly affect the Hill slope or the V̇max.
Using this relationship between cytosolic [Ca2+] and SR Ca2+ uptake rate, the total SR Ca2+ uptake during the Ca2+ transients shown in Fig. 4B can be calculated by first calculating the SR Ca2+uptake rate during the Ca2+ transient, and then integrating this signal to obtain the total SR Ca2+ uptake during the Ca2+ transient. Similarly the total sarcolemmal Ca2+extrusion can be calculated from the time integral of the tail currents in Fig. 4A. Fig. 7A shows the total SR Ca2+ uptake (labeled SR), total sarcolemmal Ca2+extrusion (labeled SL) and total Ca2+ transport (labeled TOT)without (black bars) and with 1 μmol l–1 ISO (gray bars). Notice that ISO significantly increased all three parameters. When normalizing the SR Ca2+ uptake to the total Ca2+ transport, it constituted 35±6% and 41±4% of the total Ca2+eliminated from the cytosol without and with 1 μmol l–1ISO, respectively.
Effect of β-adrenergic stimulation and low extracellular[Ca2+] on SR Ca2+ uptake
To examine whether the stimulatory effect of ISO was able to compensate for a lowering of the extracellular [Ca2+], the effect of ISO applied in the presence of a low extracellular [Ca2+] (0.3 or 0.5 mmol l–1) was compared with control values obtained in the absence of ISO with 1.8 mmol l–1 extracellular Ca2+. Fig. 8A shows representative current traces elicited by a 200 ms depolarization in control conditions and with ISO + low Ca2+. On average, ISO increased the ICaamplitude by 67±40% (N=5). Fig. 8B shows the corresponding current traces elicited by a rapid Caf application after a 10 s depolarization. Notice that SR Ca2+ uptake was strongly reduced in the presence of ISO + low Ca2+. Fig. 9 compares the relationship between the membrane potential during a 10 s depolarization and the amount of Ca2+ taken up by the SR in control conditions and with ISO + low Ca2+. The relationship between SR Ca2+uptake and membrane potential was significantly affected by application of ISO+ low Ca2+ (P<0.001, N=5) resulting in a reduced uptake at membrane potentials above –30 mV. Thus while ISO could overcome the effect of lowering extracellular Ca2+ on ICa it was unable to prevent a strong reduction in SR Ca2+ uptake at physiologically relevant membrane potentials.
The aim of the present work was to characterize the relationship between the intracellular [Ca2+] and the SR Ca2+ uptake rate and to examine the effect of β-adrenergic stimulation on SR Ca2+uptake and Ca2+ removal from the cytosol in trout atrial myocytes. To do this, the perforated-patch configuration was used to apply physiological or long depolarizations to trout atrial myocytes.
This experimental approach is based on the following assumptions: (1) SR Ca2+ uptake can be quantified as the amount of Ca2+released from the SR by Caf, (2) SR Ca2+ uptake takes place throughout a long depolarization, (3) the average SR Ca2+ uptake rate during a depolarization can be calculated because uptake is uniform throughout the depolarization. With respect to the first assumption, it has been shown that Ca2+ released from the SR by Caf can be used as a measure of the SR Ca2+ content in mammalian and trout cardiomyocytes (Hove-Madsen et al., 1998, 2000; Trafford et al., 1999). Since K is substituted with Cs and 15 mmol l–1 extracellular NaCl is replaced with CsCl in the present experiments, SR Ca2+ uptake could on the one hand be underestimated, because Cs has been shown to decrease SR Ca2+ uptake in mammalian cardiomyocytes(Kawai et al., 1998), and on the other hand be overestimated, because of the 10% reduction in the extracellular [Na+]. The effect of Cs on SR Ca2+ uptake is, however, not expected to differ in the absence and presence of ISO. Regarding the second assumption, Ca2+ uptake has been shown to increase proportionally to the length of the depolarization as long as the SR Ca2+ content is not near saturation(Hove-Madsen et al., 1998),suggesting that SR Ca2+ uptake takes place throughout the depolarizations used in the present study. Finally, Fig. 5 shows that both the Ca2+ transient and the outward current remain elevated throughout a 3 s depolarization so that the average SR Ca2+ uptake rate can be calculated as the amount of Ca2+ released by Caf divided by the duration of the preceding depolarization.
