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The release of Ca2+ from the terminal cisternae of sarcoplasmic reticulum in muscle fiber triggers muscle contraction. The signal for Ca2+ release is mediated via the triad junction, i.e. the junctional association of terminal cisternae and transverse tubule. Recently, highly purified morphologically intact triads were isolated from rabbit skeletal muscle (Mitchell, R. D., Palade, P., and Fleischer, S. (1983) J. Cell Biol. 96, 1008-1016). In this study, biochemical characterization of two variants of purified triad preparations (Pyrophosphate and Standard) is provided. Terminal cisternae of triads sequester Ca2+ at rates comparable to those of purified heavy sarcoplasmic reticulum which is referable to terminal cisternae (Meissner, G. (1975) Biochim. Biophys. Acta 389, 51-68). The permeability for calcium ions, as reflected by a 2-3-fold stimulation of (Ca2+, Mg2+)-ATPase activity in the presence of the Ca2+ ionophore A23187, and by the Ca2+ leak rate, is comparable in triads and heavy sarcoplasmic reticulum. Several transverse tubule characteristics are present in triads. Four of them, i.e. cholesterol content, ouabain binding, dihydroalprenolol binding (beta-adrenergic receptor), and ouabain-sensitive (Na+, K+)-ATPase activity, are comparably enriched in the Pyrophosphate triads and therefore appear to be quantitative indices of the amount of transverse tubule. Adenylate cyclase and basal ATPase are unreliable in this regard. Methodology for analyzing membrane integrity and sidedness was applied (adenylate cyclase activity) and modified (ouabain-sensitive (Na+, K+)-ATPase activity) to characterize the transverse tubule of the triad. In addition, a new method was developed making use of ouabain binding to study sidedness. These studies show that the transverse tubule is largely sealed and inside out in orientation, i.e. with the cytoplasmic face exposed. This report indicates that the t-tubule and sarcoplasmic reticulum components of the triads possess transport capability and retain permeability barriers for ions. Therefore, the isolated triads appear to be suitable for studying the physiological Ca2+ release process in vitro.
It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca2(+)-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, "weak," and breakage resistant, "strong," triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.
The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.
In previous work done in our laboratory, we have investigated the perceived pitch class of isolated musical triads. We have found that as the amount of musical training increased, listeners perceptions progress, from very confused percepts of pitch class, to analytic percepts corresponding to the pitch class of the highest note in the triad, and finally to synthetic percepts corresponding to the root note for the more harmonic triad types. In the present work, we used a pitch matching technique to determine the actual pitch, rather than merely the pitch class, perceived when listeners analytically "hear out" a particular note in a major triad. There was a strong tendency for the pitch of the analytically perceived note to be displaced by as much as 60 cents in the direction of the other notes in the triad. The magnitude of this effect decreased as musical training increased, and it was also affected by the relative salience of the individual triad notes. These results have implications for the mechanism of triad perception, and for claims regarding the harmonic equivalence of triad inversions.
Using biochemical and electron microscopic histochemical techniques, we studied membrane-bound alkaline phosphatase activities of rat hepatocytes and portal triads. Activity in portal triads was localized to capillaries surrounding bile ducts (peribiliary plexus) and arterioles. Despite the reputation of alkaline phosphatase as a "biliary enzyme," activity was not observed in bile ducts. Livers were separated into hepatocyte and portal triad fractions with collagenase. Enzyme from hepatocytes migrated faster during electrophoresis and eluted later during anion-exchange chromatography than that from portal triads. Thus, hepatocyte enzyme is more negatively charged (and also possibly smaller) than portal triad enzyme. Twelve hours after bile duct obstruction, new activity appeared on lateral and sinusoidal membranes of hepatocytes; appearance of portal triads did not change with obstruction. Electrophoretic mobilities of the two forms were not altered by obstruction. We conclude that two distinct liver alkaline phosphatases exist, one in hepatocytes, the other in portal triad blood vessels.
A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.
