Biomedical subjects

J J Feher

Publications and source records attributed to J J Feher.

At least 19 recordsLinked to original sources

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

The cardiac content of sarcoplasmic reticulum in the rat determined by calcium uptake rate, calcium oxalate capacity, ryanodine binding and thapsigargin titration.

The amount of cardiac sarcoplasmic reticulum in rat hearts was estimated by comparing marker activities in the isolated SR fraction with their activities in the homogenate. Four distinguishable markers were measured: the oxalate-supported rate of calcium uptake, the calcium oxalate capacity, 3H-ryanodine binding and the thapsigargin equivalents. The calcium uptake rate and capacity and thapsigargin equivalents were determined in the presence and absence of SR Ca2+ channel blockade with high concentrations of ryanodine. All of these activities are believed to be located only in the SR. However, the calculation of the heart content of SR was somewhat different for the four markers. The calcium uptake rate gave 8.4 mg SR protein per g tissue in the absence of ryanodine, and 9.6 mg per g in its presence; calcium oxalate capacity gave similar numbers, 9.9 mg per g in the absence of ryanodine and 8.0 mg per g in its presence. The thapsigargin titration gave similar equivalent with or without ryanodine, indicating that the homogenate contained about 8.0 mg of SR per g tissue. Using 3H-ranodine binding as a marker, the cardiac content of SR was calculated to be 16.7 mg per g. These differences are attributed to the non-ideal behavior of these markers. Some of the Ca2+ uptake activity is not thapsigargin sensitive, and some of the 3H-ryanodine binding does not fractionate with the SR Ca2+ uptake activity.

Animals↗

PubMed1998

Comparison of sarcoplasmic reticulum capabilities in toadfish (Opsanus tau) sonic muscle and rat fast twitch muscle.

The sonic muscle of the oyster toadfish, Opsanus tau, can produce unfused contractions at 300 Hz. Electron microscopy shows a great abundance of the Sarcoplasmic reticulum (SR) in this muscle, but no functional characterization of the capabilities of the SR has been reported. We measured the oxalate-supported Ca2+ uptake rate and capacities of homogenates of toadfish sonic muscle and rat extensor digitorum longus (EDL) muscle, and estimated the number of pump units by titration with thapsigargin, a high-affinity, specific inhibitor of the SR Ca-ATPase. The Ca2+ uptake rate averaged 70.9 +/- 9.5 mumol min -1 per g tissue for the toad fish sonic muscle, and 73.5 +/- 3.7 mumol min -1 g-1 for rat EDL. The capacity for Ca2+ -oxalate uptake was 161 +/- 20 mumol g -1 and 33 +/- 2 mumol g -1 for toadfish sonic muscle and rat EDL, respectively. Thus, the rates of Ca2+ uptake were similar in the two muscles, but the toadfish sonic muscle had about five times the capacity of the rat EDL. The number of pumps as estimated by thapsigargin titration was 68 +/- 4 nmol of Ca-ATPase per g tissue in the toadfish, and 42 +/- 5 nmol Ca-ATPase per g tissue in the rat EDL. The turnover number, defined as the Ca2+ uptake divided by the number of pumps, was 1065 +/- 150 min -1 for toadfish and 1786 +/- 230 min -1 for rat EDL (p < 0.05) at 37 degrees C. The Ca2+ uptake rate of toadfish sonic muscle at 22 degree C, a typical temperature for calling toadfish, averaged 42 +/- 1% of its rate at 37 degree C. At these operating temperatures, the toadfish SR is likely to be slower than the rat fast-twitch SR, yet the toadfish sonic muscle supports more rapid contractions. One explanation for this is that the voluminous SR provides activator Ca2+ for contraction, but the abundant parvalbumin plays a major role in relaxation.

Animals↗

PubMed1998

Ryanodine-sensitive, thapsigargin-insensitive calcium uptake in rat ventricle homogenates.