Direct stimulation of SR Ca2+ uptake by β-adrenergic stimulation
When measuring SR Ca2+ uptake as a function of membrane potential, β-adrenergic stimulation affects neither the maximal SR Ca2+ uptake nor the SR Ca2+ uptake at rest. It does,however, significantly shift the relationship between SR Ca2+uptake and membrane potential by –26 mV, so that the SR can more efficiently remove Ca2+ from the cytosol during the repolarization of the action potential and during diastole. This in turn may contribute to the faster decay of the Ca2+ transient elicited by a standard 200 ms depolarization observed in Fig. 4.
The effect of β-adrenergic stimulation on the relationship between SR Ca2+ uptake and membrane potential could either be due to a direct effect on SR Ca2+ uptake or a consequence of its stimulatory effect on ICa and the intracellular Ca2+ transient (see Fig. 4). In this respect, the voltage dependency of ICa is also shifted towards more negative membrane potentials by β-adrenergic stimulation(Brum et al., 1984) and this could potentially account for the observed shift in the relationship between membrane potential and SR Ca2+ uptake. β-adrenergic stimulation does, however, also stimulate SR Ca2+ uptake through phospholamban phosphorylation (Bassani et al., 1995; Li et al.,2000; Mattiazzi et al.,1994), and PKA-dependent phosporylation of phospholamban has been reported to shift the Kd for SR Ca2+ uptake towards lower [Ca2+] (Mattiazzi et al., 1994). The fact that β-adrenergic stimulation lowers the average [Ca2+]i during long depolarizations and shifts the K0.5 for the relationship between cytosolic[Ca2+] and SR Ca2+ uptake from 1.27 to 0.8 μmol l–1 does therefore suggest that its stimulatory effect on SR Ca2+ loading is due to a stimulation of the SR Ca2+uptake rather than being a consequence of ICa stimulation.
The SR Ca2+ uptake in trout atrial myocytes is comparable to values previously reported in trout ventricular myocytes(Hove-Madsen et al., 1998),and the uptake rate is within the range reported in intact or permeabilized mammalian cardiac myocytes at room temperature(Balke et al., 1994; Wimsatt et al., 1990). Also, a 40% reduction of the K0.5 for SR Ca2+ uptake by ISO is similar to values reported in mammals(Mattiazzi et al., 1994). The absolute value for K0.5 in control is similar to a value previously reported for permeabilized trout ventricular myocytes(Hove-Madsen et al., 1998),while values for both control and ISO are 2–3 times higher than values reported in several mammalian studies(Balke et al., 1994; Hove-Madsen and Bers, 1993; Mattiazzi et al., 1994). These differences may reflect species dependent differences in the SR Ca2+ ATPase, but could also be related to the calibration of the fluorescent Ca2+ indicator. The present work uses a Kd for fluo-3 of 1.1 μmol l–1 obtained from in situ calibration in frog skeletal muscle fibers(Harkins et al., 1993), while other studies have often used in vitro calibration of the fluorescent dyes (Balke et al., 1994; Trafford et al., 1999). Using a Kd for fluo-3 of 400 nmol l–1 from in vitro calibration would give K0.5 values of 0.46 μmol l–1 in controls and 0.29 μmol l–1 with ISO. These values are similar to those reported in mammals (Balke et al., 1994; Trafford et al., 1999),suggesting that differences in dye calibration could at least partially account for the differences in K0.5 in the present study and values measured in mammals.
Indirect effects of β-adrenergic stimulation on SR Ca2+ uptake
While the present results do show that β-adrenergic stimulation causes a direct stimulation of the SR Ca2+ uptake, they do not exclude an indirect effect of β-adrenergic stimulation on SR Ca2+ uptake with more physiological stimulation pulses. Thus, the contribution of ICa to the elevation of the [Ca2+]i during a 3 s or a 10 s depolarization is expected to be significantly smaller than its contribution during a 200 ms depolarization. In agreement with this,ICa is not reduced when the extracellular solution is switched from control to ISO plus low extracellular [Ca2+] while the SR Ca2+ uptake during a 10 s depolarization is drastically reduced at all membrane potentials examined (see Figs 8 and 9). This agrees with previous results, which suggested that the maintained outward current and tonic contraction during long depolarizations in trout cardiac myocytes(Hove-Madsen et al., 2000; Hove-Madsen and Tort, 2001)are due to reverse mode Na+–Ca2+ exchange.
Furthermore, the fact that ISO has a stimulatory effect on ICaeven when the extracellular Ca2+ is lowered while SR Ca2+ uptake during long depolarizations is strongly reduced under these conditions (see Figs 8, 9), suggests that ISO do not have any stimulatory effect on INCX.
