Telocytes in ileum of the Chinese giant salamander: ultrastructural evidence

Telocytes (TCs) and their telopodes (Tps) have been found in various organs of many mammals, including in lower animals. However, knowledge of TCs in lower animals is still very limited. This study identified TCs and their Tps in the ileum of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata), by transmission electron microscopy. The TCs/Tps were found near epithelial cells, glandular cells and unmyelinated nerve fibres. Moreover, exosomes were also found to be present in between TCs/Tps and these cells.     

Ultrastructural and immunocytochemical observations of the nervous systems of three macrodasyidan gastrotrichs

The nervous systems of three macrodasyidan gastrotrichs, Dactylopodola baltica, Macrodasys caudatus and Dolichodasys elongatus, were investigated using immunocytochemistry and electron microscopy. Labelling of neural structures against serotonin revealed the presence of two pairs of cerebral cells, a dorsal cerebral connective, and paired ventral nerve cords in D. baltica. In M. caudatus and D. elongatus serotonin immunoreactivity was present in a single pair of dorsal cerebral cells and the ventral nerve cords; the dorsal connective of D. elongatus was also immunoreactive to serotonin and acetylated α‐tubulin. The presence of paired, serotonin‐like immunoreactive cells in D. baltica and other species may represent the plesiomorphic condition in Macrodasyida. The fine structure of the photoreceptors in D. baltica was also investigated to explore the potential ground pattern for eyes in the Macrodasyida. The pigmented photoreceptors of D. baltica contain a unicellular pigment cup, sheath cell and sensory receptor. The pigment cup contains numerous osmiophilic granules that presumably function to shield the eyes from downwelling light in the red part of the spectrum. Projecting into the pigment cup and sheath cell are numerous microvilli from a bipolar sensory cell. A single sensory cell may represent the plesiomorphic condition in Macrodasyida, with multiplication of sensory cells representative of more derived taxa.     

Characterization and distribution of FMRFamide immunoreactivity in the rat central nervous system

FMRFamide immunoreactive material (irFMRFamide) was studied in rat brain and gastrointestinal tract. Highest irFMRFamide concentrations were found in tissues of the gastrointestinal tract and, in the brain, highest concentrations were found in the hippocampus, midbrain, brainstem and hypothalamus. High pressure liquid chromatographic characterization of irFMRFamide demonstrated that the immunoreactive material in brain, pancreas and duodenum was different from molluscan FMRFamide but it was also distinct from any known neuropeptide.     

Ultrastructural immunocytochemical studies on CCK-and neurotensin-like immunoreactivity in the nucleus accumbens of the rat

Using the peroxidase-antiperoxidase technique of Sternberger and collaborators the ultrastructural localization of cholecystokinin- and neurotensin-like immunoreactivity was studied in the nucleus accumbens of the albino rat. The immunoreactive material was localized to nerve terminals making probable axodendritic or axo-axonic contacts with neighbouring nerve cells. Within the nerve terminals most immuno-reactivity was confined to so-called large dense-core vesicles.     

Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum

The interrelationships between cortical efferents and terminals containing enkephalin-like immunoreactivity (ELI) were examined by combining anterograde degeneration with electron microscopic immunocytochemistry in the adult rat neostriatum. Two days following unilateral removal of the cerebral cortex, the brains were fixed by aortic arch perfusion, then sectioned and processed for the immunocytochemical localization of an antiserum directed against methionine (Met5)-enkephalin. The observed relationships between the degenerating cortical efferents and immunocytochemically labeled terminals were of two types. In the first, the degenerating and ELI containing terminals converged on the same unlabeled dendrite or dendritic spine. In the second, terminal and preterminal axons of the ELI containing neurons had one surface directly apposed to the plasma membrane of a degenerating axon terminal. These findings support the concept that neurons containing opioid peptides and cortical efferents modulate the output of common recipient neurons and may also directly interact with each other through presynaptic axonal mechanisms in the rat neostriatum.     

Ultrastructural localization of calcitonin in C-cells of dog thyroid; an immunocytochemical study

Summary Light microscopic observations of normal rat peritoneal mast cells and ultrastructural observations of human mast cells from lesions of nodular mastocytosis indicated that structural damage results in a pronounced increase in percentage of cells with a positive dopa reaction. Recent studies have indicated that the dopa reaction in mast cells is peroxidase-dependent. Enhancement of the dopa reaction by structural damage (latency) is probably related to increased substrate-enzyme interaction. Ultrastructural localization of dopa melanin to mast cell granules and the high percentage of mast cells showing a positive dopa reaction after structural damage is evidence against the possibility that dopa melanin is formed in mast cells by phagocytized enzyme.Supported by USPHS Grant Tl Am 5220, and by The General Research Support Fund, Boston City Hospital.     

Ultrastructural,cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

Summary— The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

Ultrastructural immunocytochemical distribution of tubulin in cultured cells treated with microtubule inhibitors.

