Philip W. Brandt

Sinusoidal analysis: a high resolution method for correlating biochemical reactions with physiological processes in activated skeletal muscles of rabbit, frog and crayfish.

SummaryA high resolution method for determining the complex stiffness of single muscle fibres is described. In this method the length of the fibre is oscillated sinusoidally, and the resulting force amplitude and phase shift are observed and interpreted in terms of chemo-mechanical energy transduction. In activated, fast skeletal muscles of rabbit (psoas), frog (semitendinosus) and crayfish (walking leg flexor), we resolved at least three exponential rate processes. We named these (A), (B), (C) in order of slow to fast. These processes should reflect ATP hydrolysis and concomitant energy transduction since they are absent in muscles that are relaxed, in rigor or fixed. The great similarities in the complex stiffness data from different muscles suggests that there is a common mechanism of chemo-mechanical energy transduction across a broad phylogenetic range.

Effect of different troponin T-tropomyosin combinations on thin filament activation.

The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT2f and alpha 2 Tm exhibit steeper pCa/tension relations than those in which either TnT1f or TnT3f and alpha beta Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the Tn-Tm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo.

Plasma Membrane: Substructural Changes Correlated with Electrical Resistance and Pinocytosis

Inducers of pinocytosis in amoeba cause as much as a 50-fold decrease in the electrical resistance of the plasma membrane prior to the formation of the typical tunnels and vacuoles. In this state the thickness of the electron-transparent core or lamella of the unit membrane is at least twice as thick as that of the control. The changes in structure and resistance as well as the induction of pinocytosis are dependent on the initial external concentration of calcium. These changes are rapidly reversed when the concentration of calcium in the external medium is increased.

The thin filament of vertebrate skeletal muscle co-operatively activates as a unit

We find that extraction of as little as one troponin C molecule per troponin-tropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca2+ binding protein responsible for the steep slope of the pCa/tension relation.

A Consideration of the Extraneous Coats of the Plasma Membrane

This review is concerned with the role of the “extraneous coat” of the ameba in pinocytosis. It also considers the possible role of the extraneous coat as an additional barrier to those exchanges between the cytoplasm and the environment which involve diffusion. Several examples of pinocytosis in mammalian tissues are presented and are appraised with respect to the physical-chemical properties of the extraneous coat. Types of transport that do not appear to operate by way of pinocytosis are similarly considered.

An electron microscopic study of the Malpighian tubules of the grasshopper, Dissosteira carolina

Certain specializations of the Malpighian tubules of the grasshopper are described. The epithelial, tracheolar, and muscle cells are attached to each other by thin, flat lamellar cross-bridges which extend between adjacent plasma membranes. The basement membrane separating the epithelium from the muscular and tracheolar elements is fibrous, and in some areas the fibers have a unique periodic structure. The apical polypoid processes of the epithelium appear to be engaged in pinocytosis.

The role of surface chemistry in the biology of pinocytosis

Abstract In the process of pinocytosis, cells form surface invaginations which eventually become free vacuoles in the cytoplasm surrounded by a segment of the plasma membrane. To the early observers of the phenomena, the contents of the vacuole appeared to be fluid, not particulate, and on this basis the process was distinguished from its near relative, phagocytosis. Since certain solutes which induce pinocytosis become attached to and concentrated on the cell surface prior to the formation of invaginations, we proposed earlier that pinocytosis is induced by the physical changes which take place in the cell membrane as a consequence of solute adsorption (5). The uptake of droplets, therefore, is only the visible indication of a complex response. In recent experiments we have found that pinocytosis inducers in amoeba cause as much as a fiftyfold decrease in the resistance of the plasma membrane prior to the formation of the typical tunnels and vacuoles. In the low-resistance state the electron transparent core or lipid-rich lamella of the unit membrane is doubled or more in thickness over the control. Both the resistance and structural changes are dependent in magnitude on the external calcium concentration and are rapidly reversed by increasing the calcium.

Muscle: Volume Changes in Isolated Single Fibers

Volumetric experiments on single fibers isolated from semiten-dinosus muscles of frogs, some performed in correlation with measurements of membrane potential, confirm the data obtained on whole muscles, but only for the specific range of conditions in which most of the latter experiments have been done. These conditions are restricted to media in which the anion ( Cl usually) is permanent and the K is 10 to 12.5 meqlliter, or four to five times above the normal level in Ringers solution. When other ionic conditions are employed, phenomena are disclosed which have not previously been described. The findings throw doubt upon the validity of some generally accepted views regarding the permeability properties of the membrane of frog muscle fibers and regarding the nature of the mechanisms which regulate their volume.

Force Regulation by Ca2+ in Skinned Single Cardiac Myocytes of Frog

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.

Co-operative activation of skeletal muscle thin filaments by rigor crossbridges. The effect of troponin C extraction.

When Ca2+ binds to troponin C (TnC), all 26 troponin-tropomyosin (Tn-Tm) complexes of a regulatory strand change in concert from the inactive to the active configuration. To see if the complexes respond similarly when they are activated by rigor crossbridges in the absence of Ca2+, we determined the slope (ns) of the bell-shaped pS/tension (pS = -log [MgATP], where S = MgATP2-) relationship between pS 5, where the tension is maximal, and pS 2.3, where fibers are fully relaxed. In control skinned rabbit psoas fibers the ns value is greater than 4; it progressively decreases with TnC extraction. This decrease in ns with TnC extraction is analogous to the decrease in the slope (Hill coefficient) of the pCa/tension (pCa = -log [Ca2+]) relationship with extraction. Complete TnC extraction reduces the maximum substrate-induced tension by only 25%; in contrast, it reduces the maximum Ca2+ induced tension to zero. The effects of TnC extraction on the slope of the pS/tension curve are explained by the assumptions that (1) extracted Tn-Tm complexes no longer change in concert with their neighbors but change independently of them, and (2) co-operative signals cannot cross extracted Tn-Tm complexes. The ns value, therefore, like the nH, is a direct function of the number of contiguous, intact, Tn-Tm complexes in a stretch of a regulatory strand. To describe qualitatively the bi-phasic pS/tension relationship, the mono-phasic pCa/tension relationship, and the effects of TnC extraction on them, we introduce a version of the concerted-transition formalism which includes two activating ligands, Ca2+ and rigor crossbridges.