Electrophysiological characteristics of cardiac pacemaker cells of the frog Caudiverbera caudiverbera

1. The cardiac pacemaker cells of the frog Caudiverbera caudiverbera are centrally located in the sinus venosus. These cells are rounded, smaller than contractile fibres and have large nuclei. 2. Intracellular recording confirmed the existence of primary and transitional pacemaker cells. 3. Action potentials from primary cells were resistant to blockade by tetrodotoxin (TTX), but were abolished by verapamil suggesting that their bioelectric activity is dependent on a slow inward current. 4. Transitional cells appeared to have two different inward currents contributing to the upstroke: a fast TTX-sensitive and a slow verapamil-sensitive current.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied.2. CA secretion was induced by ACh or nicotine, but not by muscarine.3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response.4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine.5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M₂, which may play a regulatory function.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad.

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied. 2. CA secretion was induced by ACh or nicotine, but not by muscarine. 3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response. 4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine. 5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M2, which may play a regulatory function.     

Presence of proenkephalin in chromaffin cells of the frog adrenal gland

Using a specific antiserum to bovine proenkephalin 1–77 (synenkephalin), the distribution of this peptide in the frog adrenal gland has been studied by means of the indirect immunofluorescence technique. Proenkephalin immunoreactivity was found in all chromaffin cells, which also demonstrated enkephalin- and vasoactive intestinal peptide-like immunoreactivity. No nerve endings containing proenkephalin-, enkephalin-, or vasoactive intestinal peptide-like material could be detected. These data suggest a precursor-product mode of biosynthesis for enkephalins in amphibian chromaffin cells. On a phylogenic point of view, they further indicate a high stability of the structure of proenkephalin during the evolution process.     

Kinetic analysis of secretion from permeabilized adrenal chromaffin cells reveals distinct components.

We have determined that there are components to the time course of Ca(2+)-dependent secretion from digitonin-permeabilized bovine adrenal chromaffin cells that can be distinguished by Ca2+ sensitivity and ATP dependence. The effects of various Ca2+ concentrations are different on the initial rates and later rates of secretion. The earliest rates (5 s) are half-maximal between 30-100 microM Ca2+ and maximal by 300 microM Ca2+. Later rates of secretion are maximal by 10 microM and decline above 30 microM Ca2+. At low Ca2+ concentrations secretion begins after a lag of several seconds. The early rates of secretion (within 1 min) are dependent on the prior effects of MgATP. MgATP primes the cells to secrete. Later rates require the continuous presence of MgATP for optimal secretion. Incubation with low concentrations of Ca2+ increases the ability of MgATP to stimulate subsequent Ca(2+)-dependent secretion. Preincubation with Ca2+ has no effect on the rapid loss of ATP-independent secretion with time after permeabilization. The data indicate that: 1) as secretion progresses in digitonin-permeabilized cells, different events become rate-limiting; 2) maximal secretion at the early times requires at least 10-fold higher Ca2+ concentrations than at later times; 3) the rate at which Ca2+ initiates secretion is concentration-dependent; and 4) Ca2+ not only triggers the final events in secretion but enhances the ability of ATP to prime secretion.     

Control of exocytosis from adrenal chromaffin cells

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.     

Digestive plasticity in tadpoles of the Chilean giant frog (Caudiverbera caudiverbera): factorial effects of diet and temperature

Anuran metamorphosis is one of the most spectacular processes in nature. Metamorphosis entails morphological transformations and extensive changes in feeding habits, such as transforming from an herbivore to a carnivore. This phenomenon is especially sensitive to environmental cues. We studied the phenotypic plasticity of intestinal morphology and enzyme activity in tadpoles of the Chilean giant frog Caudiverbera caudiverbera. We tested the effects of diet and temperature using a factorial design, which included a control of nontreated individuals. There was no significant effect of diet treatment (i.e., low- vs. high-quality diet) on any of the measured variables, including external morphology. We found significant effects of temperature on morphological traits. Temperature treatment also had significant effects on aminopeptidase-N and maltase activity. Both enzymes exhibited complex interactions with temperature along the intestine. Gut size varied significantly among temperatures, with intestines from warm-treated individuals smaller than the intestines from control and cold-treated tadpoles. Our findings suggest that phenotypic plasticity of intestinal morphology and physiology exists in larvae of this species, at least in response to temperature. However, we did not detect clear effects of diet or temperature on the timing of metamorphosis.     

Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

Inositol trisphosphate (IP3), a product of the phosphoinositide cycle, mobilizes intracellular Ca2+ in many cell types. New evidence suggests that inositol tetrakisphosphate (IP4), an IP3 derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP4 are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP4 in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine (0.4 mM) stimulated an increase in [3H]IP4 and [3H]IP3 accumulation in chromaffin cells and this effect was completely blocked by atropine (0.5 mM). [3H]IP4 accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP3 and IP4 hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP4 and calcium homeostasis.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

On the chromaffin cells in dog adrenal medulla; with special reference to the small granule chromaffin cells (SGC cells)

Small granule chromaffin cells (SGC cells) were identified in the adrenal medulla of adult dogs. They were small in size and usually showed a high nucleo-cytoplasmic ratio. Cytoplasmic projections were occasionally observed in some of these cells. They contained a variable number of small secretory granules with diameters ranging from 70 to 300 nm, but mostly from 100 to 200 nm. The densities of the secretory granules were variable, ranging from highly dense to less dense. These adrenal SGC cells were rich in free ribosomes and polysomes, but were relatively poor in other cell organelles. Chromaffin cells which were intermediate in their characteristics (IM cells) between the SGC cells and the typical A and N cells were also identified. These IM cells contained both highly electron dense and less dense granules in various proportions. The IM cells were classified into two subgroups, according to the proportions of adrenaline type granules and noradrenaline type granules. One group resembled A cells (IM-A cells) and the other resembled N cells (IM-N cells). Light microscopic histochemical studies of A cells stained with the ammoniacal silver solution demonstrated that they contained a small number of darkly stained granules. Electron microscopic cytochemistry revealed that the electron dense granuls in the SGC cells, IM cells and A cells reacted positively with both the potassium dichromate solution at pH 4.1 and the ammoniacal silver solution.     

Alpha-conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.     

Morphology and distribution of chromaffin cells in the adrenal gland of cordylidae (Reptilia,Sauria): A comparative study

The distribution of the adrenaline and noradrenaline chromaffin cells in the adrenal glands of 10 members of the family Cordylidae have been examined. In the genus Gerrhosaurus, all the catecholamine cells lie on the surface of the adrenal gland, forming a continuous envelope of one or two layers of cells that mainly contain noradrenaline (NA). In the genus Platysaurus, the chromaffin envelope is intermittent. There are relatively large tracts of interspersed interrenal tissue containing some adrenaline cells (A). Islets of chromaffin cells are scattered between these interrenal tracts. In the genus Pseudocordylus and the genus Cordylus, the superficial chromaffin cells tend to gather into a multilayered dorsal mass, containing mainly NA cells. Inside the interrenal parenchyma, there are always numerous chromaffin islets, containing mainly A cells.     

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.