Microbodies (peroxisomes) in the toad,Bufo marinus

Summary Microbodies (peroxisomes), a group of cytoplasmic organelles enriched in catalase, are demonstrated in the toad, Bufo marinus, by light and electron microscopy by means of a cytochemical staining procedure that demonstrates the peroxidatic activity of catalase with diaminobenzidine (DAB). Amphibian microbodies are similar to those of other classes in their fine structure and localization in hepatocytes and kidney, where they are prominent in the proximal tubular cells. Nucleoids are present only in renal microbodies. In the proximal renal tubule an unusual group of large brown granules are identified as lysosomes by their acid phosphatase, -glucosaminidase and -glucuronidase activities.This work was supported by U.S. Public Health Service Grants Nos. NS-06856 and HD 00674. We wish to thank Dr. Richard M. Hays who generously supplied us with toads; Dr. Alex B. Novikoff for making available facilities for ultramicrotomy, Miss Betty De Prest for technical assistance; Miss Marianne Van Hooren for preparation of the photomicrographs.     

Renal vascular anatomy of the toad (Bufo marinus)

The renal vasculature of the toad, Bufo marinus, was studied mainly by means of scanning electron microscopy of vascular corrosion casts. All arterial branches terminated in a glomerulus. Each glomerulus was supplied by only one afferent arteriole. No shunts between afferent and efferent arterioles were observed. The glomerular channels appeared to be permanent capillaries. No evidence supporting the theory of freely shifting glomerular blood channels was found. Efferent arterioles radiated out towards the dorsal surface of the kidney where they connected with peritubular vessels. The renal portal veins produced an anastomosing plexus on the dorsal surface of the kidney, giving rise to the peritubular vessels. Peritubular vessels ran radially toward the ventral surface of the kidney, where they formed the roots of the renal veins. Attention is drawn to the possibility of hairpin countercurrent exchange between the capillary-like efferent arterioles and the peritubular vessels in the dorsal kidney.     

Transferrin endocytosis and iron uptake by developing erythroid cells in the chicken (Gallus domesticus)

Summary The mechanism of iron uptake by avian erythroid cells was investigated using cells from 7 and 15-day chicken embryos, and chicken serum transferrin and conalbumin (ovotransferrin) labelled with¹²⁵I and⁵⁹Fe. Endocytosis of the protein was determined by incubation of the cells with Pronase at 4°C to distinguish internalized from surface-bound protein.Iron was taken up by the cells by receptor-mediated endocytosis of transferrin or conalbumin. The receptors had the same affinity for serum transferrin and conalbumin. Endocytosis of diferric transferrin and conalbumin and exocytosis of apo-protein occurred at the same rates, indicating that iron donation to the cells occurred during the process of intracellular cycling of the protein. The recycling time was approximately 4 min. The rate of endocytosis of diferric protein varied with incubation temperature and at each temperature the rate of endocytosis was sufficient to account for the iron accumulated by the cells. These results and experiments with a variety of inhibitors confirmed the role of endocytosis in iron uptake.The mean cell volumes, receptor numbers and iron uptake rates of 7-day embryo cells were approximately twice those of 15-day embryo cells but the protein recycling times were approximately the same. Hence, the level of transferrin receptors is probably the main determinant of the rate of iron uptake during development of chicken erythroid cells.Transferrins from a variety of mammalian species were unable to donate iron to the chicken cells, but toad (Bufo marinus) transferrin could do so at a slow rate. The mechanism of iron uptake by developing chicken erythroid cells appears to be similar to that described for mammalian cells, although receptor numbers and iron uptake rates are lower than those reported for mammalian cells at a similar stage of development.Abbreviations BSS Hanks balanced salt solution - PBS phosphate buffered saline - MCV mean corpuscular volume - CCCP carbonyl cyanide-M-chlorophenyl hydrazone     

Leptospires in the marine toad (Bufo marinus) on Barbados

Leptospires were isolated from the kidneys of four of 211 toads (Bufo marinus) caught on Barbados. Two of the isolates were identified as Leptospira interrogans serovar bim in the Autumnalis serogroup (the most common cause of leptospiral illness on Barbados), and two as possibly new serovars in the Australis serogroup. Sera from 198 of the toads were examined by the leptospire microscopic agglutination test. Forty-two (21%) were positive at titers of greater than or equal to 1:100, and 54 (27%) at greater than or equal to 1:50. The predominating serogroups were Australis (50%), Autumnalis (23%) and Panama (13%). The agglutination tests on the culture-positive toads showed that serologic studies alone may be of limited value in these animals. Bufo marinus can harbor pathogenic leptospires, and it may be a significant source of the Autumnalis serogroup infections in the Caribbean.     