Thapsigargin is a natural product that specifically inhibits all known SERCA calcium pumps with high affinity. We investigated the effects of thapsigargin on cardiac sarcoplasmic reticulum (SR) by measuring the oxalate-supported calcium uptake rate in the unfractionated homogenate and in the isolated SR fraction. The uptake rate in both the isolated SR and unfractionated homogenate are stimulated about two-fold by preincubation with high concentrations of ryanodine, which closes the SR efflux channel. Thapsigargin stoichiometrically and completely inhibited the calcium uptake rate in the isolated SR, both in the presence and absence of SR channel blockade. In contrast, thapsigargin nearly completely inhibited the homogenate calcium uptake only in the absence of SR channel blockade; in the presence of blockade, about 20% of the uptake activity was insensitive to thapsigargin. This result unmasks a thapsigargin-insensitive, ryanodine-sensitive component of calcium uptake in the heart. This activity is in an oxalate-permeable pool and is inhibited by cyclopiazonic acid, another inhibitor of the SERCA calcium pumps. There was no TG-insensitive activity in the rat EDL muscle homogenate. The absence of thapsigargin-insensitive uptake activity in the isolated SR can be attributed to its inactivation during the isolation of the SR. The oxalate permeability and ryanodine sensitivity suggest that the TG-insensitive calcium uptake activity is closely related to the classical SR. The different thapsigargin sensitivities suggests the existence of two kinds of intracellular calcium pumps in the heart.

Animals↗

PubMed1997

Effect of ischemia on the fraction of ryanodine-sensitive cardiac sarcoplasmic reticulum.

The effect of 15 min of global, normothermic ischemia on cardiac sarcoplasmic reticulum (SR) was investigated using the Ca2+ uptake rate and 3H-ryanodine binding of ventricular homogenates and isolated SR vesicles. Ischemia did not affect ryanodine binding in the homogenate, while it increased it in the isolated SR vesicles. Although ischemia decreased the homogenate oxalate-supported Ca2+ uptake rate, measured in the presence of high ryanodine to close the ryanodine-sensitive efflux pathway (+RY), its decrease of the Ca2+ uptake rate, measured in the absence of ryanodine (-RY), was more marked. This finding was also observed in the isolated SR. Although inhibition of the Ca-ATPase and its coupled Ca2+ uptake by thapsigargin proportionately decreased SR Ca2+ uptake -RY and +RY, ischemia decreased the Ca2+ uptake -RY proportionately more. This result suggested that there was a greater fraction of Ca2+ uptake activity in ryanodine-sensitive vesicles after ischemia. However, ischemia also reduced the yield of SR activity in the isolated SR fraction and the results could potentially be due to differential selection of ryanodine-sensitive and ryanodine-insensitive SR in the isolation procedure. We directly tested the hypothesis that ischemia changes the fraction of Ca2+ uptake activity in the ryanodine-sensitive vesicles by estimating the Ca-oxalate capacity measured +RY and -RY. Ischemia decreased the capacity -RY much more than +RY in the homogenate, indicating that more of the SR volume and Ca2+ uptake activity was in the ryanodine-sensitive vesicles after ischemia.

Animals↗

PubMed1996

Salbutamol changes the molecular and mechanical properties of canine skeletal muscle.

1. Salbutamol, a beta 2-agonist, increased the weight of the canine latissimus dorsi muscle. It also increased fusion frequency, and decreased time-to-peak tension, half-relaxation time, and total contraction time. These changes in twitch times and fusion frequency were associated with changes in the levels of proteins expressed in slow- and fast-twitch fibres. Salbutamol decreased the levels of the slow-twitch cardiac isoform of sarco-/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban proteins, and increased the level of the fast-twitch isoform of sarco-/endoplasmic reticulum Ca(2+)-ATPase (SERCA1a). 2. Changes in the levels of SERCA proteins, particularly SERCA1a, could account for most of the increases in calcium uptake rate observed in homogenates of muscles from the salbutamol-treated animals and could partially account for the changes in half-relaxation rates and other twitch times. 3. Changes in the levels of SERCA1a, SERCA2a and phospholamban protein did not always follow changes in the levels of their corresponding mRNAs. Divergence depended upon the SERCA isoform and muscle. The muscles studied were latissimus dorsi and vastus intermedius. 4. Salbutamol did not change the level of myosin heavy chain (HC)-I isoforms in either muscle, suggesting that it did not increase the proportion of slow-twitch fibres in these muscles. It did increase the level of HC-IIx and decrease the level of HC-IIa isoforms in the latissimus dorsi. Salbutamol did not produce these effects in the vastus intermedius. It is of particular interest that salbutamol changed the relative levels of SERCA proteins in the latissimus dorsi muscle without producing significant change in the level of HC-I isoform.

