Inhibition of the process of sugar transport stimulation in the frog sartorius muscles by amines and amides

In the experiments on isolated frog sartorius muscles, amines and amides were found to inhibit the process of stimulation of D-xylose transport induced by insulin, 2,4-dinitrophenol or potassium contracture. The inhibitory action was produced by urea, acetamide, guanidine, NH4Cl, mono-, di- and trimethyl- or ethylamines, some diamines (all the substances being, applied in the concentration range equal to 100 mM). The similar effect was obtained when cystamine (20 mM), tryptamine, 5-methoxytryptamine (2 mM) and adenine, adenosine, guanosine (1-10 mM) were used. There was no inhibitory effect of acetone, glycerol, tetraethylammonium, propilamine, butylamine, aminoacids, spermine, spermidine, ATP, AMP or cAMP. It has been suggested that the inhibitory substances may interact by producing hydrogen bonds from NH-groups with the neutrally or negatively charged groups at the external surface of the muscle membrane in the region with a slow hydrophobicity. As a result, no structural changes required for activation of the sugar transport system occur in the membrane.     

2-Deoxyglucose transport by intestinal epithelial cells isolated from the chick

Summary Characteristics of 2-deoxyglucose uptake (2DG) by intestinal epithelial cells isolated from chickens were evaluated as a means of discriminating between the concentrative transport system for monosaccharides, associated with the mucosal brush border, and other possible routes of monosaccharide entry. 2DG was chosen as it is not a substrate for the mucosal transport system. The deoxysugar enters via a saturable pathway which is not Na⁺-dependent, is not inhibited by K⁺, does not accumulate solute against a concentration gradient; exhibits a high sensitivity to inhibition by phloretin; is relatively insensitive to phlorizin inhibition; and has low affinity [but high capacity relative to Na⁺-dependent mucosal transport of 3-O-methylglucose (3-OMG) and other monosaccharides]. These characteristics confirm those established in an earlier report for Na⁺-independent uptake of 3-OMG. Complications encountered in the use of 2DG as a test sugar include significant rates of metabolic conversion to an anionic form which presumably is a phosphorylated species. Methods for distinguishing between transport and subsequent metabolism are described. Inhibition of 2DG entry by several other sugars is described and inhibitory constants (K's) given for each.     

Action of insulin on the transport of 3-O-methylglucose in frog sartorius muscles

The initial rate of entry of 3-O-methyl-D-glucose into isolated frog sartorius muscles was measured at various concentrations of substrate, at 0, 9, 19, or 29 degrees C, after prior incubation at 19 degrees C with a maximally stimulating concentration of insulin. Control muscles were treated similarly, except for the omission of insulin. A saturable transport system provided for most of the entry of 3-O-methylglucose into muscle cells, but a small amount of penetration occurred by a nonsaturable route. The major effect of insulin was to produce a large increase in activity of the suturable system. The Vmax of entry increased, but there was no significant change in the apparent Km. The ratio of insulin-stimulated to basal Vmax was 10 when transport was measured at 29 degrees C but was 22 at 0 degrees C. These findings support the hypothesis that, although a large part of the effect of insulin on sugar transport can be accounted for by an increase in the number of functional transporters in the plasma membrane, there is a separate hormonal effect that permits a relatively greater activity of transporters at lower temperatures, compared with control rates. An additional effect of insulin was to produce a small but definite increase in the entry of sugar by the nonsaturable transport system.     

Actions of some cations on the electrical properties and mechanical threshold of frog sartorius muscle fibers

With the use of a point voltage-clamp technique, the effects of Zn²⁺, UO₂ ²⁺, tetraethylammonium, and several other homologous quaternary ammonium ions on the electrical properties of the frog sartorius muscle and its mechanical threshold were studied. None of the agents separated the voltage thresholds for mechanical activation and delayed rectification. However, Zn²⁺, UO₂ ²⁺, and TEA, which are known to potentiate the twitch, caused some inhibition of the normal increase in potassium conductance during delayed rectification. Zn²⁺ and UO₂ ²⁺ also slowed the rate of development of the outward current. A strength-duration relation was studied for depolarization pulses capable of initiating contraction. With a depolarizing pulse of 2.5 msec the mechanical threshold is about -13 mv at about 20°C. UO₂ ²⁺, 0.5 µM, which markedly reduced the outward current produced by such a short pulse, did not raise the mechanical threshold. All findings indicate that there is no direct causal relation between delayed rectification and mechanical activation.     

