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The pressure-flow relationship of erythrocyte suspension in the bullfrog’s hind limb was studied by the perfusion method in which the rate of flow: pressure characteristic consists of a smooth curve convex to the pressure axis, tending to a straight line at a high rate of flow. This asymptotic line was found to have a negative intercept on the flow axis, which is independent of the haematocrit of the sample, and depended only on the radius of the vascular tube. In the light of the marginal zone theory, our finding appears appropriate.

Summary Oscillation and activated hyperpolarizing responses induced by electrical stimuli (H.A. responses) were studied in large nondividing L cells (giant L cells) under a variety of ionic conditions. When Cl- in the bathing fluid was partially replaced with SO42- at fixed external Na+ and K+ concentrations, the membrane potential depolarized transiently, but recovered to the original potential level after about 10 min. Under such a steady state in a low-Cl- medium, the amplitudes of oscillations and H.A. responses remained almost identical with those in the control medium. On exposure to a low-Na+ medium, both membrane potentials in the resting and hyperpolarized states were slightly hyperpolarized, but the pattern and the amplitude of oscillations and H.A. responses remained much the same. Changes in external K+ concentrations remarkably affected the amplitudes of oscillations and H.A. responses: the amplitudes decreased with increases in external K+ concentration. Calculation of the changes in K+, Na+ and Cl- conductances during oscillations and H.A. responses under these various ionic conditions showed that the change in K+ conductance is the only factor responsible for the oscillation and the H.A. response. The reversal potential for the potential oscillation is about -94 mV under normal conditions, this value being quite close to that of the equilibrium potential of K+. The reversal potentials in various external K+ concentrations satisfied the Nernst equation for a K+ electrode. Valinomycin induced remarkable hyperpolarization of the resting potential, resulting in an inhibition of oscillations. The level of valinomycin-induced hyperpolarization of the resting potential required to inhibit H.A. responses was the same as that of the peak potentials of the oscillation and H.A. response. In the light of these observations, it is concluded that the spontaneous potential oscillation and the H.A. response are caused solely by increase in the K+ conductance of the cell membrane.

Summary Dielectric dispersion measurements over a frequency range 0.01–100 MHz were made with the suspensions of a cultured cell line, mouse lymphoma L5178Y, and an attempt to explain the observed dielectric behavior by taking explicitly into consideration the possible involvement of cell nucleus has been presented.

Summary The membrane potentials and resistances of L cells were measured using a standard electrophysiological technique. The values obtained in physiological media were around -15 mV and 37 MO, respectively. Almost all the large nondividing L cells (giant L cells) showed spontaneous oscillations of the membrane potential between around -15 and -40 mV. Application of an appropriate electrical or mechanical stimulus was also capable of eliciting responses but such were usually induced only once. The total membrane conductance increased significantly and in parallel with such a hyperpolarizing response. Cooling of the cells and application of metabolic inhibitors to the cells completely blocked the spontaneous oscillation despite the fact that the electrically induced hyperpolarizing response remained. Intracellular K+, Na+ and Cl- concentrations were measured by means of a flame photometer and a chloridometer, and the equilibrium potential for each ion was estimated.

Addition ofd-glucose to the mucosal fluid resulted in a significant depolarization of the mucosal membrane potential (Vm) in rat duodenum, jejunum, and ileum accompanied by an increase in the transepithelial potential difference (PDt). On the other hand,l-glucose did not inducePDtandVmchanges. Glycine applied from the mucosal side also inducedVm-depolarization andPDt-increment in the ileum. Phlorizin added to the mucosal fluid or ouabain added to the serosal fluid inhibited the sugar-dependent changes inPDtandVm.Addition ofd-glucose to the mucosal fluid resulted in a significant depolarization of the mucosal membrane potential (Vm) in rat duodenum, jejunum, and ileum accompanied by an increase in the transepithelial potential difference (PDt). On the other hand,l-glucose did not inducePDtandVmchanges. Glycine applied from the mucosal side also inducedVm-depolarization andPDt-increment in the ileum. Phlorizin added to the mucosal fluid or ouabain added to the serosal fluid inhibited the sugar-dependent changes inPDtandVm.