Adrenergic beta-2 Receptor Agonists↗

PubMed1996

Salbutamol and chronic low-frequency stimulation of canine skeletal muscle.

1. The effect of simultaneous application of chronic muscle stimulation and salbutamol on the expression of mRNAs and proteins normally expressed by fast- or slow-twitch fibres was followed and the effects of changes in protein expression on mechanical performance were evaluated. Chronic low-frequency stimulation increased the myosin heavy chain (HC)-I level in the canine latissimus dorsi muscle and simultaneous administration of salbutamol partially blocked this change. Associated with the increase in HC-I level was a decrease in the velocity of shortening at zero load, VMAX. The change in VMAX was partially blocked by salbutamol. 2. Chronic low-frequency stimulation increased the levels of slow-twitch cardiac isoform sarco-/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban mRNA, and SERCA2a and phospholamban protein expression. These changes were associated with an increase in time-to-peak tension and a decrease in fusion frequency. Simultaneous administration of salbutamol blocked these changes in protein expression and muscle mechanics. Chronic stimulation of latissimus dorsi decreased the levels of the fast-twitch isoform of sarco-/endoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and increased SERCA2a protein expression and decreased calcium uptake rate by muscle homogenates. These changes were blocked by salbutamol. 3. The loss of latissimus dorsi muscle weight by chronic stimulation was partially blocked by salbutamol.

Adrenergic beta-Agonists↗

PubMed1996

Ryanodine perfusion decreases cardiac mechanical function without affecting homogenate sarcoplasmic reticulum Ca2+ uptake: comparison with the stunned heart.

This study tested the hypothesis that perfusion with low concentrations of ryanodine, which opens the sarcoplasmic reticulum (SR) Ca2+ channel in a sub-conducting state, could mimic the effects of stunning on both mechanical and SR activity. Perfusion of isolated rat hearts with 10-160 nM ryanodine progressively decreased left ventricular developed pressure (LVDP) and increased end-diastolic pressure (EDP), but LVDP decreased more and EDP increased less than in the stunned heart. The effect of ryanodine perfusion on LVDP and EDP is consistent with the opening of the SR Ca2+ channel by high-affinity ryanodine binding, reducing SR Ca2+ content and interfering with mechanical function. In contrast to stunning, ryanodine perfusion did not affect the homogenate Ca2+ uptake rates measured in the presence or absence of high [ryanodine]. Perfusion with 80 nM 3H-ryanodine resulted in a large decline in LVDP, but only a small degree of ryanodine binding. Thus, prolonged opening of only a few channels affects the SR in situ, whereas this is undetectable in the homogenate. Higher levels of ryanodine binding (0.3 pmol/mg) to the in vitro homogenate also did not affect the homogenate Ca(2+)-uptake rate in the presence or absence of high [ryanodine], whereas it reduced the stimulation of Ca2+ uptake by ruthenium red. High-affinity ryanodine binding to the SR Ca2+ channel, either by perfusion or by binding after homogenisation, did not duplicate the increased Ca2+ efflux observed in the stunned heart, suggesting that the SR defect in the stunned heart is not a prolonged opening of a sub-conducting state of the SR Ca2+ channel.

Animals↗

PubMed1995

Effect of ischemia and ischemia--reperfusion on ryanodine binding and Ca2+ uptake of cardiac sarcoplasmic reticulum.

The effect of 15 min of global, normothermic ischemia on 3H-ryanodine binding and the oxalate-supported Ca2+ uptake of cardiac sarcoplasmic reticulum (SR) was investigated in parallel using ventricular homogenates of isolated perfused rat hearts. Ischemia increased the Ca2+ efflux under the uptake assay conditions, as demonstrated by the greater stimulation of Ca2+ uptake by high concentrations of ryanodine (+RY) to close the SR Ca2+ channel. This effect was partially reversed by reperfusion. Ischemia depressed Ca2+ uptake rate -RY at free [Ca2+] of 0.4 microM and above, while the depression + RY was significant only above 10 microM Ca2+. We tested the hypothesis that inhibition of the Ca-ATPase alone, by adding thapsigargin or cyclopiazonic acid, could reproduce the effects of ischemia on the homogenate Ca2+ uptake rate. Thapsigargin or cyclopiazonic acid proportionally depressed Ca2+ uptake rate +RY and -RY and produced distinctly different effects of ischemia. Ischemia did not change the Bmax or Kd for equilibrium 3H-ryanodine binding, or the Hill coefficient or KCa for the [Ca2+]-dependence of equilibrium 3H-ryanodine binding. The rate of ryanodine binding, measured under the uptake conditions, was increased by ischemia and further increased by reperfusion. The effect of ischemia on the rate and extent of equilibrium binding to the high-affinity ryanodine binding site were unrelated to the highly reproducible effects on SR Ca2+ uptake rates measured in the homogenate.