Microtubules in tissue cultured cells are stained immunocytochemically with the PAP-method using a purified antitubulin antibody. Treatment of the cells with microtubule inhibitors (colchicine, nocodazole, vinblastine) results in the disappearance of microtubules. The diffuse cytoplasmic staining is strongly increased in the cells by colchicine and nocodazole. Vinblastine produces paracrystalline aggregates that are strongly stained and macrotubules that are unstained. The diffuse staining is much less in vinblastine-treated cells. The bundles of intermediate filaments that are induced by all microtubule inhibitors do not bind the antitubulin antibody.     

Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38°C. NT contracted the longitudinal muscle (ED₅₀, 0.3 nM), the 2–3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 μM), ≥50% of the response was blocked and the residual effect gave an ED₅₀ of 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 μM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED₅₀, 0.3 nM) and one substance P (ED₅₀, 1.4 nM). Using membrane preparations and ¹²⁵I-labeled NT, specific, high affinty receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (K_ds: 0.1 nM and 2 nM). Sodiu ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg⁺⁺ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.     

Distribution of CRF-like immunoreactivity in the rabbit

CRF-like immunoreactivity was measured by radioimmunoassay in the brains and gastroenteropancreatic tract of normal rabbits. It was detected in the brain, with the highest concentration being found in the ventral hypothalamus. The distribution of immunoreactivity was much more limited in the rabbit brain than in the rat brain, with substantial amounts of peptide detected only in areas of close proximity to the hypothalamus, e.g., thalamus, preoptic area, midbrain and amygdala. In addition, the extrahypothalamic immunoreactivity was slightly retarded on Sephadex G-50 chromatography relative to rat CRF-like immunoreactivity and synthetic ovine CRF. No apparent CRF-like immunoreactivity was detected in boiling water extracts of lung, pancreas, duodenum or antrum. These data in conjunction with a previous report of void volume immunoreactivity on Sephadex G-50 only in the hypothalamus suggest that CRF is synthesized only in the hypothalamus and is not a member of the class of peptides found throughout the gastroenteropancreatic tract and the central nervous system.     

Sulfate transport in rabbit ileum: Characterization of the serosal border anion exchange process

Summary The preceding paper [30] shows that transepithelial ileal SO₄ transport involves Na-dependent uptake across the ileal brush border, and Cl-dependent efflux across the serosal border. The present study examines more closely the serosal efflux process. Transepithelial mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) across rabbit ileal mucosa were determined under short-circuit conditions. SO₄ was present at 0.22mm. In standard Cl, HCO₃ Ringer's,J _ms ^SO4 was 81.3±5.3 (1se) andJ _ms ^SO4 was 2.5±0.2 nmol cm^–2 hr^–1 (n=20). Serosal addition of 4-acetamido-4-isothiocyanostilbene-22-disulfonate (SITS), 44-diisothiocyanostilbene-22-disulfonate (DIDS) or 1-anilino-8-naphthalene-sulfonate (ANS) inhibited SO₄ transport, SITS being the most potent. Several other inhibitors of anion exchange in erythrocytes and other cells had no effect on ileal SO₄ fluxes. In contrast to its effect on SO₄ transport, SITS (500 m) did not detectably alter Cl transport.Replacement of all Cl, HCO₃ and PO₄ with gluconate reducedJ _ms ^SO4 by 70% and increasedJ _ms ^SO4 by 400%. A small but significantJ _net ^SO4 remained.J _ms ^SO4 could be increased by addition to the serosal side of Cl, Br, I, NO₃ or SO₄. The stimulatory effect of all these anions was saturable and SITS-inhibitable. The maximalJ _ms ^SO4 in the presence of Cl was considerably higher than in the presence of SO₄ (73.1 and 42.2 nmol. cm^–2 hr^–1, respectively;p<0.001). TheK _1/2 value for Cl was 7.4mm, 10-fold higher than that for SO₄ (0.7mm). Omitting HCO₃ and PO₄ had no measurable effects on SO₄ fluxes.This study shows that (i) SO₄ crosses the serosal border of rabbit ileal mucosa by anion exchange; (ii) the exchange process is inhibited by SITS, DIDS and ANS, but not by several other inhibitors of anion exchange in other systems; (iii) SO₄ may exchange for Cl, Br, I, NO₃ and SO₄ itself, but probably not for HCO₃ or PO₄; (iv) kinetics of the exchange system suggest there is a greater affinity for SO₄ than for Cl, although the maximal rate of exchange is higher in the presence of Cl; and, finally (v) SITS has little or no effect on net Cl transport.     

Effect of morphine and acetylcholine on contractile activity and cyclic AMP in guinea-pig ileum

Neither acute nor prolonged exposure to morphine altered cAMP content or spontaneous movements of longitudinal muscle-myenteric plexus strips of the guinea-pig ileum. By contrast, exogenous acetylcholine or electrical stimulation of the strips elicited both a decrease of cAMP concentration and a twitch response. Atropine blocked the effects of stimulation on these parameters. Addition of morphine to electrically stimulated strips inhibited the twitch response but did not affect cAMP levels. Incubation with morphine led to the development of tolerance to the inhibitory effect on twitch activity and prevented the fall in cAMP normally elicited by electrical stimulation. These results suggest that muscarinic activation is associated with a reduction of cAMP content, an effect which would be impaired in opiate-tolerant tissues.     