The transepithelial resistance, the cell membrane resistance and the ratio of resistances of the serosal (baso-lateral) to the mucosal (brush border) cell membrane were measured in rat duodenum, jejunum and ileum by means of microelectrode techniques. These measured values were not affected in the presence of actively transported solutes in the mucosal bathing fluid.The transepithelial resistance, the cell membrane resistance and the ratio of resistances of the serosal (baso-lateral) to the mucosal (brush border) cell membrane were measured in rat duodenum, jejunum and ileum by means of microelectrode techniques. These measured values were not affected in the presence of actively transported solutes in the mucosal bathing fluid.

Summary As in our previous report (Kamino, Uyesaka & Inouye,J. Membrane Biol.17:13, 1974), the absorbance changes of murexide caused by Ca2+ and followed up by a dual wavelength spectrophotometer were applied to measure synaptosomal Ca2+-binding in the presence of cations such as Rb+, Mn2+ or La3+. All the cations tested showed a significant inhibition of synaptosomal Ca2+-binding except Li+. The inhibitory effects could be divided into the following three categories: (1) noncompetitive, co-operative K+-type, which includes alkali metal ions. The potency of inhibition is K+>Rb+>Cs+>Li+, Na+=0; (2) competitive Mn2+-type which includes many divalent cations. The inhibitory potency was found to be in the following order: Mn2+≫Sr2+>Cd2+, Ba2+>Mg2+; (3) nonspecific, noncompetitive La3+-type: among the cations tested, La3+ and Ce3+ were found to markedly reduce the Ca-binding capacity of synaptosomal particles, resulting in a noncompetitive inhibition, at least in the range of Ca2+ concentration used.

Summary Ca++ concentration in the synaptosomal suspension was measured to determine the liberation from and binding to isolated brain cortex synaptosomes by utilizing a dual wavelength spectrophotometer to monitor the absorbance changes of murexide raised by Ca++. When synaptosomes were suspended in isotonic solutions of a NaCl-KCl mixture containing more than 30 to 40mM of KCl, a marked liberation of calcium ion was observed in proportion to the rise in KCl concentration, whereas there was rarely any significant release of Ca++ observed with an external KCl concentration of less than 20mM. Titration of the synaptosomal suspension with Ca++ revealed that, in the absence of external K+, a part of added Ca++ was almost instantaneously bound to the synaptosomal particles so far as these particles had not been saturated with Ca++. Such a Ca++-binding was markedly depressed by a higher external K+ concentration. Ouabain and cyanide did not have that effect on such a K+-induced inhibition of Ca++-binding. The present results indicate that the inhibition of Ca++-binding by high external K+ concentration probably results from a change in synaptosomal membrane which is of a cooperative nature.

Fragmented epithelia of newborn rat small intestine were successfully cultured for periods of up to 4 weeks. Stable intracellular recordings of membrane potential were obtained from these cultured cells. Membrane resting potential varied according to cell location along a villus. The potentials ranged from -70 to -15 mV, being highest at the tip of the villus. The mean resting potential and membrane resistance were -72.4 mV and 8.6 M Ω, respectively. The membrane potential was markedly dependent on the extracellular K+ concentration ([K]0], but not significantly on [Na]0 and [Cl]0-Deprivation of Ca2+ from the surrounding medium depolarized the membrane by 20 mV. When the cells were cooled down to 6°C, membrane potential was reduced by 40 mV. Based on these data, basic mechanisms underlying the resting potential are discussed in connection with cell differentiation or maturation.

Some characteristics and the degree of intestinal absorption in the developing human fetus were examined by measuring solute evoked potentials and 14C-D-glucose uptake into the everted jejunal segments.In all segments, the Michaelis-Menten relationship was observed between D-glucose concentrations and the evoked potentials or D-glucose uptake. Increase of Na-ion concentrations enhanced both D-glucose evoked potentials and uptake.Both D-glucose and L-α-alanine evoked potentials increased in a significant correlation to the fetal age; however, the apparent Michaelis constants did not show any significant change. The structural specificity of sugar for generating evoked potentials was the same as that reported for adult mammals. Among amino acids, only the L-form of neutral and acidic amino acids generated markedly high evoked potentials, but the basic ones hardly at all. Oligopeptides such as glycyl-glycine and glycyl-glycyl-glycine also generated high evoked potentials.

