Electron microscopic and autoradiographic studies on vitellogenesis in Necturus maculosus

Electron microscope studies on Necturus maculosus oocytes ranging in size from 1.1–1.5 mm in diameter indicate the primary proteinaceous yolk to arise within structures referred to in other amphibian oocytes as yolk precursor sacs or bodies. The origin of these yolk precursor sacs appears to result from the activity of the Golgi complexes which form multivesicular and granular-vesicular bodies, the limiting membrane of which is at times incomplete. During differentiation, the yolk precursor sacs contain small vesicles similar in size to Golgi vesicles, larger vesicles similar to vesicular elements of the agranular endoplasmic reticulum and, on occasion, a portion of a mitochondrion. The interior of these sacs becomes granular, perhaps by a dissolution of the components just described, and soon becomes organized into a crystalline configuration. In oocytes 2.0–2.5 mm in diameter, an extensive micropinocytotic activity begins, continues throughout vitellogenesis, and constitutes the primary mechanism for the formation of secondary yolk protein. Numerous coated and smooth-surfaced vesicles, as well as electron-dense and electronlucent ones, fuse in the cortical ooplasm to form progressively larger yolk platelets.     

Morphological changes in tight junctions of Necturus maculosus proximal tubules undergoing saline diuresis

Tight junctions between epithelial cells are believed to control the paracellular diffusion of substances across epithelia. Epithelia in which tight junctions are poorly developed display a higher paracellular electrical conductance, while those with extensive tight junctions show lower conductance values. We described here a particular epithelium, that of the proximal tubules of the Necturus kidney, in which the development of the tight junctions varies in parallel with a change of paracellular electrical conductance. In control conditions, tight junctions between epithelial cells of the proximal tubules are more developed than in tubules undergoing saline diuresis, a situation which increases the conductance across the paracellular shunt pathway.     

Electron microscopic cytochemical localization of adenylate cyclase in amphibian urinary bladder epithelium: effects of antidiuretic hormone

A cytochemical technique for electron microscopic localization of adenylate cyclase was used to identify this enzyme in quiescent and hormone-stimulated toad urinary bladder epithelium. In the absence of vasopressin (antidiuretic hormone), adenylate cyclase was detected along the outer surface of the basolateral plasma membranes of granular cells, mitochondria-rich cells, and basal cells, the major cell types comprising the hormone-sensitive urinary epithelium. In the presence of antidiuretic hormone, the basolateral precipitates were markedly increased. The latter was true for both tissues incubated in the presence of an osmotic gradient and those stimulated in the absence of such a gradient. A significant mucosal reaction was never seen. Such data indicate that the hormone receptors for vasopressin are located along the basolateral membranes of all epithelial cells comprising the mucosal hormone-sensitive epithelium. All cells of the epithelium also demonstrate a vasopressin-sensitive adenylate cyclase. We discuss possible mechanisms that attempt to integrate the cytochemical data into an overall scheme for the physiological action of this hormone on amphibian urinary bladder.     

Electron microscopic cytochemical localization of Ca-ATPase in toad urinary bladder

The calcium-regulating enzyme calcium adenosine triphosphatase (Ca-ATPase) was localized in the epithelium of amphibian urinary bladder by the one-step electron microscopic cytochemical procedure. The enzyme was identified along the basolateral border of the epithelial cells that comprise the bladder mucosa. The electron-dense precipitate indicating Ca-ATPase activity was seen in association with the outer leaflet of the basolateral plasmalemmae. Intracellularly, Ca-ATPase activity was seen in association with the mitochondrial matrix of the mitochondria-rich cells. Ca-ATPase was not seen along the apical microvillated border. Enzyme activity was also not seen after incubation in substrate-free media, calcium-free media, or incubation in the presence of vanadate. However, Ca-ATPase activity was evident when the calcium in the standard reaction medium was deleted in favor of magnesium. Addition of antidiuretic hormone (ADH; vasopressin) increased both the basolateral Ca-ATPase reaction and the mitochondrial reaction. Such data appear to indicate further that changes in cytosolic calcium ion concentration take place during the response of amphibian urinary bladder to the polypeptide hormone vasopressin.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

Electron microscopic observations on iron-labelled secondary lysosomes in renal tubules in mice

