Ionic basis of resting membrane potential in frog taste cells

Intracellular recording of the resting membrane potential was made from taste cells of the bullfrog by replacing the interstitial fluid surrounding the cells by various physiological saline solutions. The resting potential of the taste cell was -28 +/- 4 mV (mean +/- S.E.) after replacement of the interstitial fluid by normal Ringer solution. The resting potential was very much dependent on the interstitial K+ concentration ([K+]). Tenfold increase in [K+]o decreased the resting potential by 18 mV. Total removal of interstitial Na+ increased the resting potential by 40%. Ouabain in a concentration of 0.5 nM decreased the resting potential by 36% possibly due to inhibition of the Na+-K+ exchange pump. Neither total removal nor fourfold increase of interstitial Ca2+ changed the resting potential. Complete elimination of interstitial Cl- did not change the resting potential. The mean permeability ratio PNa/PK was calculated to be 0.41. It is concluded that the resting potential of a frog taste cell consists of ionic and metabolic components, and that the ionic component is due to the permeability of the cell membrane to K+ and Na+.     

Fever and behavioural temperature regulation in the frog Rana esculenta.

The skin and colonic temperatures were recorded in frogs (Rana esculenta) which had selected a suitable microenvironment in a box filled with 2-3 cm water. The water temperatures ranged from 0 degrees C to + 40 degrees C. Such measurements were performed before and after intraperitoneal injections of killed pathogenic bacteria (M. xenopi and M. range), killed non-pathogenic bacteria (M. aquae II) and 0.9% sterile saline, intraperitoneal injections of blood plasma from frogs pre-injected with killed M. ranae, injections of PGE1 into the brain. The injections of pathogenic bacterial endotoxin caused, after latencies of 5-120 min, higher preferred water temperatures, which produced an average maximum colonic temperature increase of 6.5 degrees C +/- 1.0 degrees C (S.E.) (p less than 0.001). The non-pathogenic bacteria and sterile saline caused no temperature change. Monophasic hyperthermia of shorter latency was caused by injections of blood plasma from frog preinjected with M. ranae. Monophasic hyperthermia of the shortest latency was observed after diencephalic injections of PGE1. Based on their similarity we suggest that ectothermic and endothermic fever have a common phylogenetic origin.     

Two populations of serotonin-immunoreactive neurons in the frog (Rana esculenta) retina

Serotonin immunocytochemical staining in retina whole-mounts of Rana esculenta revealed two differently stained amacrine cell populations. Statistical investigations using nearest neighbor analysis and comparison with different theoretical cell distributions suggested that the two serotonin-immunoreactive cell populations are related to functionally different types. The analysis of type A (intensely stained somata, less numerous) revealed a good coincidence with the normal Gaussian distribution indicating a functionally homogeneous population. The distribution of the neurons of type B (weakly stained somata, more numerous) reflects rather a random dot pattern than a regular arrangement. Thus there is evidence that this last type is a heterogeneous cell population consisting of several functionally different types.     

NADPH-diaphorase positive amacrine cells in the retinae of the frog (Rana esculenta) and pigeon (Columbia livia)

The distribution of NADPH-diaphorase positive cells was examined histochemically in the retinae of the pigeon and frog. In the pigeon, three different types of amacrine cells were identified in the inner nuclear layer (INL) on the basis of cell body size and staining intensity. In the frog two types of NADPH-diaphorase positive amacrine cells have been demonstrated in the INL. Therefore, the NADPH-diaphorase method selectively stains several subtypes of amacrine cells in the retina of lower vertebrates. Although the actual function of NADPH-diaphorase activity is unknown, diaphorase histochemistry provides a convenient method for achieving Golgi-like images of amacrine cells in the retina.     

Electrophysiological analysis of sodium-transport in the colon of the frog (Rana esculenta)

Sodium transport and apical bioelectrical membrane properties were investigated in frog colonic epithelium in the absence and presence of the antidiuretic hormone arginine-vasotocin (AVT). Apical Na-permeability and intracellular Na-activity were evaluated by analysis of current-voltage relationships in the serosally K-depolarized tissue. Tissue- and apical membrane capacitance were measured by voltages step analysis. The frog colon was found to be a tight epithelium with a transepithelial resistance of 2.63±0.25 k·F (n=17). 85–90% of short circuit current (11.2±1.1 A·F·l^–1;n=17) was related to electrogenic Na-transport from mucosa to serosa. Graded doses of amiloride (<50 mol·l^–1) induced Michaelis-Menten-type inhibition kinetics. Serosal addition of 10^–6 mol·l^–1 AVT induced a significant increase in sodium current (25%), apical sodium permeability (19%) and tissue capacitance (4.3%) whereas intracellular Na-activity remained unchanged. There was a good correlation between increased Na-current and apical Na-permeability. No correlation was found between Na-current and membrane capacitance. Our results demonstrate that in contrast to other species the amphibian colon shows a natriferic reaction to AVT. We suggest that the regulation of Na-transport in frog colon is similar to that in the toad urinary bladder. It is caused by an activation of preexisting apical Na-channels and not by fusion of subapical cytoplasmic vesicles with the apical membrane.     