Light- and electron-microscopic demonstration of enkephalin-like immunoreactivity in paraganglia of the human urinary bladder

Summary The distribution of enkephalin-like immunoreactivity in paraganglia of the urinary bladder of adult humans was studied by use of immuno-electron microscopy. All paraganglionic cells were positively stained. Enkephalin-like immunoreactivity was located in chromaffin granules. Chromaffin cells in the paraganglia showed only few degenerative features, suggesting undisturbed function of the cells.     

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis

Summary The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCP_B. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCP_A and SCP_B.     

Distribution and ontogeny of VIP-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris

Summary The presence, distribution and development of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris (L.) was investigated by immunohistochemical methods utilizing mammalian VIP antisera. In the gut VIP-like immunoreactivity was observed in both nerves and endocrine cells. Endocrine cells with VIP-like material were only detected in the intestinal epithelium while nerve fibres containing VIP-like material were noted along the whole gastro-entero-pancreatic system, being more numerous in the pyloric sphincter and in the intestinal portion. Immunoreactive nerve cell bodies were encountered in the stomach and intestinal portions localized in the submucosa and in the myenteric plexus. Intestinal immunoreactive endocrine cells were already present in the first developmental stage considered (embryos aged 4 months). They grow in number and before birth reach a frequency higher than in adults. Nerves and cell bodies showing VIP-like immunoreactivity, appear later, before birth, as a few elements in the smooth muscular layer, but only after birth their distribution and frequency are similar to those found in adults. The faint immunofluorescence shown by the immunoreactive endocrine cells and their developmental pattern, which is always different from that observed in nervous elements, suggest the presence of at least two VIP-like substances in the gastro-entero-pancreatic system of S. stellaris.     

An immuno-electron-microscopic study of the localization of VIP-like immunoreactivity in the adrenal gland of the rat

Summary VIP-like immunoreactivity was revealed in a few chromaffin cells, medullary ganglion cells and a plexus of varicose nerve fibers in the superficial cortex and single varicose fibers in the juxtamedullary cortex and the medulla of the rat adrenal gland. VIP-like immunoreactive chromaffin cells were polygonal in shape without any distinct cytoplasmic processes and they appeared solitarily. Their cytoplasm contained abundant granular vesicles having a round core and the immunoreactive material was localized to the granular core. VIP-immunoreactive ganglion cells were multipolar and had large intracytoplasmic vacuoles. The immunoreactive material was localized not only in a few granular vesicles but also diffusely throughout the axoplasm. VIP-immunoreactive varicose nerve fibers in the superficial cortex were characterized by abundant small clear vesicles and some large granular vesicles, while those in the juxtamedullary cortex and medulla and the ganglionic processes were characterized by abundant large clear vesicles, as well as the same vesicular elements as contained in the nerves in the superficial cortex. The immunoreactive material was localized on the granular cores and diffusely in the axoplasm in both nerves. Based on the similarity and difference in the composition of the vesicles contained in individual nerves, it is likely that the VIP-immunoreactive nerve fibers in the medulla and the juxtamedullary cortex are derived from the medullary VIP-ganglion cells, while those in the superficial cortex are of extrinsic origin. The immunoreactive nerve fibers in both the cortex and the medulla were often in direct contact with cortical cells and chromaffin cells, where no membrane specializations were formed. The immunoreactive nerve fibers were sometimes associated with the smooth muscle cells and pericytes of small blood vessels in the superficial cortex. In addition they were often seen in close apposition to the fenestrated endothelial cells in the cortex and the medulla, only a common basal lamina intervening. Several possible mechanisms by which VIP may exert its effect in the adrenal gland are discussed.     

Release of VIP-like immunoreactivity in response to dopamine agonists, insulin hypoglycemia and feeding in conscious dogs

VIP levels were measured by radioimmunoassay in peripheral venous blood of conscious dogs. Bolus injections of the dopamine agonists apomorphine, 0.05 mg/kg, and bromocriptine, 0.2 mg/kg, were found to increase VIP levels from approximately 5 pmol/l to 150 pmol/l. The release responses were abolished by pretreatment with dopamine antagonists (haloperidol 0.1 mg/kg or halopemid 0.1 mg/kg) and by hexamethonium (1 mg/kg) a blocker of ganglionic transmission. Vagotomy did not inhibit the dopamine agonist induced output of VIP. Vagal activation by means of feeding or insulin hypoglycemia caused only minor rises of VIP levels (5-10 pmol/l). It is concluded that dopamine agonists stimulate the release of VIP from populations of neurons other than those affected by vagal and sympathetic activation. Possible sites of action for the VIP releasing effect exerted by dopamine agonists are discussed. Furthermore, it is suggested that some of the peripheral effects exerted by dopaminergic drugs are exerted via a previous release of VIP.