Nerve Stimulation and Electrical Properties of Frog Skin

The suitability of frog skin glands as a model for the study of secretory mechanisms in exocrine glands was explored. Periodic voltage clamp was used to determine continually the short-circuit current, chord conductance, and electromotive force of frog skin during neural and pharmacological activation of the skin glands. Both the chord conductance and the short-circuit current increased with glandular activation; the temporal dissociation of these increases suggests that there are at least two separate components to the secretory response. The sensitivity of the secretory electrical changes to changes in the ionic composition of the bathing solutions supports the notion of electrogenic chloride active transport as being basic to the activity of the exocrine glands.     

K Fluxes in Frog Skin

A method has been developed for measuring K influx into the epithelial cells of frog skin from the inside solution. Diffusion delay in the connective tissue has been taken into account. Ninety-four per cent of skin K was found to exchange with K⁴² in the inside solution with a single time constant. K influx showed saturation with increasing K concentration, was not altered by imposing a potential difference of ±200 mv across the skin, and was inhibited by dinitrophenol, fluoroacetate, and ouabain. Relatively low concentrations of dinitrophenol (5 x 10^-5 M) and fluoroacetate (10^-10 M) had no effect on k influx but caused a 40 per cent decrease in net Na flux. There was no correlation between the rate of K uptake at the "inner barrier" and the rate of net Na transport. Reduction of net Na transport by lowering Na concentration in the outside solution caused little change in K uptake. These observations indicate that there is not a significant Na-K exchange involved in active transport of Na across the skin. K influx was found, however, to require Na in the inside bathing solution.     

Intracellular Calcium Binding and Release in Frog Heart

The capacities and affinities of intracellular calcium-binding sites have been studied in frog ventricles, in which the concentration of Ca⁺⁺ in the sarcoplasm can be controlled as a result of treatment with EDTA. The total calcium content of calcium-depleted and nondepleted muscles at rest and muscles generating considerable tension was 0.8, 1.4, and 5.4 µmol/g of muscle, respectively. Net movement of calcium into or out of the cells occurred without change in tension when the sarcoplasmic concentration of Ca⁺⁺ was either of two values, less than 10^-7 M or approximately 5 x 10^-7 M. These data can be explained by the presence of two groups of intracellular calcium sinks which compete with the contractile proteins, one with a capacity of about 0.6 µmol/g and an affinity constant greater than 10⁷ M^-1 and a second with a capacity of 4.0 µmol/g and an affinity constant of about 2 x 10⁶ M^-1. The higher affinity calcium is released by anoxia, oligomycin, or abrupt changes in sarcoplasmic Ca⁺⁺. Muscles soaked in Sr-Ringer's contain electron densities in the sarcoplasmic reticulum and to a lesser extent in the mitochondria.     

Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

Autoradiographs consisting of a 1000 A thick tissue section and a 1400 A thick emulsion film have been prepared from frog toe muscles labeled with Ca⁴⁵. The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4°C, during relaxation following K⁺ depolarization or after prolonged depolarization. From 6 to 39 per cent of K⁺ contracture tension was produced during fixation. The grains in the autoradiographs were always concentrated in the center 0.2 to 0.3 µ of the I band and the region of the overlapping of the thick and thin filaments. The greater the tension produced during fixation, the greater was the concentration in the A band and the smaller the concentration in the I band. Autoradiographs of two muscles fixed by freeze-substitution resembled those of muscles which produced little tension during osmium fixation. Muscles which shortened during fixation produced fewer grains. In the narrow (<2.0 µ) sarcomeres of the shortened muscles, grain density decreased with decreasing sarcomere width. A theoretical analysis of the significance of these grain distributions is proposed and discussed.     