Morphohistochemical changes of the blood cells in the hibernating frog (Rana esculenta L.)

Some morphological features (degree of nuclear segmentation in neutrophils and eosinophils), histochemical patterns (DHFR, LDH, G6PDH) and, in addition, the nucleic acid distribution (DNA, RNA with acridine orange) in the peripheral blood cells of Rana esculenta, during the hibernation phase were investigated. All the observed parameters varied significantly in the frog during hibernation in comparison with the active period. The most evident changes were an increase in the nuclear segmentation of the neutrophils and a lower activity of the histochemically demonstrable DHFR and LDH, probably due to cell ageing. These findings suggest that, in hibernating frogs, there is an increase in the life span of the peripheral blood cells as a consequence of a reduced metabolic activity and a slowing of haematopoiesis.     

Ultrastructural study of the semicircular canal cells of the frog Rana esculenta

The ultrastructure of the nonsensory cells (dark cells, transitional cells, and undifferentiated cells) of the frog semicircular canal was studied by using transmission electron microscopy in an attempt to correlate the structure with the functions of these epithelial cells. All the nonsensory cells were linked by tight junctions and desmosomes; this suggested that there is little paracellular ionic transport from perilymph to endolymph. In the dark cell epithelium, the apical intercellular spaces were dilated; in the basal part, numerous basolateral plasma membrane infoldings, containing mitochondria, delimited electron-lucent spaces. The undifferentiated cells and the transitional cells were devoid of any basal membrane infolding. Surrounding the semicircular canal, very flattened and interdigitated mesothelial cells constituted a thin multilayer tissue which limited the perilymphatic space. The morphological aspect of the dark cells suggests that they may play a role in the secretion and/or in the reabsorption of endolymph, which bathes the apical pole of these cells. The undifferentiated and transitional cells can play a role in the maintenance of the endolymphatic ionic composition because of their apical tight junctions and desmosomes.     

Role of eyes and pineal gland in melanophore response in the green frog. Rana esculenta

The studies suggest that complete inhibition of MSH liberation from the pars intermedia is brought about in response to the receipt of photic cues through the eyes and, possibly, by the pineal gland. The punctuate condition of epidermal melanophores is brought about by the inhibition of MSH liberation alone, whereas the punctuate condition of dermal melanophores is brought about by the inhibition of MSH liberation and the liberation of melanin aggregating substance from the pineal gland in response to photostimulation.     

Time course and extent of mechanotransducer adaptation in mouse utricular hair cells: comparison with frog saccular hair cells

Whole cell transduction currents were recorded from hair cells in early postnatal mouse utricles in response to step deflections of the hair bundle. For displacement steps delivered by a stiff probe (1-ms rise time), half-maximal responses decayed monoexponentially with a mean time constant of 30 ms. Adaptation and other transduction properties did not vary systematically with hair cell type (I vs. II) or region (striola vs. extrastriola). Thus regional variation in the phasic properties of utricular afferents arises through other mechanisms. When bundles were deflected by a fluid jet, which delivers force steps, transduction currents decayed about 3-fold more slowly than during displacement steps. A simple model of myosin-mediated adaptation predicts such slowing through forward creep of the bundle during a force step. For a faster stiff probe (rise time 200 micros), step responses of both mouse utricular and frog saccular hair cells decayed with two exponential components, which may correspond to distinct feedback processes. For half-maximal responses, the two components had mean time constants of 5 and 45 ms (mouse) and 2 and 18 ms (frog). The fast and slow components dominated the decay of responses to small and large stimuli, respectively. Adaptation shifts the instantaneous operating range in the direction of the adapting step. In frog saccular hair cells, the operating range shift is a constant percentage of the displacement. In mouse utricular hair cells, the percentage shift increases for large displacements, extending the range of background stimuli over which adaptation can restore instantaneous sensitivity.     