Summary Effects of divalent cations on oscillations of membrane potentials (i.e., spontaneous repetitive hyperpolarizing responses) and on hyperpolarizing responses induced by electrical stimuli as well as on resting potentials were studied in large nondividing L cells. Deprivation of Ca2+ from the external medium inhibited these hyperpolarizing responses accompanying slight depolarization of the resting potential. Sr2+ or Mn2+ applied to the external medium in place of Ca2+ was able to substitute for Ca2+ in the generation of hyperpolarizing responses, while Mg2+, Ba2+ or La3+ suppressed hyperpolarizing responses. The addition of A23187 to the bathing medium or intracellular injection of Ca2+, Sr2+, Mn2+ or La3+ induced membrane hyperpolarization. When the external Ca2+, Sr2+ or Mn2+ concentration was increased, the resting potential also hyperpolarized, in a saturating manner. The amplitude of maximum hyperpolarization produced by high external Ca2+ was of the same order of magnitude as those of hyperpolarizing responses and was dependent on the external K+ concentration. In the light of these experimental observations, it was deduced that the K+ conductance increase associated with the hyperpolarizing excitation is the result of an increase in the intracellular concentration of free Ca2+ mainly derived from the external solution.

Summary Using nitroxide fatty acid spin labels, the effects of some cations such as La3+, Cd2+ and Hg2+ on synaptosomal membranes were studied by observing changes in their ESR spectra. The labels were incorporated almost instantaneously into synaptosomes isolated from rat brain cortex. ESR spectra of the spin-labeled synaptosomes were significantly broadened immediately upon adding La3+, Ce3+, Cd2+ or Hg2+ but hardly affected by Ca2+, Sr2+ and Ba2+. The magnitude of the change in the separation of the outer two peaks in ESR spectra (2T?') depends on the number (n) of methylene units between the polar head group and the spin-label (nitroxide) group; that is, it increases with decreasingn. Among these ions, the effect of La3+ was the greatest and appeared to be in parallel with the amount of La3+ bound with the synaptosomes. On the other hand, K+, Rb+ or Li+ causes hardly any significant changes.

Summary Ruthenium red combines with isolated synaptosomes, resulting in strong inhibition of their Ca2+-binding. In isotonic saline media, however, the dye-induced inhibition of Ca2+-binding is significantly greater than that expected for the amount of bound dye and Hill's exponent of the Ca2+-binding decreases to 1 with an increase in the amount of the dye bound. On the other hand in isotonic mannitol-sucrose solution, inhibition of synaptosomal Ca2+-binding brought about by the dye is proportional to the amount of dye bound. Based on these results, the effects of the dye on the co-operative nature of synaptosomal Ca2+-binding is discussed.

Summary Free ion concentration of some divalent heavy metal ions such as Mn2+, Co2+, Ni2+, Cd2+ and Zn2+ in the synaptosomal suspension was measured to determine binding with synaptosomes isolated from rat brain cortex. A dual wavelength spectrophotometer was utilized to monitor the absorbance changes of murexide raised by stepwise addition of these ions (as chloride salts). Such titration experiments of the synaptosomal suspension revealed that a part of the added divalent cation such as Mn2+, Co2+ or Ni2+ was almost instantaneously bound to synaptosomes in isotonic NaCl media. Our previous study (Kamino, Uyesaka & Inouye,J. Membrane Biol.17:13, 1974) demonstrated that raised external K+ resulted in a specific noncompetitive inhibition of synaptosomal Ca-binding. Just like the Ca-binding, Mn-, Co- or Ni-binding was almost completely depressed by high external K+ or ruthenium red when the free concentration of the cations was 10 µm or less, while at higher concentrations the binding was not affected. The present results indicate that tested divalent cations bind with both “Ca-binding sites” and “non-Ca-binding sites” of synaptosomal membrane, the nature of the binding sites of both being quite different: the former is sensitive to high external K+ and to ruthenium red but the latter is not.

