Immunohistochemical study on the development of the adenohypophysial cells in the lizard Gallotia galloti

Summary Immunohistochemical methods have been used to study the embryonic and postnatal development of the hormone-producing cells in the adenohypophysis of the lizard Gallotia galloti. In this species, Rathke's pouch is formed between stages 30 to 32 of the embryonic development, although the first sign of immunoreactivity to antisera against adenohypophysial hormones occurs in stage 33 in the pars distalis anlage. These cells derive from the dorsal face of Rathke's pouch and are immunoreactive to anti-ACTH serum. The cytodifferentiation of ACTH and MSH cells occurs in the pars intermedia in stage 34. The TSH cells appear at stage 35 and the gonadotrope cells at stage 37. These cells derive from both the dorsal and ventral face of the Rathke's pouch. The LTH cells are revealed at stage 39 and are only originated from the dorsal face. The STH cells, which come from the dorsal as well as ventral face, are the last secretory cells differentiated just before hatching. During postnatal development an increase and also a redistribution of the immunoreactive cells occur until acquiring the adult distribution.     

Small granule-containing cells in the heart of the toad (Bufo melanostictus)

This study reveals the presence of small granule-containing cells in the heart of the toad, Bufo melanostictus. The cells were small in size (5-25 microns in diameter) and located in the interatrial septum. The small cells occurred singly or in small clusters within the cardiac ganglia or near the myocardial cells. They were characterized by numerous large dense core vesicles (80-300 nm) in their cytoplasm. With 5-hydroxydopamine treatment, the granularity of the large dense core vesicles was greatly intensified. The large dense core vesicles were variable in size and shape and extended into the long and short processes of the cells. Some of these processes were in close contact with myocardial cells. Other cytoplasmic organelles included rough endoplasmic reticulum, free ribosomes, randomly distributed mitochondria and glycogen particles. Individual cells or cell clusters were usually ensheathed by a thin layer of cytoplasm from sheath cells. The small granule-containing cells in the present study correspond to the catecholamine-containing (SIF) cells described by earlier workers. These cells presumably regulate muscular and ganglionic activities by virtue of their close association.     

The osmotic behaviour of toad skin epithelium (Bufo viridis)

The effect of saline adaptation on the intracellular Na, K, Cl, P concentrations and dry weight content of the toad skin epithelium (Bufo viridis) was studied using the technique of electron microprobe analysis. The measurements were performed on isolated abdominal skins either directly after dissection or after additional incubation in Ussing-type chambers.Adaptations of the toads to increasing NaCl concentrations for 7 days resulted in increased blood plasma osmolarity and a parallel increase in the cellular electrolyte, P and dry weight concentrations of the epithelium, the K increase representing the most significant fraction of the intracellular osmolarity increase. No evidence was obtained to show that the nucleus and cytoplasm reacted differently from each other and all living epithelial cell types basically showed the same response.Incubation of the isolated skins under control conditions showed a drastic inhibition of the transepithelial Na transport after adaptation to high salinities. In spite of the large variations in the transport rate almost identical intracellular electrolyte concentrations were observed. In tap water adapted toads the average cellular concentrations were 8.8 mmole/kg wet weight for Na, 109.6 for K, 41.5 for Cl, and 135.3 for P, respectively. Incubation of the skin with Ringer's solution of different osmolarities demonstrated that the epithelial cells are in osmotic equilibrium with the inner bathing solution. The results are consistent with the view that the osmotic adaptation is mainly accomplished by the movement of water.This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk     

An immunohistochemical study on the mouse adenohypophysis with reference to the spatial relationship between GH cells and other types of hormone-producing cells

Summary The objective of the present immunohistochemical study was to determine whether the close spatial relationship between hormone-producing cells as described in rats also exists in the mouse adenohypophysis. In both immature and mature mice, GH cells were the only cell type that had a round shape throughout the pars distalis. All other types of secretory cells had angular or irregular shapes and were closely apposed to round GH cells. Thus, between GH and ACTH cells the same intimate relationship pertains as in rats. Unlike in rats, however, the juxtaposition of LH and Prl cells was observed only occasionally in mature female mice. The salient features of the mouse adenohypophysis were that most LH cells closely surrounded GH cells. These findings show that the cytoarchitectural interrelationship between adenohypophysial cells of mice differs from that of rats.     