Ion Secretion and Isotonic Transport in Frog Skin Glands

The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion by the glands was studied by measuring unidirectional fluxes of ²⁴Na⁺, ⁴²K⁺, and carrier-free ¹³⁴Cs⁺ in paired frog skins bathed on both sides with Ringer's solution, and with 10⁻⁵ m noradrenaline on the inside and 10⁻⁴ m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the ¹³⁴Cs⁺ flux ratio, J ^out _Cs/J ⁱⁿ _Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free ¹³⁴Cs⁺ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux of ¹³⁴Cs⁺ indicates that a paracellular flow of water is dragging ¹³⁴Cs⁺ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving the secretory flux of Cs⁺ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na⁺ fluxes were measured as well, and it was found that also Na⁺ was subjected to secretion. The ratio of unidirectional Na⁺ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular route dragged by the flow of water. This result indicates that Na⁺ and Cs⁺ do not take the same pathway through the glands. The flux ratio of unidirectional K⁺ fluxes indicated active secretion of K⁺. The time it takes for steady-state K⁺ fluxes to be established was significantly longer than that of the simultaneously measured Cs⁺ fluxes. These results allow the conclusion that — in addition to being transported between cells — K⁺ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which accounts for stationary Na⁺, K⁺, Cl⁻ and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na⁺ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction of Na⁺ entering the lateral space from returning to the serosal bath. Thus, Na⁺ is secreted into the outside bath. It has to be assumed then that the Na⁺ permeability of the basement membrane barrier (P ^BM _Na) is smaller than the Na⁺ permeability of the junctional membrane (P ^JM _Na), i.e., P ^JM _Na/P ^BM _Na > 1. The secretory paracellular flow of water further requires that the Na⁺ reflection coefficients (σ_Na) of the two barriers are governed by the conditions, σ^BM _Na > 0, and σ^BM _Na > σ^JM _Na. (iii) Na⁺ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na⁺ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would be hydrolyzed for every Na⁺ secreted to the outside bath. Received: 30 January 1996/Revised: 12 March 1996     

Electrical Potentials during Gravitropism in Bean Epicotyls

An apparatus for measuring simultaneously the surface electrical potentials along epicotyl was developed to study the largely bending situation by gravity. Potentials increased on the upper side and decreased on the lower side after the horizontal placement. The time course of electrical changes consisted of two components which correspond to growth movements observed during gravitropism.     

Electrical Inexcitability of the Frog Neuromuscular Synapse

Frog muscle endplates were explored with an extracellular microelectrode. An intracellular microelectrode nearby simultaneously monitored invasion of the endplate by a spike directly evoked by a third microelectrode placed away from the endplate in the same fiber. External positivities were seen only at sites generating miniature endplate potentials. The external positivity reached a maximum prior to the internally recorded potential and was followed by a small late negativity. Small movements away from active synaptic sites resulted in positive-negative-positive potential sequences characteristic of activity and propagation. Since the external potential is a function of membrane current, the absence of negativity associated with the rising phase of the spike indicates the absence of inward current at synaptic sites. Thus, the synaptic membrane appears not to be excited by a depolarization of the magnitude of an action potential. In an Appendix it is shown that the late negativity and earlier maximum of the external potential can be accounted for by capacitative current through passive membrane.     

Contraction and Action Potentials of Frog Heart Muscles Soaked in Sucrose Solution

Isolated auricles or ventricles from the frog continue to contract, either spontaneously or when stimulated, for from 2 to 4 hours after they are placed in isotonic sucrose solution. After the muscles stop contracting in sucrose solution, contractility is partially restored when the muscles are placed in chloride Ringer's. However, contractility is usually not restored if the muscles are placed in sulfate Ringer's. Ventricles soaked in sucrose solution at 4–7°C continue to contract for 12 to 24 hours and during the first few hours in sucrose solution the contractions often are enhanced. Several types of experiment indicate that the sucrose solution does replace the Ringer's in the extracellular space. Auricles and ventricles also continue to conduct action potentials, with an overshoot, for from 30 to 360 minutes after being placed in sucrose solution. Muscles soaked in sucrose until they are inexcitable rapidly recover in chloride Ringer's but often fail to recover in sulfate Ringer's. The results are discussed in relation to theories about the generation of the action potential in cardiac muscle, and the role of the extracellular fluid in contraction.     

The Nature of Water Transport across Frog Skin

A method has been developed for determining simultaneously shortcircuit currents and net water fluxes across frog skin. The basis of the water flux measurement is the determination of changes in weight of a plastic chamber containing the skin and external solution. The accuracy of this method permits net water flows larger than 0.5 mg cm^-2hr.^-1 to be detected, and the apparatus has been used to investigate the relationship between active Na transport and non-osmotic water flow across the skin. Measurement of Na transport and net water influx across completely short-circuited skins provides no good correlation between the two flows. However, skins exhibiting no net water movement in sulfate Ringer displayed an apparent electroosmotic flow of about 40 water molecules per Na ion when depolarizing current densities of 50 and 100 μA cm^-2 are used. It is concluded from this and other evidence that the net water influx across frog skin may be partially electroosmotic in character and that there remains another component of water flow unrelated to active Na transport. A theoretical model, based on irreversible thermodynamics, has been developed to explain the non-osmotic water flow across frog skin.     