Calcium in the toad (Bufo marinus) cornea: binding by the stroma

1. The Ca concentration in the toad (Bufo marinus) cornea was 2.6 mmol/kg wet wt compared at 1.0 mmol/l in the bathing aqueous humor and 2.8 mmol/kg wet wt in the separated corneal stromal layer. Cell Ca content was calculated to be about 1.8 mmol/kg wet wt. 2. About 80% of the total Ca appears to be sequestered or bound to tissue components most of which (68% of the total) is associated with the stroma (2.2 mmol/kg wet wt stroma). 3. About 85-90% of the Ca in the stroma is readily exchangeable with external 45Ca. 4. The loss of accumulated 45Ca from the stroma was measured in vitro. This efflux of the isotope was enhanced by multivalent ions and was greatest when Ca2+ or La3+ was present in the external media. Other alkaline earth metal ions were not as effective. The relative effectiveness of this displacement of 45Ca was Ca = La greater than Sr greater than Ba greater than Mg. 5. The results suggest that the Ca2+ is bound by the amphibian stroma at sites that have a preference or specificity for this divalent ion as compared to the other alkaline earth metals. 6. The possible functional role of this bound Ca is discussed.     

Innervation of the renal vasculature of the toad (Bufo marinus)

The innervation of the dorsal aorta and renal vasculature in the toad (Bufo marinus) has been studied with both fluorescence and ultrastructural histochemistry. The innervation consists primarily of a dense plexus of adrenergic nerves associated with all levels of the preglomerular vasculature. Non-adrenergic nerves are occasionally found in the renal artery, and even more rarely near the afferent arterioles. Many of the adrenergic nerve profiles in the dorsal aorta and renal vasculature are distinguished by high proportions of chromaffin-negative, large, filled vesicles. Close neuromuscular contacts are common in both the renal arteries and afferent arterioles. Possibly every smooth muscle cell in the afferent arterioles is multiply innervated. The glomerular capillaries and peritubular vessels are not innervated, and only 3-5% of efferent arterioles are accompanied by single adrenergic nerve fibres. Thus, nervous control of glomerular blood flow must be exerted primarily by adrenergic nerves acting on the preglomerular vasculature. The adrenergic innervation of the renal portal veins and efferent renal veins may play a role in regulating peritubular blood flow. In addition, glomerular and postglomerular control of renal blood flow could be achieved by circulating agents acting via contractile elements in the glomerular mesangial cells, and in the endothelial cells and pericytes of the efferent arterioles. Some adrenergic nerve profiles near afferent arterioles are as close as 70 nm to distal tubule cells, indicating that tubular function may be directly controlled by adrenergic nerves.     

Transferrin and ferritin endocytosis and recycling in guinea-pig reticulocytes

Transferrin and ferritin endocytosis and exocytosis by guinea-pig reticulocytes were studied using incubation with pronase at 4 degrees C to distinguish internalized and membrane-bound protein. Internalization of both transferrin and ferritin occurred in a time- and temperature-dependent fashion. Transferrin endocytosis was more rapid than that of ferritin. Transferrin binding to receptors was not altered, but transferrin endocytosis was decreased in the presence of ferritin. Iron accumulation from transferrin was inhibited by ferritin to a greater extent than could be accounted for by the decreased rate of endocytosis. In pulse-chase experiments, almost all of the transferrin was released intact from reticulocytes, but only about 50% of the total internalized ferritin was released, of which 85% was intact. The endocytosis of transferrin by rabbit reticulocytes was 2- to 2.5-times faster than guinea-pig reticulocytes. These data suggest that ferritin and transferrin are internalized by receptor-mediated endocytosis, possibly involving the same coated pits and vesicles, but that the proteins are recycled only partly in common.     