Summary Labelling of renal tubule cytosomes with electron dense iron granules can be attained by daily intramuscular injections to mice of an iron sorbitol citric-acid compound in a total of approximately 50 mg Fe⁺⁺⁺/100 g of body weight. The labelled cytosomes correspond to secondary lysosomes and represent heterolysosomes or ambilysosomes. The evidence suggests that tubule and lysosome function are undisturbed by the labelling procedure. The use of this method for fine structural studies of the interaction between secondary lysosomes and other cytoplasmic organelles and elements is indicated.Microbodies do not incorporate administered Fe⁺⁺⁺. The morphological observations support the opinion that these bodies are formed in specialized portions of the smooth surfaced endoplasmic reticulum of proximal tubule cells.Supported in part by grants from the Swedish Medical Research Council (Projects No. K 67-12x-1006-2, B 67-12x-1006-02K, K 68-12x-1006-03, and B 69-12x-1006-04A). The assistance of Miss Silwa Mengarelli and Miss Britt-Marie Pettersson is gratefully acknowledged.     

Electron microscopic observations on absorption in the epithelium of the guinea pig gall bladder

Summary The fine structure of the epithelium of the gall bladder in the guinea pig has been examined after the resorption of Thorotrast. Vesicles at the apex of the epithelial cells containing Thorotrast give evidence of pinocytosis and particles are also found in the intercellular spaces and in dense cytoplasmic bodies. The epithelium exhibits interdigitating lateral cell membranes which are correlated with its function of resorbing large amounts of water.     

Is there functional cholinergic innervation in the frog duodenum (Rana catesbeiana)?

To determine if functional cholinergic innervation occurs in the frog duodenum or not, the effects of exogenous acetylcholine and electrical transmural stimulation, the contractile activity of an acid extract from the frog duodenum, and the distribution of acetylcholinesterase (AChE) activity in the wall of the frog duodenum were investigated. Acetylcholine caused non-sustained contraction in a dose-dependent manner (100 nM-1 mM). The ED50 value was 17 +/- 2.4 microM. Atropine (500 nM) shifted the dose-response curve for acetylcholine parallel to the right. Transmural stimulation of the frog duodenum caused frequency-dependent (0.5-50 Hz) contraction which was not decreased by atropine (500 nM) at all. The acid extract from the frog duodenum caused contraction of a longitudinal muscle strip of guinea-pig ileum but atropine (500 nM) had no significant effect on the contraction. Only a little AChE activity was found in Auerbach's plexus of the frog duodenum compared with that of the rat ileum. These results suggest that a cholinergic nerve is present in the frog duodenum but its physiological significance is very small.     

Norepinephrine stimulation of sodium transport in Necturus urinary bladder

Norepinephrine alters the transepithelial electrical properties of an open-circuited urinary bladder from the mud puppy, Necturus maculosus. When 10(-5) M norepinephrine is superfused over the serosa of the epithelium, the transepithelial voltage (Vt) and short-circuit current (Isc) increase as the resistance (Rt) decreases. The norepinephrine-mediated changes are reversed by the addition of amiloride (5.10(-5) M) to the mucosal Ringer's solution. The serosal adrenoceptors mediating the Na+ transport are more sensitive to norepinephrine (EC50 = 1.2.10(-6) M) than to epinephrine or isoproterenol. Since the Isc is blocked selectively by the antagonist, phenoxybenzamine, stimulation of active transepithelial Na(+)-flux by catecholamines is mediated by an alpha-adrenoceptor. The apical cell membrane voltage (Va) and fractional resistance (fRa) were recorded using conventional KCl-filled microelectrodes. Untreated tissues have Va close to 0 mV while the basolateral membrane voltage (Vb) is between -85 and -95 mV. About 90% of Rt is apical cell membrane resistance (fRa). When amiloride inhibits sodium transport, Va becomes negative, Vb hyperpolarizes slightly and fRa increases to 97%. On the other hand, if the bladders are treated with norepinephrine, fRa decreases to 79% as Va becomes positive and Vb depolarizes. When Rt changes, the resistance of the paracellular pathway (Rp) is unaltered. Changes in the electrical properties of the tissue appear to be mediated primarily by alterations in Ra. Since the Necturus bladder does not respond to antidiuretic hormone, this study implies that biogenic amines regulate Na+ transport in the epithelium.     

Characterization of the basolateral membrane conductance of Necturus urinary bladder

Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that permitted rapid changes in the ion composition of the serosal solution. The transepithelial electrical properties exhibited a marked seasonal variation that could be attributed to variations in the conductance of the shunt pathway, apical membrane selectivity, and basolateral Na+ transport. In contrast, the passive electrical properties of the basolateral membrane remained constant throughout the year. The apparent transference numbers (Ti) of the basolateral membrane for K+ and Cl- were determined from the effect on the basolateral membrane equivalent electromotive force of a sudden increase in the serosal K+ concentration from 2.5 to 50 mM/liter or a decrease in the Cl- concentration from 101 to 10 mM/liter. TK and TCl were 0.71 +/- 0.05 and 0.04 +/- 0.01, respectively. The basolateral K+ conductance could be blocked by Ba2+ (0.5 mM), Cs+ (10 mM), or Rb+ (10 mM), but was unaffected by 3,4-diaminopyridine (100 microM), decamethonium (100 microM), or tetraethylammonium (10 mM). We conclude that a highly selective K+ conductance dominates the electrical properties of the basolateral membrane and that this conductance is different from those found in nerve and muscle membranes.     