Correlation between chloride flux via the mitochondria-rich cells and transepithelial water movement in isolated frog skin (Rana esculenta)

The coupling between net transepithelial Cl- influx and net water flow was investigated. Experiments were performed on isolated frog skin bathed in isotonic Cl- Ringer's solution in the presence of the Na⁺ channel blocking agent amiloride in the mucosal solution. The skins were voltage-clamped at -80 or - 100 mV (with the serosal solution as reference). Under these conditions the current across the skin is carried by an influx of Cl-. In the absence of antidiuretic hormone the correlation between current and net water flow was low, but in the presence of the antidiuretic hormone, arginine vasotocin, there was a highly significant correlation between current and net water flow. The data presented here indicate that under steady state conditions about 70 molecules of water follow each Cl- ion across the skin. If the water influx is driven by electroosmosis one would expect that a change in current should result in an immediate change in the water flow. There was, however, a considerable time delay between the change in current and water flow. This indicates that the observed coupling between Cl- flux and water flow is caused by current-induced local osmosis and not electroosmosis.     

Molecular identification of an N-type Ca2+ channel in saccular hair cells

We report new molecular evidence for the presence of an N-type (Ca(v)2.2, alpha1B) voltage-gated Ca(2+) channel in hair cells of the saccular epithelium of the rainbow trout. The Ca(v)2.2 amino-acid sequence shows 68% and 63% identity compared with chick and human Ca(v)2.2, respectively. This channel reveals features that are characteristic of an N-type Ca(2+) channel: an omega-conotoxin GVIA binding domain, G(betagamma) binding regions, and a synaptic protein interaction site. Immunohistochemical studies with a custom antibody show that immunoreactivity for the Ca(v)2.2 is concentrated in the basolateral and apical regions of hair cells. Whereas trout brain and saccular macula express an 11-amino-acid insert in the second G(betagamma) binding domain of the Ca(v)2.2 I-II loop, isolated hair cells appear not to express this variant. We constructed fusion polypeptides representing portions of the I-II loop, beta1 and beta2a auxiliary subunits, the II-III loop, and syntaxin, and examined their intermolecular interactions via immunoprecipitation and surface plasmon resonance. The I-II loop polypeptides bound both beta1 and beta2a subunits with a preference for beta1, and the II-III loop exhibited Ca(2+)-dependent syntaxin binding. We demonstrated syntaxin immunoreactivity near afferent endings in hair cells, at hair-cell apices, and in efferent endings on hair cells, the former two sites consistent with binding of syntaxin to Ca(v)2.2. The present molecular characterization of the Ca(v)2.2 channel provides novel biochemical evidence for an N-type channel in hair cells, and details molecular interactions of this channel that reflect hair-cell function, such as spontaneous activity and vesicular trafficking. The current work, to our knowledge, represents the first demonstration of a putative N-type channel in hair cells as documented by tissue-specific antibody immunoreactivity and hair-cell-specific cDNA sequence.     

The effect of an intraperitoneal injection of capsaicin on the thermopreferendum in the frog (Rana esculenta)

In 7 frogs (Rana esculenta) weighing 70 to 180 g, thermopreferendum (Thp), measured by recording cutaneous temperature (Ts) in the animal placed in the warm end of an aqueous temperature gradient (0 degree C-40 degrees C), equalled 25 +/- 2 degrees C. After an intraperitoneal (IP) injection of 20 mg/kg of capsaicin, Thp was significantly decreased and equalled 3 +/- 1 degree C. The frogs then remained in the cold end of the gradient for 60 minutes. When the time of observation was extended to 3 hours, one frog died from hypothermia. Seven to 24 days after the capsaicin injection, Thp was still decreased in 4 surviving frogs (Thp = 15 +/- 2 degrees C). Capsaicin or isotonic saline solution injected in frogs maintained at 25 degrees C ambient temperature had no effect on Ts or on cloacal temperature. According to results previously obtained in homeothermic species, small doses of capsaicin activated heat-loss responses in the frog.     

NADPH-diaphorase-positive neurons and pathways in the brain of the frog Rana esculenta