1.Some properties of the open-tipped, uninsulated, pH-sensitive glass microelectrode were examined in several electrical experiments.2.Based on these observations, technical and theoretical problems were considered for application to the pH measurement in small cells.3.The intracellular pH, (pH)i, of the epithelial cell in rat duodenum measured was approximately 7.0. A reduction in (pH)i was apparent (about 0.3) with the addition of 20 mM-glucose to the bathing fluid.4.It was concluded, that with certain limitations such uninsulated, open-tipped microelectrodes may be successfully utilized for intracellular pH measurements.

1.Tip potential (TP) of glass microelectrodes filled with 3 M KCl increased remarkably with the increase in the storage period in 3 M KCl solution at 37‡ C, while the electrode resistances decreased gradually.2.The electrical conductivity through the thin glass wall near the tip was found to increase in parallel with the TP increase.3.The e.m.f. across the thin glass wall in the tip region was directly measured. This seems to contribute to the TP generation of the microelectrode when the conductivity of the glass wall is significantly high in the tip region.4.Effects of the acid treatment of glass employed and the acidification of fillant electrolyte solution suggested that fixed negative charges on the glass wall play a fundamental role in the TP formation.5.Based on these experimental results, it was concluded that not only the diffusion potential through the tip pore but also the interfacial potential through the thin glass wall near the tip contributes to the TP generation, and the contribution of the latter increases with a long exposure period of the electrodes to electrolyte solution.6.In this connection, technical problems related to reduction of the tip potential were also discussed.

Dielectric measurements were performed on the suspensions of synaptosomes isolated from rat brain cortex. The synaptosomes in buffered salt media showed typical dielectric dispersions caused by the presence of a thin limiting membrane of sufficiently low conductivity. An analysis of the dielectric data revealed that the electric conductivity of the synaptosome interior was about 37 % of the external medium conductivity under isotonic conditions and that the dielectric constant for the interior phase was about 35. The membrane capacitance (0.7 ?F cm-2) remained constant irrespective of nature and concentration of the univalent salts examined. Significant reduction in both the conductivity and the dielectric constant of the internal phase can be explained theoretically provided that some intra-synaptosomal structure (synaptic vesicles and/or small mitochondria) of non-conducting nature occupies about 50 % of the particulate volume, the remainder being in equilibrium with the external salt medium.

1. Applying the frontier electron method of Fukui et al., it was attempted to explain the effectiveness of nicotinic action of some choline phenyl ether derivatives and the substrate specificity of some phenyl acetates in the enzymatic hydrolysis.2. A fairly good correlation could be proved between the frontier electron density (fr) or its superdelocalizability (Sr) of a certain position of these compounds and their potency or susceptibility to hydrolysis.3. Assuming that the active site of receptor (or enzyme) has also π-electron systems and a drug-receptor (or -enzyme) complex is formed by hyperconjugation through π-electron systems, a general formulation of the effect of such a hyperconjugation on the perturbation energy of their system was attempted. It was demonstrated, qualitatively at least, by our calculations, that the larger the frof drug molecules or their Srthe larger the perturbation energy and so a possibility of above-mentioned correlation was predicted to some extent from the quantum-chemical basis.

IN previous experiments on the particulate 5-hydroxytryptamine (5-HT) of rabbit brain1, we found that the sub-cellular particles of rabbit did not take up appreciably the 5-HT added to the suspension media. Our observations were, however, made at a low temperature (4° C). Moreover, the 5-HT concentration used was very low compared with that utilized in work on the 5-HT uptake of blood platelet by Hunphry et al.2or Hardisty et al.3. Using higher incubation temperature (37°–60° C) and higher 5-HT concentration (up to 2,000 ng/ml.), the observations were repeated and a remarkable incorporation of added 5-HT into the sub-cellular particles of rabbit brain could be confirmed.