Electron microscopic study on the innervation of the pancreas of the domestic fowl

Summary The innervation of the pancreas of the domestic fowl was studied electron microscopically. The extrapancreatic nerve is composed mostly of unmyelinated nerve fibers with a smaller component of myelinated nerve fibers. The latter are not found in the parenchyma. The pancreas contains ganglion cells in the interlobular connective tissue. The unmyelinated nerve fibers branch off along blood vessels. Their synaptic terminals contact with the exocrine and endocrine tissues. The synaptic terminals can be divided into four types based on a combination of three kinds of synaptic vesicles. Type I synaptic terminals contain only small clear vesicles about 600 Å in diameter. Type II terminals are characterized by small clear and large dense core vesicles 1,000 Å in diameter. Type III terminals contain small clear vesicles and small dense core vesicles 500 Å in diameter. Type IV terminals are characterized by small and large dense core vesicles. The exocrine tissue receives a richer nervous supply than the endocrine tissue. Type II and IV terminals are distributed in the acinus, and they contact A and D cells of the islets. B cells and pancreatic ducts are supplied mainly by Type II terminals, the blood vessels by Type IV terminals.This work was supported by a scientific research grant (No. 144017) and (No. 136031) from the Ministry of Education of Japan to Prof. M. Yasuda     

Electron microscopic study of the innervation of the heart conduction system in rats]

A systemic quantitative electron microscopic analysis on innervation of the sinus node, the atrioventricular node, the bundle of His and its pedicles within the interventricular septum has been performed in intact hearts of mature rats. The data have been obtained on the size of nonmyelinated and myelinated nerve fibres, efferent and afferent terminals within different parts of the cardiac conductive system, their interconnection with specialized cardiomyocytes have been described. Application of certain methods for electron microscopic investigation on the innervation of mammalian cardiac conductive system has been discussed.     

Electron microscopic study on the innervation of Peyer's patches of the Syrian hamster

Summary Electron microscopic investigations have been carried out on the innervation of Peyer's patches. Bundles of axons partly devoid of Schwann satelite cells have been observed. The intercellular space between free axons which contain synaptic vesicles and lymphatic respectively reticulum cells is 200 Å.Apart from the possibility of neuronal transmission at these contact points the hypothesis is established that a temporary contact between lymphocytes and the surface of nerve cells of the own body is necessary for maintainance of immune tolerance of some specific neuronal antigenic structures. Peyer's patches may grant a suitable environment for this purpose.For stimulating discussions we are much obliged to Prof. Dr. med. K. Lennert and to Prof. Dr. med. Dr. med. dent W. Müller-Ruchholtz.     

Peroxidase activity in the epithelium of the digestive tract of the bullfrog, Rana catesbeiana

Peroxidase activity was examined cytochemically in the mucosal epithelium along the length of the digestive tract from the esophagus through the large intestine during the development of the bullfrog, Rana catesbeiana. In the tadpole of this species, cells with peroxidase activity were found abundantly in the esophagus, stomach, and large intestine; and the types of such cells differed according to the region: ciliated cells and mucous cells in the esophagus; ciliated cells in the stomach; and brush cells, absorptive cells, and goblet cells in the large intestine, respectively. After metamorphosis, however, peroxidase activity was observed exclusively in absorptive cells and goblet cells in the large intestine. Peroxidase activity was commonly demonstrated in apical vesicles or granules, to some degree in rough endoplasmic reticulum, and in some elements of the Golgi apparatus. Furthermore, reaction product was also found in mucus covering the luminal surface of such epithelial cells. These findings indicate that peroxidase-positive cells, which may have the ability to synthesize peroxidase as a secretory product, were distributed mainly in three regions of the digestive tract in tadpoles (esophagus, stomach, and large intestine), but were centered in one specific region, the large intestine, after metamorphosis. Concomitantly, the variety of types of peroxidase-positive cells decreased during metamorphosis. Our results indicate that some of the peroxidase in the digestive tract may have a secretory origin and may play a role in the defense against microorganisms.