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The Journal of Experimental Biology 202, 2531–2537 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JEB2195
Ca2+ UPTAKE IN THE SARCOPLASMIC RETICULUM FROM THE SYSTEMIC HEART OF OCTOPOD CEPHALOPODS JORDI ALTIMIRAS1,*, LEIF HOVE-MADSEN2 AND HANS GESSER1 Center for Respiratory Adaptation, Department of Zoophysiology, University of Aarhus, DK-8000 Aarhus C, Denmark and 2Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
1Danish
*Present address: Department of Zoophysiology, University of Göteborg, Box 463, SE 405 30 Göteborg, Sweden (e-mail:
[email protected])
Accepted 23 June; published on WWW 25 August 1999 Summary We have measured Ca2+
uptake in crude homogenates of heart tissue, as well as cell shortening and ionic currents in isolated myocytes exposed to caffeine, to characterize Ca2+ uptake in the sarcoplasmic reticulum (SR) of the systemic heart of octopus. The maximal rate of SR Ca2+ uptake in crude homogenates of octopus heart was 43±4 (mean ± S.E.M., N=7), compared with 28±2 nmol min−1 mg−1 protein (N=4) in homogenates of rat heart. The Ca2+-dependency of SR Ca2+ uptake was similar for the two species, with a Ca2+ activity at half-maximal uptake rate (pCa50) of 6.04±0.02 for octopus and 6.02±0.05 for rat. Exposure of isolated myocytes to 10 mmol l−1 caffeine resulted in cell shortening to 53±2 % of the resting cell length and an inward trans-sarcolemmal ionic current. The charge carried by this current was 3.28±0.70 pC pF−1 (mean ± S.E.M., N=5) corresponding to extrusion of
34.0±0.7 amol Ca2+ pF−1 from the cell by Na+/Ca2+ exchange. This is approximately 50 times more than the Ca2+ carried by the Ca2+ current elicited by a 200 ms depolarization from −80 to 0 mV and corresponds to an increase in the total intracellular [Ca2+] of 404±86 µmol l−1 non-mitochondrial volume due to Ca2+ release from the SR. Thus, we find that at 20 °C in the SR both Ca2+ content and Ca2+ uptake rate in the systemic heart of octopus are comparable with or larger than the corresponding values obtained in the rat heart. These results support the argument that the SR may play an important role in the regulation of contraction in the systemic heart of cephalopods. Key words: ryanodine, caffeine contracture, Na+/Ca2+ exchange, oxalate-supported calcium uptake, Octopus vulgaris
Introduction Coleoid cephalopods (octopus, cuttlefish and squid) have high metabolic rates and a relatively low oxygen-carrying capacity in the blood, which implies large cardiac outputs by the standards of invertebrate animals or fish (Wells, 1992). Furthermore, during periods of exercise, the increased rate of oxygen uptake (2.3-fold in Octopus vulgaris; Wells et al., 1983) is met by an increased cardiac output, since there is little or no scope for increasing oxygen extraction (already approximately 80 % in resting conditions; Houlihan et al., 1986). The high demand on the cardiac pump is also reflected in the cellular characteristics of the systemic cardiac muscle. Isolated cardiac muscle strips from Octopus vulgaris show a regular twitch force development at stimulation frequencies at which the cardiac muscle of other ectothermic vertebrates, such as the rainbow trout, commonly fail (Gesser et al., 1997). Furthermore, ryanodine strongly inhibits twitch-force development and increases resting tension, suggesting that excitation–contraction coupling is highly dependent on Ca2+ cycling via the sarcoplasmic reticulum (SR), as is known to occur in the cardiac muscle of the rat.