ROUGHLY speaking, RNA of high molecular weight could be regarded as a polymer composed of nearly the same units as in the synthetic high polymer, if the minor differences in the structure of its base components are assumed to be negligible and hence the relation such as [η] = KMαwould be expected between its intrinsic viscosity [η] and its molecular weight, M, which has been well known for the synthetic high polymers since the pioneer works of Staudinger. Indeed, Kurland1proved it on the ribosomal RNA, reporting that the value of α is nearly 0.5. Summarizing the data reported hitherto, Iso2also pointed out the following relation: is applicable to the RNA of the high molecular weight, where S0is the sedimentation constant in Svedberg units and M the molecular weight. The value of the exponent in these formations is very close to those calculated theoretically on the model of spherical molecules of matted chains without free drain, a fact which provides us with an indirect support for the view of Hall and Doty3that RNA molecules could be regarded as an ellipsoid of revolution hydrodynamically.

IN the course of experiments on the sub-cellular distribution of substance P in the rabbit brain1, we have confirmed Whittaker's finding2that the rat fundus stimulating action of his “synaptic vesicle fraction” was higher than that of the mitochondrial or supernatant fraction. As pointed out by him, however, the total amount of brain 5-hydroxytryptamine (5HT) thus estimated was about several times as much as that reported by previous workers3. This should be attributable to his omitting extracting 5HT by acetone. Using the acetone extraction3and Vane's rat fundus preparation4, therefore, we attempted to compare the sub-cellular distribution of brain 5HT with and without acetone treatment. Methods of preparation of the rabbit brain homogenates and of sub-cellular fraction were as described previously1. As illustrated in Fig. 1, the amount of 5HT estimated is lowest in the vesicle fraction, while that in the mitochondrial fraction is relatively high. It is apparent at a glance that the 5HT distribution obtained by omitting acetone treatment as in Whittaker's experiment is quite similar to that of substance P1,9. In fact, it was found that the rat's fundus was considerably sensitive to substance P, and the 5HT equivalence of acetone-insoluble aliquot of each fraction measured with the fundus preparation ran parallel to their substance P content (Fig. 1).

Ribosomes from liver are known to exist in a number of different stable forms, depending foremost on the concentration of free magnesium ions, and characterized by sedimentation coefficients of 120s, 80s, 60s and 40s. These different forms have been investigated by electron microscopy, using phosphotungstic acid staining. It was found that the 120s particles consisted of two 80s particles jointed together as a dimer. The 80s particles could be seen to contain two subunits of unequal size with a well-marked cleft between them. Electron microscope observations, after EDTA and urea treatment on the preparations of calf liver ribosomes, suggest the existence of a protein shell around a nucleic acid core in the particles.

Ribosomes from beef brain cortex were purified and examined in an electron microscope and in a ultracentrifuge. The ultraviolet absorption spectrum indicated the ribonucleoprotein character of the ribosome preparation. The nearly constant ratio of RNA/protein was found to be close to 50/50. The preparation of brain cortex ribosomes was characterized by the sedimentation coefficients of 120 s, 80 s, 60 s and 40 s. Electron microscope observations showed the different forms of brain ribosomes, of which stability depended on the Mg++ concentration of the medium. A 80 s particle could dissociate to form a 60 s particle and a 40 s particle.

RNA of high molecular weight was isolated from fresh calf livers and studied electron-microscopically by means of spraying-shadowing method. The RNA molecules were found polymorphic, but grossly classified into two categories; the longer rod-like ones and the shorter, rather oval ones. The dimension of the former was about 1,000 Å in length, 40–50 Å in width and about 30 Å in thickness, its molecular weight estimates from the dimension being about 1.2×106. This value was in good agreement with the sedimentation-viscosity molecular weight of the 26 S component found by ultracentrifugal analysis. The latter component was supposed to correspond to the 16 S component found by ultracentrifugation.

Hebb and Whittaker1have described the isolation of a sub-cellular fraction of brain tissue which contains bound acetylcholine and choline acetylase. This fraction was examined further by Whittaker2with equilibrium centrifugation in a density gradient. His results appear to provide evidence for physiological significance of his “synaptic vesicle fraction”. We have also attempted to apply his fractionation method to study sub-cellular distribution of substance P in the nervous system.