云南昆明地区三种蛙皮肤显微结构的比较

对昆明地区的多疣狭口蛙、昭觉林蛙及黑斑蛙的背腹皮肤切片的显微结构进行了观察和比较,尤其对它们皮肤腺体的类型和功能进行了比较分析.结果 表明:多疣狭口蛙皮肤比昭觉林蛙及黑斑蛙皮肤含有更多的腺体,尤其是颗粒腺比后两者含量更为丰富,并且在皮下存在许多腺体团,背腹皮肤较后者厚.昭觉林蛙皮肤较黑斑蛙的含有更多的颗粒腺和粘液腺.3种蛙的背部皮肤都含有色素细胞,而除了在多疣狭口蛙雌性腹部观察到少量色素细胞外,另两种蛙在腹部皮肤中均未见色素层.相比之下,昭觉林蛙和黑斑蛙皮肤结构较相似,两者同多疣狭口蛙在皮肤腺体分布及数量上差别较大,这也体现了三者不同的生态适应机制.     

Hyperosmolarity and the Net Transport of Nonelectrolytes in Frog Skin

The permeability of frog skin to a series of nonelectrolytes (thiourea, urea, mannitol, and sucrose) under the influence of 2.5 times normal osmolarity in the outer bathing solution has been investigated. Although the flux of the tracer nonelectrolytes across the skin in either direction is greatly increased by hyperosmolarity, the influx is found to be increased to a significantly greater extent than the outflux. Flux ratios as high as 3:1 can be observed. The net inward movement of the nonelectrolyte proceeds in spite of a sizeable bulk flow of water in the opposite direction. Possible driving forces for this phenomenon are discussed.     

The Hyperpolarizing Region of the Current-Voltage Curve in Frog Skin

Excitability (action potential and refractory period) has been described by A. Finkelstein in the depolarizing region of the current-voltage (I-V) curve of the isolated frog skin. Recently Fishman and Macey interpreted this phenomenon as a consequence of a region with negative resistance that confers to the I-V curve an N shape. We have studied the I-V relation of the isolated frog skin in the hyperpolarizing region with a current-ramp system. It was found that in Na₂SO₄ Ringer's, the resistance continuously increases in the hyperpolarizing direction. When hyperpolarization reaches 300 mv an electrical breakdown occurs, occasionally followed by a region of negative resistance. In NaCl Ringer's the breakdown was also found although the I-V relation was reasonably linear. Unidirectional Na⁺ outflux was measured at different levels of voltage clamping across the skin and with different Na⁺ concentrations in the solutions. The Na⁺ outflux was found to be relatively independent of these parameters. Based on these results a Na⁺ rectifying structure is postulated. An electrical model for active Na⁺ transport including a diode and an oscillator is proposed. The effects of CO₂, nitrogen, amiloride, and ouabain on the I-V relation are described.     

The Intracellular Site of Calcium Activation of Contraction in Frog Skeletal Muscle

Radioautography has been used to localize ⁴⁵Ca in isotopically labeled frog skeletal muscle fibers which had been quickly frozen during a maintained tetanus, a declining tetanus, or during the period immediately following a tetanus or a contracture. During a tetanus almost all of the myofibrillar ⁴⁵Ca is localized in the region of the sarcomere occupied by the thin filaments. The amount varies with the tension being developed by the muscle. The movement of calcium within the reticulum from the tubular portion to the terminal cisternae during the posttetanic period has a half-time of about 9 sec at room temperature and a Q₁₀ of about 1.7. Repolarization is not necessary for this movement. Evidence is given to support the notion that most calcium efflux from the cell occurs from the terminal cisternae into the transverse tubules.     