Resistive properties of the epithelial membranes of the urinary bladder of the toad,Bufo marinus,determined using the fluorescent dye,RH160

A technique is described for quantitative epifluorescence studies of the apical membrane of the epithelial cells of the urinary bladder of the toad, Bufo marinus, using the lipid-soluble dye, RH160. When the urinary bladder is appropriately mounted, fluorescence signals, in response to a transepithelial voltage pulse, can be recorded from the epithelium immediately after the addition of the dye to the mucosal bath, and for some hours subsequently. The optical signal, recorded as the change in fluorescence in response to a transepithelial voltage pulse, as a fraction of resting fluorescence, was found to be a linear function of the applied voltage over the range ±200 mV, and was approximately 3% for a 100 mV change in transepithelial potential. The signal was enhanced by amiloride (10 μmol · 1⁻¹), reduced by bretylium (5 mmol · 1⁻¹) and abolished in the presence of nystatin (730 U · ml⁻¹). Calculations based on these data permitted estimation of the fractional resistance of the apical membrane, which was found to be 0.85 under control conditions. Apical membrane resistance was 8.6 kΩ · μF, and the basolateral membrane resistance was 1.5 kΩ · μF. These findings support the conclusion that the apical membrane of toad urinary bladder epithelial cells is of high resistance, thus resembling other sodium-transporting epithelia.     

Microelectrode studies of toad urinary bladder epithelial cells using a novel mounting method

To optimise the conditions for recording stable membrane potentials, epithelial cells of short-circuited toad bladders were impaled via either their apical or basolateral membranes. Microeletrode impalements via the apical membrane were affected by impalement damage and were typically biphasic, consisting of an initial sharp increase in apical membrane potential (V _sc of around –26mV), followed by a rapid depolarization of V _sc towards 0 mV in the next 10–20 s. To facilitate basolateral impalement two different methods for mounting bladders were tested. Both mounting methods yielded similar values for V _sc and R _a/R _b (the ratio of apical to basolateral membrane resistance) of around –57 mV and 5, respectively, which were larger than those recorded via the apical membrane and consistent with potential measurements from other tight epithelial tissues. Of the two basolateral mounting methods tested, the agar method gave the most stable impalements, making it possible to use amiloride and Ba²⁺ to assess for impalement damage. In conclusion, basolateral impalements of agar-mounted toad bladders makes this traditionally difficult tissue amenable to microelectrode studies.     

The innervation of the pars intermedia of the pituitary gland in the toad

Summary The pars intermedia of the pituitary in Bufo arenarum Hensel is innervated by two types of fibers, both coming from the adjacent neurohypophysis: 1. The neurosecretory nerve endings, originating in the neurohypophysis and found along the limit of the pars intermedia with the neurohypophysis. 2. The common nerve endings, which penetrate the pars intermedia as a single bundle from the neurohypophysis and distribute throughout the gland.The present observations report morphological evidence that in the pars intermedia of the pituitary gland in the toad a double nervous control might be involved.     

Effects of ADH on the apical and basolateral membranes of toad urinary bladder epithelial cells

Short-circuited urinary bladders from Bufo marinus were supported on their apical surface by an agar mounting method and impaled with microelectrodes via their basolateral membrane. This arrangement provided stable and long-lasting impalements of epithelial cells and yielded reliable membrane potentials and voltage divider ratios (R _a/R _b), where R _a and R _b are apical and basolateral membrane resistances respectively. The membrane potential under short-circuit conditions (V _sc) was –51.4±2.2 mV (n=59), while under open-circuit conditions apical membrane potential (V _a) and basolateral membrane potential (V _b) were –31.0±2.4 and 59.5±2.4 mV, respectively. This yields a well-shaped potential profile across the toad urinary bladder, where V _a is inversely related to the rate of transport, I _sc. Antidiuretic hormone (ADH) produced a hyperpolarisation of V _sc and V _b but had no significant effect on V _a. In addition, R _a/R _b was significantly increased by ADH (4.6±0.5 to 10.2±3.6). Calculation of individual membrane resistances following the addition of amiloride showed that ADH produced a parallel decrease in R _a and R _b membrane resistance, with the observed increase in R _a/R _b being due to a greater percentage decrease in R _b than in R _a. The ability of ADH to effect parallel changes in apical and basolateral membrane conductance helps to maintain a constant cellular volume despite an increase in transepithelial transport.     

Immunohistochemical and electron microscopical studies of mitotic adenohypophysial cells in different ages of rats

Summary Mitotic rates of the six types of immunohistochemically identifiable adenohypophysial cells were histometrically calculated in colchicine-pretrated male rats 5, 17, 30 and 70 days old. Sections were stained with the antisera against rLH, rFSH, rTSH, oGH, rPRL and pACTH^1–39. The mitotic growth rate of the anterior pituitary gland at 30 days of age was much higher than at other times. Mitotic growth rates of GH and PRL cells increased with advancing age, while those of ACTH-, TSH- and immunonegative cells decreased with advancing age. LH/FSH cells showed no variation in mitotic growth rate with age. Mitotic cells can be classified into six cell types based on their fine structural properties: (1) agranular cells associated with the folliculo-stellate cells; (2) ambiguous cells with scanty minute secretory granules (50–150 nm in diameter); (3) basophils with a number of small secretory granules (130–200 nm); (4) immature acidophils whose large secretory granules (130–300 nm) are sporadically scattered; (5) acidophils with numerous spherical larger secretory granules (200–300 nm); and (6) prolactin cells with large polymorphic granules. At day 5 there was a high mitotic rate of the agranular and ambiguous cells [types (1) and (2)]; at day 70 a high mitotic rate was found in immature and mature acidophils [types (4) and (5)]. The mitotic rate of basophils (type 3) was high only at day 17 and low at all other times. The mitotic rate of prolactin cells (type 6) showed a slight increment with advancing age. It is concluded that the mitotic rates of the six cell types are age-dependent.Supported by a grant from Ministry of Education, Science and Culture of Japan (No. 337002)     

