Joseph J. Feher

Mechanism of action of ryanodine on cardiac sarcoplasmic reticulum

Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This initial depression was followed by a later marked stimulation of calcium uptake. These effects were noted when calcium uptake was measured in the presence or absence of oxalate. The requirement for preincubation with ryanodine was highly dependent on ryanodine concentration and temperature. The mechanism of action of ryanodine clearly was not an effect on oxalate entry or calcium oxalate precipitation because the effects were also observed in the absence of oxalate. Ryanodine also had no effect on passive calcium efflux from actively loaded vesicles. Because ryanodine had no effect on Ca2+-ATPase activity under defined conditions of an ATP-regenerating system and no calcium gradient, we suggest ryanodine does not change the stoichiometry of the pump. Our results are consistent with the hypothesis that ryanodine closes a calcium channel in a subpopulation of the vesicles.

Differential effect of global ischemia on the ryanodine-sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum.

The effect of ischemia on the function of cardiac sarcoplasmic reticulum (SR) was assessed by the calcium uptake rate of rat whole-heart homogenates In the presence of 10 mM oxalate. Previous studies have shown that this uptake is restricted to the SR. The contribution of the ryanodine-sensitive fractions of the SR to the total homogenate uptake was assessed by using 20 μM ruthenium red and 625 μM ryanodine to close the SR calcium release channel under previously established optimal conditions. Global ischemia of 10, 15, 30, and 60 minutes depressed homogenate calcium uptake rate 19 ± 2%, 50 ± 6%, 65 ± 3%, and 81 ± 5%, respectively. This decrease was not observed when the uptake rates were measured after closure of the calcium channel with ryanodine or ruthenium red. Similar results were obtained with a Langendorff in vitro perfusion preparation, in which calcium uptake was decreased 35 ± 5%, 37 ± 8%, 58 ± 7%, and 64 ± 4% after 10, 15, 30, and 60 minutes of ischemia, but no significant decrease was observed when homogenate uptake rates were measured in the presence of ryanodine. Thus, ischemia caused a depression in the calcium uptake rate of cardiac SR only when this activity was measured in the absence of SR calcium channel blockers. Reperfuslon of ischemic hearts in a Langendorff preparation resulted in recovery of homogenate calcium uptake activity that correlated well with the return to sinus rhythm of the reperfused hearts. These reperfused hearts showed no change in the calcium uptake rate measured in the presence of ryanodine. These results suggest that the decrease in homogenate calcium uptake caused by ischemia is not due to a defect in calcium pumping capabilities but is due to an increased efflux through the ryanodine-sensitive calcium release channel of cardiac SR. {Circulation Research 1989;65:1400-1408)

Effects of ischemia on the isolation and function of canine cardiac sarcoplasmic reticulum

Normothermic global ischemia of 7, 10, 15 and 60 min was found to depress oxalate supported calcium uptake rate measured either in unfractionated homogenates or isolated sarcoplasmic reticulum. The degree of depression increased with the duration of ischemia. Comparison of the isolated sarcoplasmic reticulum with unfractionated homogenates showed that the isolated sarcoplasmic reticulum was more damaged by ischemia than the unfractionated homogenate. The cause of this discrepancy was not due to inactivation of sarcoplasmic reticulum during isolation but was due to the discard of greater portions of undamaged sarcoplasmic reticulum as the ischemic period increased. Ischemia preferentially affected that sarcoplasmic reticulum most easily fragmented by homogenization. To determine if the depression of sarcoplasmic reticulum function is uniform throughout the isolated fraction, we compared several properties of the isolated fractions. After 10 min of ischemia, extensive properties such as calcium oxalate uptake rate, calcium ATPase rate, calcium oxalate capacity and steady-state calcium loading were depressed 50, 41, 48 and 24% respectively. In contrast, intensive properties such as permeability, calcium-ATPase turnover rate, and ratio of forward nucleotide flux to reverse nucleotide flux were unaffected by ischemia. However, one intensive property, the coupling ratio, was depressed 20%. We conclude from this difference in the effects of ischemia on extensive and intensive properties that the major effect of ischemia is to inactivate the Ca-ATPase.

Ca-ATPase isozyme expression in sarcoplasmic reticulum is altered by chronic stimulation of skeletal muscle

Chronic stimulation of a predominantly fast skeletal muscle enhanced the expression of type I (slow muscle) Ca‐ATPase and suppressed the expression of the type II (fast muscle) Ca‐ATPase. Monoclonal antibodies IID8 and IIH11 against type I (slow) and type II (fast) isozymes respectively, were used to type the Ca‐ATPases of the isolated SR (sarcoplasmic reticulum) by Western blots, and the Ca‐ATPases of the muscle fibers by immunohistochemistry. Of the fibers from control muscles 80% stained for the type II isozyme and 20% for the type I isozyme. Following chronic stimulation all fibers stained for type I isozyme and none stained for type II isozyme. Ca‐ATPase isozyme distribution in isolated SR confirmed this effect of chronic stimulation. The calcium uptake activities of homogenates of stimulated muscles were 22% of the control muscles. The Ca‐ATPase and calcium‐uptake activities of the isolated SR from stimulated muscles were, respectively, 32 and 45% of the control muscles.