Normal CFTR Activity and Reversed Skin Potentials in Pseudohypoaldosteronism

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl⁻ channel function is required for activating amiloride-sensitive epithelial Na⁺ channels (ENaC) in salt-absorbing human sweat duct. It is unclear whether ENaC channel function is also required for CFTR activation. The dysfunctional ENaC mutations in type-1 pseudohypoaldosteronism (PHA-1) provided a good opportunity to study this phenomenon of ion channel interaction between CFTR and ENaC. The PHA-1 ducts completely lacked spontaneous ENaC conductance (gENaC). In contrast, the normal ducts showed large spontaneous gENaC (46 ± 10 ms, mean ± SE). After permeabilization of the basolateral membrane with α-toxin, cAMP + ATP activation of CFTR Cl⁻ conductance (gCFTR) or alkalinization of cytosolic pH (6.8 to 8.5) stimulated gENaC of normal but not PHA-1 ducts. In contrast, both spontaneous gCFTR in intact ducts and (cAMP + ATP)-activated gCFTR of permeabilized ducts appeared to be similar in normal and PHA-1 subjects. Lack of gENaC completely blocked salt absorption and caused dramatic reversal of skin potentials associated with pilocarpine-induced sweat secretion from significantly negative in normal subjects (−13 ± 7.0 mV) to significantly positive (+22 ± 11.0 mV) in PHA-1 patients. We conclude that virtual lack of ENaC in PHA-1 ducts had little effect on CFTR activity and that the positive skin potentials could potentially serve as a diagnostic tool to identify type-1 pseudohypoaldosteronism. An erratum to this article is available at .     

Intracellular Calcium Movements of Frog Skeletal Muscle during Recovery from Tetanus

Radioautographs of ⁴⁵Ca-labeled frog skeletal muscles have been prepared using freeze-dry and vapor fixation techniques to avoid displacement of the isotope during the preparation of the radioautographs. ⁴⁵Ca has been localized in resting muscles exposed to ⁴⁵Ca Ringer's for 5 min or 5 hr and in isotopically labeled muscles recovering from tetanic stimulation at room temperature or at 4°C. In muscles soaked at rest for 5 min ⁴⁵Ca was present almost exclusively in the terminal cisternae. In all other muscles there were three sites at which the isotope was concentrated: (a) the terminal cisternae, (b) the intermediate cisternae and the longitudinal tubules, and (c) the A band portion of the myofibrils. The terminal cisternae were labeled more rapidly than the myofibrils, but both exchanges were accelerated by electrical stimulation. The amount of ⁴⁵Ca in the longitudinal tubules and the intermediate cisternae decreased with time after a tetanus as the amount in the terminal cisternae increased. It is proposed that electrical stimulation releases calcium from the terminal cisternae and that relaxation occurs from the binding of the released calcium by the longitudinal tubules and the intermediate cisternae. Complete recovery from mechanical activity involves the transport of this bound calcium into the reticulum and its subsequent binding by the terminal cisternae. Resting exchange of calcium occurs primarily between the terminal cisternae and the transverse tubules.     

Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of providing precise and controlled stimulation. This article introduces a technique for recording somatosensory event-related potentials (ERP) that uses a novel mechanical stimulation method involving skin deformation using a robotic device. Controlled deformation of the facial skin is used to modulate kinesthetic inputs through excitation of cutaneous mechanoreceptors. By combining somatosensory stimulation with electroencephalographic recording, somatosensory evoked responses can be successfully measured at the level of the cortex. Somatosensory stimulation can be combined with the stimulation of other sensory modalities to assess multisensory interactions. For speech, orofacial stimulation is combined with speech sound stimulation to assess the contribution of multi-sensory processing including the effects of timing differences. The ability to precisely control orofacial somatosensory stimulation during speech perception and speech production with ERP recording is an important tool that provides new insight into the neural organization and neural representations for speech.     

Nuclear Magnetic Resonance Studies on Intracellular Sodium in Human Erythrocytes and Frog Muscle

The nuclear magnetic resonance (NMR) spectrum of sodium was determined in muscle and erythrocytes using conventional continuous wave techniques. NMR spectra of fresh intact muscle revealed a single line with a width of about 38 Hz equivalent in intensity to about 53% of the total muscle sodium, in general agreement with previous work. Prolonged washing with sodium-free solutions led to a marked loss of both total and NMR-detectable sodium. The NMR-visible sodium remaining in the muscle was somewhat larger than the fraction calculated to remain extracellular and, presumably, was intracellular. The original sodium signal is thus interpreted as arising from both extracellular sodium and the narrow line portion of the signal from intracellular sodium. NMR spectra of sodium were also obtained for human erythrocytes under conditions preserving the sodium transport system. The intensity of the sodium signal in fresh cells was 98% of that present in the same samples after complete hemolysis of the cells. The NMR sodium present in intact cells was 92% of the sodium recovered by flame photometric determination of sodium from ashed samples. It is concluded that no NMR-“invisible” sodium occurs in human erythrocytes and that the presence of such sodium is not necessary for the normal functioning of the sodium transport system in erythrocytes.