The strong response of O. vulgaris systemic heart tissue to ryanodine is rather surprising, since it far exceeds that described for cardiac tissue of ectothermic vertebrates. In ectothermic vertebrates, heart tissue is either weakly responsive or unresponsive to ryanodine at physiological heart rates and temperatures (Driedzic and Gesser, 1988; HoveMadsen, 1992; Møller-Nielsen and Gesser, 1992) although trout ventricular myocytes have recently been shown to possess an SR with a significant Ca2+-accumulating capacity and high Ca2+ maximal uptake rate (Hove-Madsen et al., 1998). Since the cellular aspects of cardiac muscle function have been poorly studied in cephalopods, the possibility that ryanodine affects mechanisms other than SR-dependent Ca2+ cycling in this tissue cannot be excluded. In addition, ultrastructural studies suggest that in Octopus vulgaris the heart possesses a richly developed SR (Schipp, 1987; Dykens and Mangum, 1979), but no quantitative analysis has been performed. The importance of the SR in cardiac tissue of cephalopods was therefore examined using two alternative methods. In one approach, the Ca2+ uptake rate of the SR in
2532 J. ALTIMIRAS, L. HOVE-MADSEN AND H. GESSER crude cardiac homogenates from octopus and rat was compared in terms of the maximum rate of uptake and its Ca2+ dependence. Rat heart was chosen because its excitation–contraction coupling has been shown to be extremely dependent on the SR (Bers, 1985). It also has the advantage that this dependence on the SR is retained at lower temperatures, so that ryanodine has virtually the same effect at 20 °C and at 37 °C (Shattock and Bers, 1987). The comparison was therefore carried out at 20 °C, a temperature within the range experienced by the living octopus but below the body temperature of rat. In a second approach, the importance of the SR was assessed by measuring cell shortening and the ionic current elicited by exposure of isolated cardiomyocytes to caffeine. This substance is known to induce Ca2+ release from the SR, which in turn gives rise to a cell contracture and a trans-sarcolemmal ionic current due to Ca2+ extrusion by the electrogenic Na+/Ca2+ exchanger. This approach has previously been used to quantify the Ca2+ content of the SR in single cardiac myocytes from both mammalian (Varro et al., 1993) and teleost (Hove-Madsen et al., 1998) species. Materials and methods Animals Three different species of octopuses were used in the study: Octopus vulgaris Cuvier, Eledone cirrhosa Lamarck and Eledone moschata Lamarck. The animals were caught with trawl-netting by commercial fishermen in fishing grounds near Barcelona (North West Mediterranean, Spain). While at sea, the animals were kept in clean aerated water. They were later transported to the aquarium facilities of the Universitat Autònoma of Barcelona (UAB), where they were maintained in a closed seawater system (3.6 %) at 16 °C and fed live crayfish (Carcinus maenas) every other day. Sprague-Dawley rats were housed in the Animal Facility at UAB and taken to the laboratory on the day of the study. Tissue homogenization and cell isolation Thirteen octopuses (306±46 g) and seven rats (402±4 g) (means ± S.E.M.) were used in the study. Octopuses were killed by decerebration and rats by cervical dislocation. The systemic hearts of octopuses and the ventricles of rats were quickly dissected free and placed in chilled homogenization solution (see below). Subsequently, the tissue was cut into small pieces and homogenized using a Polytron homogenizer in 25 volumes (50 volumes for rat tissue) of ice-cold homogenization buffer. The final homogenate was treated with a ground-glass homogenizer. The homogenization solution contained (in mmol l−1): 20 Tris, 2 MgCl2, 0.01 leupeptin, 0.01 phenylmethylsulfonyl fluoride, 1 dithiothreitol, 2 benzamidine and 250 (rat) or 1000 (octopus) sucrose. The difference in sucrose concentration reflects the different osmolality of the body fluids, which in Octopus are nearly iso-osmotic with sea water (Wells, 1978). The pH of the homogenization solution was adjusted to 7.2 (at 20 °C) using HCl. Cardiomyocytes from the systemic heart of E. cirhosa were
