Comparative study of respiration kinetics and protein composition of skinned fibers from various types of rat muscle.

The respiration parameters of mitochondria from rat heart muscle and from fast-twitch and slow-twitch skeletal muscle skinned fibers were comparatively analyzed. Electrophoretic patterns of fiber protein composition were also compared. It was found that fibers with low affinity of mitochondria for ADP (i.e., heart and slow-twitch skeletal muscle soleus) contain a 27.5-kD protein that is absent from the fibers that exert high affinity for ADP (i.e., fast-twitch skeletal muscle gastrocnemius). Partial proteolysis, which increases the affinity of mitochondria of the heart and slow-twitch skeletal muscles for ADP, results in the disappearance of this protein. The results suggest that this protein may be an intracellular factor that controls the permeability of the outer mitochondrial membrane for ADP.

Potassium channels in aortic microsomes: conductance, selectivity, barium-induced blockage and subconductance states.

The activity of potassium channels of canine aortic sarcoplasmic reticulum was measured using the planar lipid bilayer-fusion technique. The channels have a conductance of 208 pS (400/100 mM K+ in cis/trans solutions) and potassium-to-sodium permeability ratio of 7.7 Ba2+ ions produced two main effects: one is the interruption of channel currents for tens to hundreds of milliseconds in a voltage-dependent manner, and the other is the appearance of a second conductance level with amplitude about 60% of the main level.

Monoclonal antibodies to dog heart sarcoplasmic reticulum as markers of endoplasmic reticulum.

A monoclonal antibody (mAb 4B4) was raised against purified sarcoplasmic reticulum vesicles from canine myocardium, and shown to inhibit Ca2+ uptake by microsomes isolated from cardiac, skeletal, and smooth muscle. The amount of mAb 4B4 needed to inhibit the Ca2+ uptake 50% at a given membrane concentration correlated with the amount of Ca2+ pump protein in the microsomal preparation. This is consistent with the observation the mAb 4B4 binds specifically to the sarcoplasmic/endoplasmic reticulum Ca2+ pump (Mr 100 kDa), but has no effect on the T-tubule Mg2+-ATPase. Changes in the binding of mAb 4B4 to crude microsomes isolated from dog heart after various durations of global ischemia showed that the decrease in microsomal Ca2+ transport during the first 15 min of ischemia correlated with a loss of active Ca2+ pump molecules. The monoclonal antibody mAb 4B4 may therefore serve as a specific marker for the sarcoplasmic/endoplasmic reticulum Ca2+ pump system in various cells, and can provide quantitative information about the loss of active Ca2+ pump proteins under pathological conditions.

Characterization of Ca2+ release from the sarcoplasmic reticulum of myocardium and vascular smooth muscle.

The effects of Ca2+ and inositol-1,4,5-trisphosphate (IP3) as putative inducers of Ca2+ release from sarcoplasmic reticulum (SR) vesicles were studied. Addition of Ca2+ (5-50 microM) or caffeine (5 mM) to calcium loaded SR vesicles from canine ventricular myocardium caused immediate release of Ca2+; whereas, no such Ca2+ release was observed in canine aortic SR vesicles. Fractionation of the cardiac SR by zonal centrifugation of calcium/oxalate-loaded SR vesicles showed that Ca2+-induced Ca2+ release was present in a caffeine-sensitive, ryanodine-sensitive, calsequestrin-enriched fraction, probably derived from the junctional SR. By contrast, IP3 (greater than 0.1 microM) stimulated Ca2+ release from aortic SR, but had no such effect on cardiac SR (even with 50 microM IP3. These data indicate a difference in Ca2+ release mechanisms in SR from the heart and vascular smooth muscle.

Effects of changing Ca2+-to-H+ ratio on Ca2+ uptake by cardiac sarcoplasmic reticulum.

Ca uptake by fragments of cardiac sarcoplasmic reticulum (SR) in the absence of oxalate depends on the ratios between concentrations of free Ca2+ and H+ and on the level of Ca loading. Using a rapid quenching technique, we showed that at [Ca2+]f less than 1 microM or in short time intervals after ATP addition (less than or equal to 1 s), pH decrease led to a significant inhibition of Ca accumulation. If Ca transport was quenched in 1 s, [Ca2+]f for half-maximal stimulation of the process would be 0.3, 1.0, and 4.0 microM at pH 7.2, 6.8, and 6.2, respectively. At higher [Ca2+]f and at longer intervals, low pH increases the amount of Ca taken up by SR. These phenomena could not be explained by an interaction of ethyleneglycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) with the Ca2+-pump system since they were also demonstrated without application of Ca-EGTA buffers. Scatchard plot analysis of ATP-independent Ca binding to SR shows that apparent Kd for Ca increases from 0.3 microM at pH 7.2 to 7.4 microM at pH 6.2. pH shifts do not change the number of high-affinity Ca-binding sites. The results suggest a competition between Ca2+ and H+ for high-affinity binding sites on Ca2+-ATPase molecules. This may cause a delay of myocardial relaxation during ischemia when a decrease in intracellular pH takes place.

The cardiac relaxing system. Its nature, calcium ion capacity, and influence of hydrogen and magnesium ions on initial velocity of calcium binding.

The functional and structural properties of Ca2+ ATPases isolated from heart and skeletal muscles were compared. The pH and Ca2+ dependences of the activities as well as amino acid and phospholipid composition of the enzymes are similar. On the other hand, specific activities of Ca2+ ATPases and their abilities to pump calcium in the reconstituted proteoliposomes differ. The sarcoplasmic reticulum (SR) extracted completely from 1 g of pigeon and guinea pig hearts is able to bind up to 65 and 76 nmol Ca2+/sec, respectively, at 37 degrees (24 microM concentration of free Ca2+ ions). In the absence of oxalate, the process of calcium binding is nonlinear in a time interval of 100 msec to 5 min. One-third and half of the quantity of calcium consumed at 1 sec is bound at 100 and 200 msec, respectively. Judging by steady-state levels of the phosphorylated intermediate of Ca2+ ATPase in highly purified and completely extracted preparations of SR, an estimate was made that 1 g of heart contains from 2 to 3 mg of SR protein. The rate of energy-dependent calcium binding by isolated cardiac SR depends on pH and the concentrations of free calcium and magnesium. At calcium concentrations above 5 microM, the rate of calcium accumulation is higher at pH 6.2 than at pH 7.2. Increase in magnesium ion concentration from 0.5 to 6.0 mM leads to a significant inhibition of calcium binding at calcium concentrations 0.1 to 10 microM. The data obtained show that at physiological concentrations of calcium, the ability of SR to accumulate calcium is close to that postulated for a system of calcium transport providing relaxation for heart muscle.