Structure and function of ciliated peritoneal funnels in the toad kidney (Bufo marinus)

Scanning electron microscopy revealed 600-800 ciliated peritoneal funnels opening onto the ventral surface of each kidney in Bufo marinus. The size and configuration of funnel apertures vary greatly, but individuals course beneath the kidney surface before opening into peritubular blood vessels. Injections of India ink into the peritoneal cavity demonstrate that cilia lining the peritoneal funnels create a current carrying peritoneal fluid into the renal vasculature. Clearance of fluid by the funnels was dependent on pressure in the peritubular vessels, and was increased by arginine vasotocin. Ciliated peritoneal funnels may provide an important route for return of lymphatic fluid from the peritoneal cavity to the vasculature.     

Studies on the mechanism of transferrin iron uptake by rat reticulocytes

Mechanism of transferrin iron uptake by rat reticulocytes was studied using ⁵⁹Fe- and ¹²⁵I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound ⁵⁹Fe was located in the cytoplasmic fraction, only 25–30% of ¹²⁵I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic ¹²⁵I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ¹²⁵I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering ⁵⁹Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.     

Inward rectification in muscle fibres of the toad Bufo marinus

Inward rectification of the resting potassium conductance was studied in skeletal muscle fibres of the toad Bufo marinus. This conductance was shown to be blocked by Ba and Cs and located both in the surface membrane and the membranes of the tubular system. Some differences were found between the properties of this conductance channel in Bufo marinus and those reported for various species of Rana. The possible adaptative value of these differences is pointed out.     

Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons

Summary The innervation of the major arteries and heart of the toad (Bufo marinus) was examined by use of glyoxylic acid-induced catecholamine fluorescence and peptide immunohistochemistry. All arteries possessed a moderate to dense plexus of adrenergic axons, which also showed neuropeptide Y-like immunoreactivity (NPY-LI). Some adrenergic axons in the intracardiac vagal trunks showed NPY-LI, but the varicose adrenergic axons innervating the cardiac muscle of the atria and ventricle, and the coronary blood vessels did not display NPY-LI. About half of the nerve cell bodies in the anterior sympathetic chain ganglia with dopamine--hydroxylase-LI (DBH-LI) also contained NPY-LI. The nerve cell bodies with DBH-LI alone were generally larger (median diameter 30 m) than those with both DBH-LI and NPY-LI (median diameter 20 m). Some cell bodies showing DBH-LI alone were surrounded by boutons with NPY-LI but not DBH-LI. Axons that displayed simultaneously both substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI) also formed a plexus around all arteries studied, being particularly dense around the mesenteric and pulmonary arteries. These axons are most likely sensory since SP-LI was reduced by capsaicin treatment, and nerve cell bodies with both SP-LI and CGRP-LI were found in dorsal root ganglia and the vagal ganglion. A dense plexus of axons showing somatostatin-LI was located around the pulmonary artery and its main intrapulmonary branches. A few nerves with vasoactive intestinal polypeptide-LI were found around the dorsal aorta and pulmonary artery. No perivascular nerves with enkephalin-LI were observed. Reversed-phase, high-pressure liquid chromatography of acid extracts of the large arteries showed that the major peaks of NPY-LI and SP-LI coeluted with porcine NPY (1–36) and synthetic SP (1–11), respectively. Thus, the location and structure of these peptides in perivascular nerves has been highly conserved during vertebrate evolution.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

Weber and noise adaptation in the retina of the toad Bufo marinus

Responses to flashes and steps of light were recorded intracellularly from rods and horizontal cells, and extracellularly from ganglion cells, in toad eyecups which were either dark adapted or exposed to various levels of background light. The average background intensities needed to depress the dark-adapted flash sensitivity by half in the three cell types, determined under identical conditions, were 0.9 Rh*s-1 (rods), 0.8 Rh*s-1 (horizontal cells), and 0.17 Rh*s-1 (ganglion cells), where Rh* denotes one isomerization per rod. Thus, there is a range (approximately 0.7 log units) of weak backgrounds where the sensitivity (response amplitude/Rh*) of rods is not significantly affected, but where that of ganglion cells (1/threshold) is substantially reduced, which implies that the gain of the transmission from rods to the ganglion cell output is decreased. In this range, the ganglion cell threshold rises approximately as the square root of background intensity (i.e. in proportion to the quantal noise from the background), while the maintained rate of discharge stays constant. The threshold response of the cell will then signal light deviations (from a mean level) of constant statistical significance. We propose that this type of ganglion cell desensitization under dim backgrounds is due to a post-receptoral gain control driven by quantal fluctuations, and term it noise adaptation in contrast to the Weber adaptation (desensitization proportional to the mean background intensity) of rods, horizontal cells, and ganglion cells at higher background intensities.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Parallel metabotropic pathways in the heart of the toad, Bufo marinus