obtained by perfusing the systemic heart with an extracellular solution containing collagenase and trypsin. The composition of this solution was 419 mmol l−1 NaCl, 20 mmol l−1 KCl, 20 mmol l−1 MgCl2, 10 mmol l−1 Hepes, 0.05 mmol l−1 EGTA, 0.0375 mmol l−1 CaCl2, 0.25 mg ml−1 collagenase, 0.2 mg ml−1 trypsin and 0.5 mg ml−1 bovine serum albumin (BSA). Ca2+ uptake assays Portions of the crude homogenate obtained were added to different uptake solutions containing (in mmol l−1): 80 imidazole, 8 potassium oxalate, 6 MgCl2, 6 Na2-ATP, 0.58 EGTA, 0.02 Ruthenium Red, 3 sodium azide and 120 (rat) or 360 (octopus) KCl. The pH was adjusted to 7.4 with HCl. These solutions also contained varying concentrations of CaCl2, all with 45Ca2+ (3.7×10−3 Bq ml−1). The resulting Ca2+ activities were calculated with respect to the concentrations of EGTA, ATP and oxalate, ionic strength and pH using MaxChelator (Bers et al., 1994). Ruthenium Red, which blocks the Ca2+ efflux channels of the SR, was included to prohibit the escape of Ca2+ taken up by the SR vesicles (Feher et al., 1988). Sodium azide was applied to block mitochondrial accumulation of Ca2+ (Ito et al., 1974). To assess the SRindependent Ca2+ uptake, control experiments were performed in the presence of cyclopiazonic acid (30 µmol l−1), a blocker of the SR Ca2+-ATPase (Takahashi et al., 1995). At different times after the addition of the crude homogenate, 0.5 ml of the solution, directly followed by 2.5 ml of unlabelled uptake solution, were sucked through membrane filters (0.45 µm, HAWP 025 Millipore). The filter paper was incubated overnight in 4 ml of scintillation solution (Optiphase HiSafe 2, Wallac, Milton Keynes, UK) before counting 45Ca2+ activity in a scintillation counter. To relate radioactivity to mmoles of Ca2+, 50 µl of 45Ca2+-labelled uptake solution was counted under identical conditions. All the assays were carried out at 20 °C. Protein-specific Ca2+ uptake was calculated after determining the protein concentration of the homogenate (Lowry et al., 1951). Ca2+ uptake is expressed as nmol Ca2+ min−1 mg−1 protein. The uptake measurements involved two protocols. In the maximum uptake rate protocol, the uptake solution had a constant pCa of 4.96, and samples were taken every second minute for rat or third minute for octopus to assess the maximal uptake rate, up to a maximum time of 6 min for rat or 9 min for octopus. To assess the dependence of uptake rate on the SR, some of these experiments were performed in the presence of cyclopiazonic acid. The second protocol aimed to quantify the effect of Ca2+ availability, and uptake rate was determined after 8 min for rat and 12 min for octopus in a series of solutions with varying pCa. Electrophysiological measurements Ionic currents were measured using the patch-clamp technique in the whole-cell configuration using an EPC-9 software-driven amplifier (HEKA, Germany). The extracellular medium contained (in mmol l−1): 419 NaCl, 20
Cardiac sarcoplasmic reticulum Ca2+ uptake in octopuses 2533
Effect of caffeine on cell shortening Maximal cell shortening in isolated myocytes from the systemic heart was measured manually ‘off-line’ by replaying individual video frames from recordings of the caffeineinduced cell contractures. Maximal cell shortening was normalized to resting cell length. Calculations and statistics Cell volume was calculated from a cell model with an elliptical cross section as: Vc = (W/2)(W/4)L ,
(1)
where Vc is cell volume, W is cell width and L is cell length. The ratio C/V was used as a conversion factor to calculate the cell volume from capacitance measurements in patchclamp experiments. Values are given as mean ± S.E.M. Differences were analyzed for statistical significance using Student’s t-test with the level of significance set at P=0.05. Results In the first set of experiments the rate of uptake of Ca2+ was measured in conditions of saturating Ca2+ activity (pCa=4.96) with or without cyclopiazonic acid (CPA). The amount of Ca2+ recovered in the particulate fraction with time was well described by linear regression. Furthermore, Ca2+ accumulation appeared to be exclusively related to the uptake by the SR as it was abolished in the presence of CPA (Fig. 1). The maximal uptake rate of the cardiac homogenate was 43±4 nmol min−1 mg−1 protein for octopus (N=7) and 28±2 nmol min−1 mg−1 protein for rat (N=4). Hence, at 20 °C,
0.6
A
Ca2+ uptake (µmol mg-1 protein)
0.4 0.2 0 0 0.6
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9
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4 6 Time (min)
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Fig. 1. Ca2+ uptake rates by heart homogenates with (open symbols) and without (filled symbols) cyclopiazonic acid in (A) octopus (N=7) and (B) rat (N=4). Values are means ± S.E.M.
the maximal uptake rate is significantly higher for octopus than for rat (P