Animals↗

PubMed1995

A simple student laboratory on osmotic flow, osmotic pressure, and the reflection coefficient.

Osmosis is usually taught from the point of view of the osmotic pressure developed when solutions of different concentrations of solute are separated by an ideal semipermeable membrane. The osmotic pressure is defined at equilibrium when there is no net flow, and it takes some time to reach this equilibrium. Although the osmotic pressure is certainly important, teaching only this point of view implicitly diminishes the importance of osmotic flow, which begins almost instantaneously across a membrane. A device was constructed with which students could measure the flow across a model membrane (dialysis tubing) as a function of concentration for solutes of different sizes. The device produced flows that were linearly proportional to the concentration, providing a confirmation of van't Hoff's law. Separate student groups repeated these experiments using both different solutes and different dialysis membranes. The combined results of four student groups showed that the flow across these nonideal membranes depends on the solute and membrane as well as the concentration of solute. Given a value for area times filtration coefficient (A x Lp) for the membranes (determined beforehand by their instructor), the students could calculate the reflection coefficient (sigma) for three solutes and two membranes. The results showed that large solutes had large sigma and that less porous membranes had larger sigma. A concurrent demonstration using this device and membranes showed that the osmotic flow can generate large pressures. These experiments and demonstration provide a balanced view of osmotic flow and pressure.

Dialysis↗

PubMed1994

Effect of low perfusate [Ca2+] and diltiazem on cardiac sarcoplasmic reticulum in myocardial stunning.

The ability of low perfusate Ca2+ concentration ([Ca2+]) or diltiazem to improve sarcoplasmic reticulum (SR) function and mechanical performance after ischemia-reperfusion was investigated using an isovolumic Langendorff preparation. SR function was evaluated by the oxalate-supported Ca2+ uptake rates of ventricular homogenates. Influx of Ca2+ was estimated from the rate of Ca2+ uptake in the presence of high concentrations of ryanodine (500 microM) to close the Ca2+ efflux channel. Ca2+ efflux under the assay conditions was estimated as the difference in Ca2+ uptake rate in the presence and absence of ryanodine. Ten and fifteen min of global, normothermic ischemia decreased the Ca2+ uptake rate in the presence of ryanodine, suggesting that Ca2+ influx was decreased. The effect of ischemia on Ca2+ influx was not altered by preperfusion with low [Ca2+] (0.2 mM) or with 1.0 microM diltiazem. Ischemia decreased SR Ca2+ uptake rate twice as much in the absence of ryanodine as in its presence, indicating an increased efflux of Ca2+. This increased efflux was reduced by preperfusion with either low [Ca2+] or diltiazem. The decreased Ca2+ influx was completely reversed by 15 min of reperfusion, whereas the increased Ca2+ efflux was only partially reversed. These results indicate that ischemia exerts independent effects on the SR Ca2+ influx and efflux pathways. The results also suggest that one site of cardiac protection by low [Ca2+] or diltiazem is the ryanodine-sensitive Ca2+ efflux pathway of the SR, rather than the Ca2+ influx pathway.

Animals↗

PubMed1994

Cooling and pH jump-induced calcium release from isolated cardiac sarcoplasmic reticulum.

Rapid-cooling contracture in cardiac muscle preparations is thought to be caused by the release of Ca2+ from the sarcoplasmic reticulum (SR), but the mechanism of this release is unknown. Cooling of isolated canine cardiac SR from 37 to 4 degrees C resulted in the net release of enough Ca2+ to account for rapid-cooling contracture. The release of Ca2+ on cooling appeared to be a relaxation between different steady-state levels of Ca2+ uptake. Cooling release of Ca2+ was also observed in the presence of ryanodine or ruthenium red to block the ryanodine-sensitive Ca2+ efflux pathway. In the presence of ryanodine, the extent and initial rate of rapid-cooling release were increased due to increased steady-state uptake of Ca2+ by the SR. The first-order rate constant of rapid-cooling Ca2+ release was unchanged by ryanodine or ruthenium red, suggesting that the rapid-cooling release does not occur through the ryanodine-sensitive pathway. The alkalinization on cooling was not the major cause of the Ca2+ release, as comparable Ca2+ release was observed with cooling and no alkalinization. However, alkalinization without cooling produced a rapid net Ca2+ release, which was also observed in the presence of ryanodine.