We described the NADPH-diaphorase-containing neurons and fibres in the brain of the frog Rana esculenta. In the telencephalon stained cells occurred in the olfactory bulb, all subdivisions of the pallium, the diagonal band, the medial septum and the striatum. The olfactory glomeruli showed the most intense enzyme reaction. The neuropil of the accessory olfactory bulb was also heavily stained and this staining extended to the rostral diencephalon through the ventral lateral pallium. Fibre staining was less intense in the medial pallium and the medial septum. In the diencephalon, NADPH-diaphorase staining was concentrated in the middle third of this part of the brain. The stained cells were embedded in a dense network of thin, stained fibres and terminals in the lateral anterior and central thalamic nuclei. Faintly stained cells were present also in the posterior preoptic nucleus, anterior thalamic nucleus, nucleus of Bellonci, corpus geniculatum thalamicum and the suprachismatic nucleus. In the mesencephalon, heavily stained cells occurred in the nucleus profundus mesencephali, anterodorsal, anteroventral and especially in the posterodorsal tegmental nuclei. Neuronal staining was less intense in the optic tectum and the torus semicircularis. Thick, intensely stained fibres occupied the lateral part of the tegmentum and the 7th layer of the tectum. A loose network of thin fibres occupied the periventricular area and all tegmental nuclei. In the rhombencephalon, the reticular nuclei and the inferior raphe nucleus showed the most intense staining, while some cells in the nucleus of the solitary tract and the dorsal column nuclei were less intensely stained. Heavy staining of fibres was characteristic of the spinal trigeminal tract, the solitary tract and the reticulospinal pathway.     

Caffeine-evoked contractures in single slow (tonic) muscle fibres of the frog (Rana temporaria and R. esculenta)

Single slow (tonic) muscle fibres were dissected from cruralis muscles of Rana temporaria and R. esculenta. Increasing concentrations of caffeine were applied in Ringer solution, and contractures were measured isometrically. Sigmoid caffeine concentration-response curves were obtained, the threshold value being near 1.2 mmol/l, and maximum contractures being obtained with 10 to 20 mmol/l concentrations of caffeine. Contracture solutions were modified by varying the Ca²⁺ concentration or by replacing Ca²⁺ with 1.8 mmol/l Mg²⁺, Ni²⁺, Co²⁺ or with 0.1–5.0 mmol/l La³⁺. The effects of low pH (5.3), K⁺ (6,10 and 95 mmol/l), adenosine (10 mmol/l) and gallopamil (D600; 30 μmol/l) were examined too. The caffeine threshold was lowered by Mg²⁺, K⁺, 0 .1 mmol/l La³⁺ and D600, while all other substances including 0.5–5.0 mmol/l La³⁺ increased it. The amplitude of contractures evoked by high caffeine concentrations was unaffected. Caffeine (1–40 mmol/l) was also pressure injected into slow fibres. The composition of the solution was modified in a number of ways, but a contractile response was not observed or measured. Extracellular application of caffeine from the same pipettes evoked local contractures. Similar injection experiments in twitch fibres revealed the same results. These observations suggest that an extracellular binding site seems to be involved in the initiation of caffeine-evoked contractures in intact frog muscle fibres. Possible reasons for the ineffectiveness of intracellular caffeine are discussed. Received: 2 September 1995/Received after revision: 22 December 1995/Accepted: 4 January 1996     

A voltage-dependent K+ channel controlling the membrane potential in frog oocytes

Full-grown frog ovarian oocytes (Rana esculenta), were voltage clamped with a conventional two-microelectrode system. Depolarizations from a holding potential of –60 mV produced slowly developing outward currents. Two-step clamp experiments showed that, in Ringer's solution, this current has a reversal potential at about –84 mV. Substitution of either sodium or chloride with impermeant ions in the external solution did not alter significantly the activation of the current nor its reversal potential. Increasing the potassium ions concentration caused a shift on the reversal potential in the positive direction with a slope of about 48 mV per decade. The presence of TEA ions (50 mM) in the external solution partially reduced the current. It is concluded that the membrane of full-grown frog ovarian oocytes possesses voltage-dependent ionic channels permeated mainly by potassium. They appear to play an important role in the control of membrane potential.     

TRK neurotrophin receptor-like proteins in the kidney of frog (Rana esculenta) and lizard (Podarcis sicula): an immunohistochemical study

The present study investigates the occurrence of Trk-like neurotrophin receptor proteins in the lizard and frog kidney. In lizard rare TrkB-like immunoreactive cells in intermediate and distal tubules were found. TrkC-like immunoreactive cells were numerous in collecting tubules and became less numerous in collecting ducts. No TrkC-like immunoreactivity was detected in the ureteric duct. In the frog, we observed numerous TrkC-like immunoreactive cells in collecting tubules and ducts while they were scattered among negative epithelial cells in the wolffian duct. TrkB- and TrkA-like immunoreactivity was never found. Western blot analysis demonstrated that the frog and lizard kidney contains TrkC-like protein; TrkB-like protein was present only in the lizard kidney. These results demonstrate for the first time the occurrence of Trk-like proteins in the kidney of amphibians and reptiles, and aid in the assessment of the role of Trk receptor-like proteins in the kidney physiology of vertebrates.