In this respect the average Ca2+ transient is smaller in the presence of ISO, while the corresponding outward current is larger in Fig. 5. Assuming that the outward current is reverse mode INCX, this suggests that under these conditions the amplitude of reverse mode INCX during long depolarizations is being determined by the [Ca2+]irather than being the amplitude of reverse mode INCX that determines the [Ca2+]i. Therefore, ISO not only stimulates SR Ca2+ uptake as shown in Figs 2, 3, 5, 6, 7, but the SR Ca2+uptake relative to reverse mode NCX does in fact become faster in the presence of ISO. The relatively faster SR Ca2+ uptake in turn leads to a reduction in [Ca2+]i, an increase in the transsarcolemmal Ca2+ gradient and as a consequence a larger reverse mode INCX as observed in Fig. 5. Thus, the apparent stimulation of reverse mode INCX in Fig. 5 is more likely to be an indirect effect of ISO on SR Ca2+ uptake. The absence ofβ-adrenergic stimulation of the INCX agrees with data from the toad heart (Ju and Allen,1999). It would also be in agreement with the absence, in both the trout and mammalian NCX protein (Nicoll et al., 1990; Xue et al.,1999), of the putative PKA-dependent phosporylation site described in frog (Fan et al., 1996). It is, however, contrary to results from the frog heart, where a putative PKA-dependent phosporylation site on the NCX has been described(Fan et al., 1996) and a PKA-dependent inhibition of NCX activity has been reported in both frog and shark cardiac myocytes (Fan et al.,1996; Woo and Morad,2001). The present results also show that the importance of SR Ca2+ uptake relative to sarcolemmal Ca2+ extrusion by the NCX is unchanged with β-adrenergic stimulation in trout atrial myocytes. This is contrary to conclusions reached from measurements of SR function in tissue preparations (Shiels and Farrell, 1997) and the difference is most likely related to the indirect nature of the measurements in the tissue preparation. Also, the stimulation of l-type Ca2+ current by ISO in isolated trout myocytes has been taken as evidence for a larger contribution of sarcolemmal Ca2+ entry to the activation of contraction in the presence of β-adrenergic stimulation(Vornanen, 1998). This conclusion is not easily compatible with an unchanged relative contribution of forward mode INCX to relaxation with β-adrenergic stimulation found in the present study, since it indirectly implies a similar contribution of sarcolemmal Ca2+ entry at steady state.
Finally, it should be noticed that the present results were obtained at room temperature, while the acclimation temperature was 16°C and the preferred temperature for rainbow trout is around 14°C. We cannot rule out that this may have consequences for the measured effects of β-adrenergic stimulation on SR Ca2+ content and uptake rates, but we recently found that neither SR Ca2+ uptake nor SR Ca2+ release were significantly changed when the experimental temperature was lowered from 21 to 7°C (Hove-Madsen et al.,2001). Furthermore, it is not clear that a difference between acclimation and experimental temperature of 4°C would selectively alter the SR Ca2+ uptake under basal or phosporylating conditions. An unequivocal answer to this question will, however, have to await further experimentation that directly addresses it.
In conclusion, the present study has characterized the relationship between the membrane potential, cytosolic [Ca2+] and SR Ca2+uptake rate in trout atrial myocytes in the absence and presence ofβ-adrenergic stimulation. The results show that β-adrenergic stimulation shifts the relationship between membrane potential and SR Ca2+ uptake by –26 mV and decreases the K0.5 for SR Ca2+ uptake from 1.27 to 0.8μmol l–1, while it has no effect on resting or maximal SR Ca2+ loading. This may contribute to the faster decay of the Ca2+ transient with β-adrenergic stimulation in these cells. Furthermore, β-adrenergic stimulation does not alter the importance of SR Ca2+ uptake relative to Ca2+ extrusion by the INCX, and the characterization of the dependency of SR Ca2+ uptake on membrane potential and [Ca2+]ishould be useful for quantitative comparisons of SR Ca2+ uptake and Ca2+ extrusion by the INCX under specific experimental conditions.
This work was supported by a grants from The Spanish Ministry of Education and Culture (Incorporación de Doctores) to L. Hove-Madsen and Generalitat de Catalunya (FPI grant) to A. Llach, (MAR97-402-C02) to L. Tort,and the NSERC of Canada to G. F. Tibbits.