Gossypol interferes selectively with water and urea permeability of toad bladder

Summary Gossypol, a polyphenol compound with antifertility properties in human male, has been found to interfere with the response of toad bladder to vasopressin in terms of permeability to water and to urea; on the other hand, ion movement is spared for several hours as judged from short-circuit current and conductance data. Action of the drug lies distal to the generation of cyclic AMP since bladder hydrosmotic response to the latter was affected to the same extent as when the hormone was applied.     

Structural and functional response of the isolated toad skin to mucosal lithium

Structural and functional changes induced by long-term Li-exposure of the outer surface of toad skin was studied. Electron microscopy revealed that total Na by Li replacement in the outer compartment of short-circuited toad skin promotes a conspicuous cellular damage expressed as focal swollen cells with altered intercellular spaces and nuclear morphology. Short-circuit current (SCC) decreases by about 70% over the first 60 min after 115 mmol/l Li-exposure. An amiloride sensitive transepithelial Li transport remains intact over a further 150 min despite the epithelial damage, indicating that the pathways across the apical barrier are functioning. Increase of the paracellular permeability is detected by elevation of Na-efflux. Partial 50% or 10% Na by Li replacements induce minor structural alterations and are not sufficient to trigger appreciable Na-efflux and SCC alterations. Therefore, low Li concentrations are less effective than 115 mmol/l in promoting morphofunctional responses. Although ouabain and Li reduce the SCC, ouabain does not promote structural lesions, showing that Li-inhibition of Na transport by itself is not responsible for the observed morphological alterations. In the light of this study, Li utilization as a tool to investigate transepithelial Na transport requires careful judgement.     

Salt sensitivity and hydration behavior of the toad, Bufo marinus

Toads, Bufo marinus, were placed on laboratory tissue saturated with water or with hyperosmotic (250 or 500 mM NaCl or KCl) solutions, and their behavior was observed for 5 min. Toads placed on water initially allowed their ventral skin to touch the surface without abducting the hind limbs. During this "seat patch down" (SPD) behavior toads appeared to be evaluating the suitability of a hydration source prior to initiating "water absorption response" (WR) behavior with the hind limbs fully abducted and the ventral skin pressed to the moist surface. Toads dehydrated by more than 10% showed significantly shorter periods of SPD behavior and initiated WR behavior more frequently than did hydrated toads. Dehydrated toads placed on 250 mM NaCl initiated WR behavior in only 18% of the trials, but spent significantly more time showing SPD behavior than they did on water, indicating that this concentration is marginally acceptable to them. Recordings from spinal nerve #6 showed an increase in activity when 250 mM NaCl or KCl solutions were perfused over the outer surface of the ventral skin. The response to KCl was significantly greater than NaCl. The addition of 10 microM amiloride to 250 mM NaCl resulted in a higher frequency of WR behavior and reversibly inhibited the neural response to 250 mM NaCl. These results suggest that epithelial Na+ channels in the skin serve a sensory function in this species. Neither the hydrated nor dehydrated toads initiated WR behavior on 250 or 500 mM KCl solutions, indicating that toads have a lower tolerance of K+ than of Na+ salts.     

离体蛙心灌流实验方法的比较研究

目的：比较和评价两种离体蟾蜍心脏灌流实验方法的应用及效果。 方法： 分别采用单管法和双管法制备离体蟾蜍心脏灌流标本，观察和分析两种方法在心脏插管所需时间、从处死动物到心脏离体所用时间、离体后心脏存活时间、操作中动脉瓣受损率及心律失常发生率等方面的差异。 结果： 单管法与双管法相比较，二者在心脏离体所需时间上无明显差别。使用双管法对心脏和动脉瓣受损率及心律失常发生率都明显低于单管法，心脏离体后的存活时间也较单管法长。单管法优点是心脏插管所用时间较双管法短。另外,双管法还可用于测定回心血量及心脏前、后负荷改变对心输出量的影响的研究，而单管法则不能实现这些功能。 结论： 在离体心脏研究实验中采用双管法明显优于单管法。