Characteristic synaptic complexes, the triadic synapses, were investigated in long series of sections of parvicellular and magnocellular laminae of the monkey lateral geniculate nucleus. Electron microscopic observations revealed the presence of different triadic types, the intercalated element being in all cases a presynaptic dendrite or soma of an interneuron (I-cell), and the output component being constantly a dendrite or soma of a geniculocortical projection or principal neuron (P-cell). The axonal input to the triads, however, was found to be of three different types: (1) the majority were retinal axon terminals; (2) a smaller fraction were the axonal endings of corticogeniculate fibers, always connected to thin, distal P-cell dendrites; (3) others were terminals with pleomorphic or flattened, small synaptic vesicles, probably belonging to axons of I-cells and/or of thalamic reticular nucleus origin. It was observed also that the retinal terminals established multiple synaptic contacts with both P-cell and I-cell dendrites. Essentially, two types of triadic arrangements with retinal input were recognized: the "simple" unit, frequent in parvicellular laminae, in which the retinal axon was accompanied by only 1-2 presynaptic dendrites; and the "complex" unit, found mostly in the magnocellular laminae, characterized by the presence of up to eight presynaptic dendrites. In the glomerular "complex" units, "closely packed" classical triads, with the three synaptic junctions localized close to each other, coexisted with triads "at a distance" where the synapses were distributed relatively far from each other. The coupling by presynaptic dendrites of "closely-packed" and "at a distance" triads resulted in the formation of multiple triadic arrangements. Since cortical and inhibitory triads were never seen to be involved in multiple triadic complexes, the latter appeared exclusively retinal in nature. The possible functional significance of multiple triads in "ON-gating" operation is briefly discussed.
Isolated triads from rabbit skeletal muscle were shown to contain an intrinsic protein kinase which was neither Ca2+/calmodulin-dependent nor cAMP-dependent. The protein substrates phosphorylated by this protein kinase exhibited apparent molecular weights of 300,000, 170,000, 90,000, 80,000, 65,000, 56,000, 52,000, 51,000, 40,000, 25,000, 22,000, and 15,000. Purification of the 1,4-dihydropyridine receptor from phosphorylated triads has demonstrated that the 170,000- and 52,000-Da subunits of the 1,4-dihydropyridine receptor are phosphorylated by this intrinsic protein kinase in isolated triads. Monoclonal antibodies to the 170,000-Da subunit of the dihydropyridine receptor immunoprecipitated the 170,000-Da phosphoprotein from detergent extracts of phosphorylated triads. The mobility of the 170,000-Da phosphoprotein in sodium dodecyl sulfate-polyacrylamide gels was not changed with or without reduction, demonstrating that the 170,000-Da phosphoprotein is not the glycoprotein subunit of the receptor. Our results demonstrate that the 170,000- and 52,000-Da subunits of the dihydropyridine receptor are phosphorylated by an intrinsic protein kinase in isolated triads. In addition, our results also demonstrate that the 175,000-Da glycoprotein subunit of the dihydropyridine receptor is not phosphorylated in isolated triads by the intrinsic protein kinase, cAMP-dependent protein kinase, or endogenous Ca2+/calmodulin-dependent protein kinase.
The effectiveness of the nonmetabolizable second messenger analogue DL-myo-inositol 1,4,5-trisphosphorothioate (IPS3) described by Cooke, A. M., R. Gigg, and B. V. L. Potter, (1987b. Jour. Chem. Soc. Chem. Commun. 1525-1526.) was examined in triads purified from rabbit skeletal muscle. A Ca2+ electrode uptake-release assay was used to determine the size and sensitivity of the IPS3-releasable pool of Ca2+ in isolated triads. Uptake was initiated by 1 mM MgATP, pCa 5.8, pH 7.5 Release was initiated when the free Ca2+ had lowered to pCa approximately 7. We found that 5-25 microM myo-inositol 1,4,5-trisphosphate (IP3), and separately IPS3, consistently released 5-20% of the Ca2+ pool actively loaded into triads. Single channel recording was used to determine if ryanodine receptor Ca2+ release channels were affected by IPS3 at the same myoplasmic Ca2+ and IPS3 concentrations. Open probability of ryanodine receptor Ca2+ release channels was monitored in triads fused to bilayers over long periods (200 s) in the absence and following addition of 30 microM IPS3 to the same channel. At myoplasmic pCa approximately 7, IPS3 had no effect in the absence of MgATP (Po = 0.0094 +/- 0.001 in control and Po = 0.01 +/- 0.006 after IPS3) and slightly increased activity in the presence of 1 mM MgATP (Po = 0.024 +/- 0.03 in control and Po = 0.05 +/- 0.03 after IPS3). Equally small effects were observed at higher myoplasmic Ca2+. The onset of channel activation by IPS3 or IP3 was slow, on the time scale 20-60 s. We suggest that in isolated triads of rabbit skeletal muscle, IP3-induced release of stored Ca2+ is probably not mediated by the opening of Ca2+ release channels.
