Abstract. We have recently described a preparation of junctional terminal cisternae (JTC) from fast skeletal muscle of rabbit hind leg. The fraction differs from other heavy sarcoplasmic reticulum (SR) fractions in that it contains a substantial amount of junctional face membrane (JFM) (15-20% of the membrane) with morphologically well-defined junctional feet structures. In common with other heavy SR preparations, it contains predominantly the calcium pump membrane (80-85 % of the membrane) and compartmental contents (CC), consisting mainly of calcium-binding protein (calsequestrin). In this study, a modified procedure for the preparation of JTC from frozen rabbit back muscle is described. The yield is substantially greater (threefold per weight of muscle), yet retaining characteristics similar to JTC from fresh hind leg muscles.Methodology has been developed for the disassembly of the JTC. This is achieved by selectively extracting the calcium pump membrane with 0.5 % Triton X-100 in the presence of 1 mM CaCI2 to yield a complex of JFM with CC. The CC are then solubilized in the presence of EDTA to yield JFM. This fraction contains unidirectionally aligned junctional feet structures protruding from the cytoplasmic face of the membrane with repeat spacings comparable to that observed in JTC. The JFM contains 0.16 lxmol phosphorus (lipid) per milligram protein. Characteristic proteins include 340 and 79-kD bands, a doublet at 28 kD, and a component that migrates somewhat slower than or equivalent to the calcium pump protein. Approximately 10% of the calcium-binding protein remains bound to the JFM after EDTA extraction, indicating the presence of a specific binding component in the JFM. The JFM, which is involved in junctional association with transverse tubule and likely in the Ca 2÷ release process in excitation-contraction coupling, is now available in the test tube.
We have studied factors controlling message levels for the neuronal growth- and plasticity-associated protein, GAP-43. Following exposure of PC12 cells to various effectors, cytoplasmic RNA was isolated and analyzed by Northern transfer and autoradiography using a GAP-43 cDNA probe. Induction by NGF is apparent after 3 hr exposure and reaches maximal levels at 24 hr. Beyond 24 hr, levels remain constant in the continued presence of NGF. Induction is insensitive to variations in culture conditions, such as plating density or substrate, which influence NGF-induced neurite outgrowth. Other inducers, in order of decreasing efficacy, are FGF, dBcAMP, TPA, K+, and EGF. Insulin and retinoic acid are ineffective. Dexamethasone partially inhibited basal expression as well as induction by NGF, FGF, dBcAMP, and TPA. The methyltransferase inhibitor 5'-S-(2-methyl-propyl)adenosine completely inhibited induction by NGF, FGF, and dBcAMP. Inhibition of protein synthesis by cycloheximide partially decreased induction by NGF, FGF, and TPA but slightly enhanced dBcAMP induction. Complete down-regulation of protein kinase C by chronic TPA treatment completely eliminated the TPA response but slightly enhanced induction by NGF. These findings and the results of additivity experiments in which cells were stimulated with various combinations of NGF, dBcAMP and TPA suggest that NGF induction of GAP-43 RNA (1) does not involve activation of protein kinase C but (2) may be mediated partially via activation of protein kinase A.
Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)
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