Restoration of limb regeneration ability in frog tadpoles by electrical stimulation

Electrical stimulation of advanced tadpoles of Rana breviceps for 24 hr with 3 microA current after hindlimb amputation through shank and ankle slightly increased the number of perfect regenerates as compared to controls. When amputation was made through thigh in tadpoles of the same stage regeneration failed in all cases of the controls but 24 hr stimulation of these tadpoles after similar operation with 3 and 10 microA currents induced fair degree of regeneration in 9 and 40% cases, respectively.     

Effect of amines on insulin-stimulated processes: the transport of sugars and synthesis of glycogen in frog sartorius muscles

Amines and amides were found to inhibit the process of stimulation of sugar transport in muscle tissue (N. A. Vinogradova et al., 1978). The present paper reports results of experiments on frog sartorius muscles acted upon by amines and other substances that inhibit induction of cultured cell proliferation. The stimulation of sugar transport induced by insulin, 2,4-dinitrophenol, or KCl was found to be inhibited by dansylcadaverine (1 mM), 5-methoxytryptamine (2 mM), or methylamine (100 mM). Such substances as chloroquine, bacitracin, or monensin exerted no effect. Besides, dansylcadaverine (1 mM), and 5-methoxytryptamine inhibited the stimulation of insulin induced glycogen synthesis. Dansylcadaverine was ineffective at concentrations lower than 0.5 mM. It is suggested that the inhibitory action of amines depends on their influence on the processes of membrane protein phosphorylation.     

Effects of caffeine on the intracellular distribution of calcium in frog sartorius muscle

The influence of caffeine on the intracellular distribution of calcium in the frog sartorius muscle was studied by differential centrifugation in an attempt to identify the locus of action of this alkaloid. The problem was approached in two ways. In the first, the locus of action was sought by relating the kinetic functions of ⁴⁵Ca washout curves of muscles to changes in the distribution of ⁴⁵Ca in the isolated fractions from the same muscles. It was not possible to make any correlation of the ⁴⁵Ca-washout curves to the activity in the fractions; the relative distribution of this nuclide remained essentially unchanged at 1-, 2-, and 3-hour intervals along the curve. The washout curves appear to be the net effect of a complex interaction of the calcium in pools containing both readily exchangeable calcium and calcium which has a slow exchange or turnover rate. The second approach centered upon the examination of the effect of caffeine on the intracellular distribution of ⁴⁵Ca and of calcium among the cellular fractions. Caffeine treatment resulted in a distinct increase in the calcium content of the mitochondrial fraction and a decrease in the calcium of the microsomal fraction. Electron micrographic studies revealed significant morphological changes in the whole muscle and in the isolated mitochondrial fraction after the muscle had been exposed to caffeine in a concentration producing irreversible contracture or rigor (10 mM). The increase in calcium content of the mitochondrial fraction after caffeine treatment may be due to an actual accumulation of calcium by the mitochondria or may be the consequence of the appearance of granular vesicles in the fraction.     

Activation of the frog sartorius acetylcholine receptor by a covalently attached group

Summary The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester ofp-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor inElectrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorius motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic bond.     

Intracellular pH of frog sartorius muscle.

A weak base, morpholine, has been labelled with 3H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20 degrees C in a solution of the same ionic strength as frog Ringer was found to be 8.45 +/- 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indcator. Intracellular pH was then measured by distribution of morpholine (6.985 +/- 0.08), nicotine (6.915 +/- 0.03) and the weak acid 5,5'-dimethyl-2,4-oxazolidinedione (7.10 +/- 0.05) and the pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and all ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H+ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Satellite and invasive cells in frog sartorius muscle

The occurrence and distribution of two cell types associated with normal and denervated frog skeletal muscle fibers are described. The first is the satellite cell. The general appearance and the number of satellite cells are not affected by long-term denervation. The second type of cell is the invasive cell. Invasive cells penetrate across the basal lamina and up to the core of the muscle fiber, without fusing with it. It is suggested that the origin of invasive cells is extramuscular, probably circulatory. Although invasive cells are more numerous in some denervated muscle, it is established that this is not a direct effect of denervation.