Animals↗

PubMed1992

Modulation of rabbit ventricular cell volume and Na+/K+/2Cl- cotransport by cGMP and atrial natriuretic factor.

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2Cl- cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volume(test)/volume(control)) were determined. Exposure to 10 microM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 was 0.77 +/- 0.33 microM. Activating guanylate cyclase with 100 microM sodium nitroprusside also decreased cell volume to 0.889 +/- 0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 microM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2Cl- cotransport was tested by blocking the cotransporter with 10 microM bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 microM 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2Cl-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 microM 8-Br-cGMP prevented the effect of 1 microM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 microM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100 microM) amplified the volume decrease caused by a low dose of ANF (0.01 microM) approximately fivefold. In contrast, neither 100 microM 8-Br-cAMP nor 50 microM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also appear to modulate cell volume.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1992

Role of facilitated diffusion of calcium by calbindin in intestinal calcium absorption.

Computer simulations of transcellular Ca2+ transport in enterocytes were carried out using the simulation program SPICE. The program incorporated a negative-feedback entry of Ca2+ at the brush-border membrane that was characterized by an inhibitor constant of 0.5 microM cytosolic Ca2+ concentration ([Ca2+]). The basolateral Ca(2+)-ATPase was simulated by a four-step mechanism that resulted in Michaelis-Menten kinetics with a Michaelis constant of 0.24 microM [Ca2+]. The cytosolic diffusion of Ca2+ was simulated by dividing the cytosol into 10 slabs of equal width. Ca2+ binding to calbindin-D9K was simulated in each slab, and diffusion of free Ca2+, free calbindin, and Ca(2+)-laden calbindin was simulated between each slab. The cytosolic [Ca2+] of the simulated cells was regulated within the physiological range. Calbindin-D9K reduced the cytosolic [Ca2+] gradient, increased Ca2+ entry into the cell by removing the negative-feedback inhibition of Ca2+ entry, increased cytosolic Ca2+ flow, and increased the efflux of Ca2+ across the basolateral membrane by increasing the free [Ca2+] immediately adjacent to the pump. The enhancement of transcellular Ca2+ transport was nearly linearly dependent on calbindin-D9K concentration. The values of the dissociation constant (Kd) for calbindin-D9K were previously obtained experimentally in the presence and absence of KCl. Calbindin with the Kd obtained in the presence of KCl enhanced the simulated Ca2+ transport more than with the Kd obtained in the absence of KCl. This result suggests that the physiological Kd of calbindin is optimal for the enhancement of transcellular Ca2+ transport. The simulated Ca2+ flow was less than that predicted from the "near-equilibrium" analytic solution of the reaction-diffusion problem.

Absorption↗

PubMed1992

Reversibility of the effects of normothermic global ischemia on the ryanodine-sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum.

The effect of normothermic ischemia and ischemia/reperfusion on the function of cardiac sarcoplasmic reticulum (CSR) was investigated using a modified Langendorff perfusion of isolated rat hearts. The function of the CSR was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates. The contribution of the ryanodine-sensitive portion of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The Ca2+ uptake rate of the CSR decreased progressively with increasing duration of ischemia, but this depression was much less when uptake was assayed in the presence of ryanodine. The depression in CSR Ca2+ uptake preceded ischemic contracture. Ryanodine and ruthenium red stimulated uptake almost equally in control hearts, but ruthenium red was much less effective than ryanodine after ischemia. This difference could not be overcome by increasing the ruthenium red concentration. These results confirm the suggestion that the Ca2+ release channel is inappropriately opened after ischemia. The CSR uptake rates were almost completely restored at 15 minutes of reperfusion after 5 and 10 minutes of ischemia but were only partially restored after 15 minutes of ischemia. At reperfusion, mechanical function (end-diastolic pressure and peak systolic developed pressure) was markedly depressed after only 15 minutes of ischemia. The degree of "stunning" correlated well with the depression of CSR function in individual hearts. The decreased Ca2+ uptake of the CSR was not due to a buildup of ADP in the homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Effect of perfusate [Ca2+] on cardiac sarcoplasmic reticulum Ca2+ release channel in isolated rat hearts.