A paired-comparisons task was used to determine which note of a pure-tone triad sounded most similar to the triad. Musically inexperienced Ss showed no systematic preference, experienced Ss consistently preferred the highest note in the triad, and professional musicians split equally between preferring the highest note and the root note. Preference for the root note shifted to preference for the highest note as the triad type became increasingly inharmonic, suggesting that the former depended on inference of a missing fundamental. When Ss were asked to vocally reproduce the pitch they heard when listening to a triad, similar results were obtained, except that a root-note preference was not detectable in Ss with less musical experience. Preference for the root note was also facilitated by use of octave-replicated tones, and this increase was shown to be due to obscuring of pitch-height cues, rather than harmonic complexity.
It has recently been demonstrated that the tonic level of accommodation is related to the loading of accommodative work. The aim of the present study was to investigate the effect of a sustained accommodative stimulus on the tonic level of accommodation, pupil, and convergence, i.e., the near triad, in three different groups of young (20-39 years) subjects, viz., 1. 19 professional visual display unit (VDU) workers; 2. 12 ordinary hard copy (non-VDU) workers; and 3. 19 control subjects doing different jobs from the two former groups. All had worked 25-30 h per week for over two years. Only Groups 1 and 2 had eyestrain after office work. Except for mild myopia, subjects had no eye or systemic disease upon routine examination. Accommodation was measured with an objective infrared optometer, the pupil was measured by an infrared video pupillography, and convergence was measured from the contralateral eye by photoelectronic oculography. In order to elicit tonic responses, the velocity of the target stimulus was 0.2 D (diopters) per second. Based on the data obtained from the controls, the near triad was analysed. Asymmetric response of the near triad in the right and the left eye and the existence of a hysteresis curve, especially in the pupil and in convergence, were the major findings. No abnormality was found in the control group. In the VDU group, 34.6% showed an abnormal near triad, but this also occurred in 18.5% of the non-VDU group. Even with the same complaint of eyestrain, it was concluded that VDU work produced abnormalities 1.8 times higher in the near triad produced by tonic stimulus.
Carney's Triad comprises a triad of neoplasms: gastric stromal tumor, extra-adrenal paraganglioma (usually functional), and pulmonary chondroma. At least two of these are needed for the presumptive diagnosis of the Triad. This report presents a patient who had resected a gastric tumor and nonfunctional extra-adrenal paraganglioma. The gastric tumor resembled a gastric leiomyosarcoma by light microscopy, but electron microscopy revealed it to be a gastric autonomic nerve (GAN) tumor. Based on this evidence it appears that both the gastric lesions and the paragangliomata of Carney's Triad are tumors of the autonomic nervous system. Thus, the Triad may be a disorder of the autonomic nervous system rather than a multiple endocrine neoplasia syndrome or multiple hamartoma syndrome.
The effects of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which has been hypothesized to be a chemical transmitter in excitation-contraction coupling in skeletal muscle, on aldolase bound to isolated triad junctions were investigated. Fructose-1,6-bisphosphate aldolase was identified as the major specific binding protein for the Ins(1,4,5)P3 analogue glycolaldehyde (2)-1-phospho-D-myo-inositol 4,5-bisphosphate which can form covalent bonds with protein amino groups by reduction of the Schiff's base intermediate with [3H]NaCNBH3. This analogue, Ins(1,4,5) P3, and the inositol polyphosphates inositol 1,3,4,5-tetrakisphosphate and inositol 1,4-bisphosphate were nearly equipotent in selectively releasing membrane bound aldolase with a K0.5 of about 3 microM. The rank order of the K0.5 values was identical to the KI values for inhibition of aldolase. Aldolase was also released by its substrate fructose 1,6-bisphosphate and by 2,3-bisphosphoglycerate. Ins(1,4,5)P3-induced aldolase release did not disrupt the triad junction; glyceraldehyde-3-phosphate dehydrogenase, a known junctional constituent, was displaced only at much higher Ins(1,4,5)P3 concentrations. Ins(1,4,5)P3 was as effective as fructose 1,6-bisphosphate in releasing aldolase from myofibrils. A finite number of binding sites for aldolase exist on triads (Bmax = 43-47 pmol of tetrameric aldolase exist on triads (Bmax = 43-47 pmol of tetrameric aldolase/mg of triad protein, KD = 23 nM). The junctional foot protein was implicated as an aldolase binding site by affinity chromatography with the junctional foot protein immobilized on Sepharose 4B. The potential consequences of aldolase being bound in the gap between the terminal cisternae and the transverse tubule to inositol polyphosphate and glycolytic metabolism in that local region are discussed.