This study examined the transduction pathways activated by epinephrine in the pacemaker region of the toad heart. Recordings of membrane potential, force, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were made from arrested toad sinus venosus. Sympathetic nerve stimulation activated non-alpha-, non-beta-adrenoceptors to evoke a membrane depolarization and a transient increase in [Ca(2+)](i). In contrast, the beta-adrenoceptor agonist isoprenaline (10 microM) caused membrane hyperpolarization and decreased [Ca(2+)](i). The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) mimicked the isoprenaline-evoked membrane hyperpolarization. Epinephrine (10-50 microM) caused an initial membrane depolarization and an increase in [Ca(2+)](i) followed by membrane hyperpolarization and decreased [Ca(2+)](i). The membrane depolarizations evoked by sympathetic nerve stimulation or epinephrine were abolished either by the phospholipase C inhibitor U-73122 (20 microM) or by the blocker of D-myo-inositol 1,4,5,-trisphosphate-induced Ca(2+) release, 2-aminoethoxydiphenyl borate (2-APB, 60 microM). Neither U-73122 nor 2-APB had an affect on the membrane hyperpolarization evoked by beta-adrenoceptor activation. These results suggest that in the toad sinus venosus, two distinct transduction pathways can be activated by epinephrine to cause an increase in heart rate.     

Peptide-containing nerves in the urinary bladder of the toad,Bufo marinus

The distributions of peptide-containing nerves in the urinary bladder of the toad, Bufo marinus, were studied by means of fluorescence immunohistochemistry of whole-mount preparations. The bundles of smooth muscle in the bladder are well supplied by varicose nerve fibres displaying somatostatin-like immunoreactivity; these fibres probably arise from intrinsic perikarya. The urinary bladder also has a well-developed plexus of nerves containing substance P-like immunoreactive material; these elements probably represent sensory nerves of extrinsic origin. Nerve fibres showing immunoreactivity to vasoactive intestinal polypeptide (VIP) or enkephalin are rare within the urinary bladder of the toad. It is considered unlikely that any of these peptides directly mediates the hyoscine-resistant excitatory response of the smooth muscle to nerve stimulation in the toad bladder.     

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus)

Summary Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibres containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which form distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P_{CO 2} and so leading to modulation of activity in afferent nerve fibres (type containing agranular vesicles). The presence of NEBs solely with an adrenergic (efferent) innervation poses a problem with this interpretation.     

Osmoregulation of the cane toad, Bufo marinus, in salt water

Adult cane toads, B. marinus, survived in salinities up to 40% sea-water (SW). Pre-exposure to 30, then 40% SW, increased the survival time of toads in 50% SW. Plasma from toads acclimated to salt water is hyperosmotic to the environment--a result of increased plasma sodium, chloride and urea concentrations. When toads were placed in tap-water and 20% SW, all significant changes to plasma sodium, chloride, urea and osmotic pressure occurred within the first 2 days of exposure. When toads were placed in 30 and 40% SW environments, the increases in plasma sodium and chloride concentrations occurred within the first 2 days of exposure while urea and total osmotic pressure continued to rise until some time between 2 and 7 days exposure.     

Amino acid transport by corneal epithelial cells from the toad, Bufo marinus

alpha-Aminoisobutyric acid is actively accumulated by the epithelium of the cornea of the toad, Bufo marinus, resulting in a tissue to medium ratio of 4 to 1 after 40 min of incubation. The accumulation of alpha-aminoisobutyric acid is ouabain-sensitive and dependent upon the presence of extracellular sodium. Transport is inhibited by carbon monoxide, 6-aminonicotinamide, arsenite and n-heptyl-3-hydroxy-quinoline-n-oxide and stimulated by diamide.