The interaction of calcium and ryanodine with cardiac sarcoplasmic reticulum

The binding of [3H]ryanodine with cardiac sarcoplasmic reticulum vesicles depends on the calcium concentration. Binding in the absence of calcium appears to be non-specific because it shows no saturation up to 20 microM ryanodine. The apparent Km value for calcium varied between 2 and 0.8 microM when the ryanodine concentration varied between 10 and 265 nM. The Hill coefficient for the calcium dependence of [3H]ryanodine binding was near two. Scatchard analysis of ryanodine binding indicated a high-affinity site with a Bmax of 5.2 +/- 0.4 pmol/mg with a Kd of 6.8 +/- 0.1 nM. Preincubation under conditions in which the high-affinity sites were saturated did not result in stimulation of the calcium uptake rate indicative of closure of the calcium channel. Stimulation of calcium uptake rate occurred only at higher concentrations of ryanodine (apparent Km = 17 microM). This stimulation of the calcium uptake rate also required calcium in the submicromolar range. The data obtained support the hypothesis that ryanodine binding to the low-affinity site (Km about 17 microM) is responsible for closure of the calcium release channel and the subsequent increase in the calcium uptake rate of the sarcoplasmic reticulum. Because the number of ryanodine-binding sites is much less than the number of calcium transport pumps the channel is probably distinct from the pump.

Isolation of rat cardiac sarcoplasmic reticulum with improved Ca2+ uptake and ryanodine binding

The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 M KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mM oxalate at 37 degrees C was 0.45 mumols/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 mumols/min-mg in the presence of 10 microM ruthenium red. The Ca(2+)-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 mumols/min-mg and 0.84 mumols/min-mg in the absence and presence of 10 microM ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nM and a Bmax of 3.7 pmol mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values Bmax, EP and Ca(2+)-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.

Determinants of calcium loading at steady state in sarcoplasmic reticulum

The determinants of steady-state calcium loading by sarcoplasmic reticulum vesicles were evaluated by measuring the contribution of different pathways of calcium flux to the total calcium flux at steady state. The diffusional passive pathway was least significant at all calcium loads studied. Diffusional passive calcium flux was evaluated by a number of methods which gave comparable results and support its designation as passive and diffusional. These methods included (a) flux measurements with the simple pump-leak system which pertains when acetyl phosphate is used to load the vesicles; (b) flux measurements made after quenching the pump with EGTA; (c) flux measurements made after quenching the pump with glucose plus hexokinase; and (d) evaluation of the effect of pump activity on the efflux of mannitol. The calcium efflux not accounted for by the diffusional pathway was assigned to non-diffusional pathways. Efflux through the non-diffusional pathways required ATP, ADP and extravesicular Ca2+. The ADP-dependent, phosphoenzyme-independent pathway described by Beirao and DeMeis (Biochim. Biophys. Acta (1976) 433, 520-530) was not significantly involved in efflux. We propose that the level of calcium loading achieved at steady state is determined by the levels of the intermediates of the calcium pump which are established at this pseudo-equilibrium condition, these levels being determined by the concentrations of intravesicular and extravesicular calcium ([Ca2+]i and [Ca2+]), ATP and ADP. The different levels of calcium loading achieved by skeletal and cardiac sarcoplasmic reticulum are attributed to different nucleotide and calcium kinetics in these two types of sarcoplasmic reticulum and possibly to different intravesicular volumes. Differences in diffusional permeability are not responsible for differences in calcium loading.

Flanking Proline Residues Identify the L-Type Ca2+Channel Binding Site of Calciseptine and FS2†

Calciseptine and FS2 are 60-amino acid polypeptides, isolated from venom of the black mamba (Dendroaspis polylepis polylepis), that block voltage-dependent L-type Ca2+ channels. We predicted that these polypeptides contain an identical functional site between residues 43 and 46 by searching for proline residues that mark the flanks of protein-protein interaction sites [Kini, R. M., and Evans, H. J. (1966) FEBS Lett. 385, 81-86]. The predicted Ca2+ channel binding site also occurs in closely related toxins, C10S2C2 and S4C8. Therefore, it is likely that these toxins also will block L-type Ca2+ channels. To test the proposed binding site on calciseptine and FS2, an eight-residue peptide, named L-calchin (L-type calcium channel inhibitor), was synthesized and examined for biological activity. As expected for an L-type Ca2+ channel blocker, L-calchin reduced peak systolic and developed pressure in isolated rat heart Langendorff preparations without affecting diastolic pressure or heart rate. Furthermore, L-calchin caused a voltage-independent block of L-type Ca2+ channel currents in whole-cell patch-clamped rabbit ventricular myocytes. Thus the synthetic peptide exhibits the L-type Ca2+ channel blocking properties of the parent molecules, calciseptine and FS2, but with a lower potency. These results strongly support the identification of a site in calciseptine and FS2 that is important for binding to L-type Ca2+ channels and reinforce the importance of proline brackets flanking protein-protein interaction sites.

The rate and capacity of calcium uptake by sarcoplasmic reticulum in fast, slow, and cardiac muscle: Effects of ryanodine and ruthenium red

The rate and capacity of oxalate-supported calcium uptake was measured in homogenates of rat fast, slow, and cardiac muscle. The contribution of the releasing fraction of the sarcoplasmic reticulum (SR) to the calcium uptake abilities was estimated using ruthenium red or ryanodine to block the release channel. A relatively small fraction (12-20%) of the calcium pumping activity was associated with the release channel in skeletal muscle compared to 50% or more in cardiac muscle. The total capacity of the SR in the muscle types was in the ratio 1:0.75:1.5 for cardiac, slow, and fast muscle, respectively, while the rates of uptake were in the ratio 1:3.8:14.4. The major difference in the muscle types appears to be the density of pumping activity in the SR rather than the volume of the SR. The difference in the density of pumping activity is due to intrinsic differences in the kinetics of the calcium pump units and in their surface density.

Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum

Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 mumol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 mumol/mg, but stayed constant at about 0.7 mumol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.