1. Owing to differing structural characteristics of the contractile substance, the muscle fibres have been divided into the three types A, B and C in former papers. This distinction seems to be corroborated by our investigations into the different structure regarding the traids. As for the A-fibres, they are structured in terms of the T-system being connected in its entire length with the SR-cisternae and circling the myofibrils at the level of the Z-layer. In the B-fibres, this permanent couping of the two membrane systems is partially interrupted so that the T-tubules are arranged round the myofibrils in such a way that they are isolated or only coupled on one side with the SR-cisternae. Apart from the triads we also find diads. There is a totally different arrangement of the membrane systems in the C-fibres. In this instance the T-tubules are not only arranged transversally but also vertically along the contractile elements. They are surrounded by an "SR-labyrinth" which forms individual minor cisternae which are lateraly coupled with the T-tubules. So the axis of these triads is turned by 90 degrees as compared to the A and B fibres. As a result of this different arrangement, these triads always appear in cross-sections, not however, in longitudinal sections as is the case with the A and B fibres. The tirads have a varying shape in the cross-sections depending on the level of the section and due to the fact that the cisternae are not always coupled congruently with the T-tubules. 2. In our discussion we have tried to related these differing shapes and arrangements of the triads in the fibre types A, B and C to known physiological findings. Therefore we deduced that the excitation transmittance and calcium release can be correlated with the anomal rectification of the triads, which has been localized in the region where the T-tubules and SR-cisternae are coupled. However, we can only reckon with a solution once the morphology and function of the "feet" and the eletronmicroscopically "blank" spaces which fill the gap-junction between the T-tubules and the SR-cisternae have been explained. Whatever function the free surface of the T-tubules has remains open. It is directly adjoining the sarcoplasm and we are tryping to relate it to the delayed rectification which appears on the fibre membrane. 3. Moreover from the arrangement of the SR-cisternae i- the individual fibre types we can deduce th intrafibrillar directions of expansion of the calcium after its release and thus the process of the excitation in the A, B and C fibres. It appears that calcium is being directed homogeneously from the SR-cisternae of the A-fibres to the actinfilaments. here the morphological appearance of the twitch fibre presents itself to us. In principle this pattern of expansion of calcium in the B-fibres remains consistent. Owing to the interruption between the T-system and the SR-cisternae we may assume that the process of contraction is delayed in contrast to the A-fibres...
The Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m)[125I]iodophenyl) diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to Mr 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa fragment was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin.
The junction of isolated triads can be mechanically broken by passage through a French press and subsequently reformed by incubation of the isolated organelles with certain salts of weak acids (e.g., K cacodylate, K propionate, and K butyrate). In contrast, other salts (e.g., KCl, K phosphate, and K benzoate) are ineffective in promoting triad formation. An endogenous factor obtained from a muscle homogenate acts in the same manner as these artificial compounds. When rabbit skeletal muscle is homogenized in a KCl solution and centrifuged to remove large cellular components and membrane fractions, an endogenous factor is extracted into the high speed supernatant which promotes the reformation of mechanically broken triads. A three-stage purification of this factor has been achieved using: ammonium sulfate fractionation, adsorption chromatography, and molecular sieve chromatography. SDS-PAGE showed that the protein was purified to homogeneity and had a subunit Mr of 34,000 daltons. This protein has the following characteristics: it exists in 0.1 M KCl as a polymeric substance with an estimated Mr = 123,000 on molecular sieve chromatography and a Mr = 155,000 on sedimentation equilibrium; it promotes the formation of triadic vesicles from isolated organelles in a low ionic strength medium; Both this protein and cacodylate share the property of specifically catalyzing the association and aggregation of junctional proteins which had previously been dissolved by neutral detergent and salt; it appears to be identical to an extrinsic constituent of terminal cisternae, which has been described as a protein of Mr = 34K. It is not clear, however, whether this protein is a necessary and integral component of the junctional feet or whether it exerts predominantly a catalytic role in the formation of the triad junction.