The effect of perfusate [Ca2+] on the function of cardiac sarcoplasmic reticulum (CSR) was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates of isolated rat hearts maintained in a modified Langendorff preparation. The total Ca2+ pumping activity of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The homogenate Ca2+ uptake rate in the absence of ruthenium red or ryanodine decreased progressively with increasing perfusate [Ca2+] (25.7 +/- 1.2, 21.4 +/- 1.5, 17.2 +/- 1.1, and 16.3 +/- 1.2 [mean +/- SEM] nmol Ca2+.min-1.mg-1 for hearts perfused for 5 minutes with 0.2, 1.4, 2.8, and 5.6 mM Ca2+, respectively; p = 0.0001; n = 8). This depression was not observed when Ca2+ uptake was assayed in the presence of ryanodine or ruthenium red. Since the Ca2+ uptake in the presence of ryanodine or ruthenium red is determined by the Ca(2+)-ATPase, this result suggests that perfusion with varying [Ca2+] did not affect the Ca(2+)-ATPase. The observed decrease in Ca2+ uptake in the absence of ryanodine or ruthenium red is caused by an increased efflux through the ryanodine-sensitive Ca2+ release channel. When hearts perfused for 5 minutes with 0.2 or 5.6 mM Ca2+ were reperfused for 10 minutes with 1.4 mM Ca2+, homogenate Ca2+ uptake rates were restored to near control levels. These effects of perfusate Ca2+ were not direct effects, because changes in the [Ca2+] of the homogenization medium did not alter the homogenate Ca2+ uptake activity in either the presence or absence of ryanodine. The homogenate Ca2+ uptake rates were unaffected by prior active loading of the CSR with Ca2+. These results suggest a regulatory role of perfusate Ca2+ in increasing the open state of the ryanodine-sensitive Ca2+ release channel that is distinct from the beat-to-beat regulation of Ca2+ release from the CSR by Ca2+ (Ca(2+)-induced Ca2+ release).

Adenosine Triphosphate↗

PubMed1991

Effect of brief regional ischemia followed by reperfusion with or without superoxide dismutase and catalase administration on myocardial sarcoplasmic reticulum and contractile function.

The effect of reperfusion with and without free radical scavengers on sarcoplasmic reticulum and contractile function was examined in a canine model of 15-minute coronary artery occlusion followed by reperfusion. Dogs were reperfused with (n = 13) or without (n = 16) superoxide dismutase and catalase or were killed at 15 minutes of ischemia (n = 17). Superoxide dismutase and catalase were administered as a bolus (20,000 and 12,500 U/kg, respectively) beginning 1.25 minutes before reperfusion followed by infusion of 16,000 and 12,500 U/kg/hr, respectively. Sarcoplasmic reticulum function was evaluated from the rate of calcium uptake of unfractionated subepicardial, subendocardial, and transmural homogenates determined with and without ruthenium red to close the calcium release channel. Mechanical function was evaluated by means of sonomicrometry. Fifteen minutes of ischemia significantly (p less than 0.05) depressed the sarcoplasmic reticulum calcium uptake rate only in the subendocardium (from 25 +/- 2 to 14 +/- 1 nmol/min/mg without ruthenium red and from 60 +/- 3 to 49 +/- 3 nmol/min/mg with ruthenium red). Reperfusion with or without superoxide dismutase and catalase restored homogenate calcium uptake rates to normal, although severe contractile dysfunction persisted. This indicates that damage to the sarcoplasmic reticulum may not be the major cause of postreperfusion contractile dysfunction. Ischemia-reperfusion caused a decrease in systolic shortening from 19 +/- 2% to 1 +/- 2% with and from 18 +/- 1% to 4 +/- 1% without free radical scavengers (p = NS between groups). Thus administration of superoxide dismutase and catalase beginning shortly before reperfusion had no effect on postreperfusion contractile dysfunction or sarcoplasmic reticulum function.

Animals↗

PubMed1991

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.

Animals↗