Histochemical studies on catecholaminergic cells in the carp retina

Histochemical studies on catecholaminergic cells were conducted with the carp (Cyprinus carpio) retina. Catecholamine (CA)-containing cell bodies appear sparsely distributed among amacrine cells in the innermost cellular row of the inner nuclear layer (INL) and occasionally in the outer half part of the inner plexiform layer (IPL); only exceptionally are they found among ganglion cells. The fluorescent cells interspersed with the amacrine cells and in the IPL send their fiber processes toward both the outer plexiform layer (OPL) and the IPL; the fine fibers form dense networks in the INL and IPL. Pretreatment of the fish with intramuscular injection of reserpine (20 hr prior to enucleation) completely depleted CA from the retina. The fluorescence of catecholaminergic cells was enhanced, and the number of fluorescent cells visible was increased, by intravitreous injection ofl-DOPA, DA, and NA (3 hr prior to enucleation). A combination of pretreatment with intramuscular reserpine and intravitreous NA was particularly effective. These results indicate that catecholamines may play an important role in the modulation of the membrane potential of horizontal cells.     

Distribution of indolealkylamine nerve terminals in the ventricles of the rat brain

Summary The density, distribution and the pharmacologically produced changes of a formaldehyde-induced yellow supra-ependymal fluorescence in the lateral and third ventricles and in the aqueduct of the rat brain are described. The fluorescence consists of small spots or a thin spotted layer just above the ependymal cells. The highest fluorescence densities occur in the areas near the tela chorioidea of the third ventricle and in the interventricular foramen. A high to moderate density occurs in the lateral ventricles and in the aqueduct. Little or no fluorescence is seen above the hypothalamic areas bordering the third ventricle. The fluorescence rapidly fades upon irradiation with violet-blue light, disappears after treatment of the rats with reserpine or p-chlorophenylalanine, is intensified after nialamide or reserpine + nialamide, and does not change after -methyl-p-tyrosine.Electron microscopically supra-ependymal varicose nerves containing small (500 Å) and large (1000 Å) vesicles in the varicosities are observed in areas with supra-ependymal yellow fluorescence. A fine-structural cytochemical technique reveals the presence of a specific, chromaffine, reserpine-sensitive electron dense core in the small and large vesicles.The conclusion is drawn that a characteristically distributed population of supra-ependymal efferent nerve terminals containing an indolealkylamine, most probably 5-hydroxytryptamine, exists in the cerebral ventricles of the rat brain.The skilful assistance of Mr. R. Wybrecht and Mrs. G. Gschwind is gratefully acknowledged.     

Radioimmunoassay for reserpine

Antisera against reserpine have been developed in rabbits immunized with reserpine conjugated to bovine serum albumin. These antisera were used in a radioimmunoassay procedure for reserpine. No appreciable cross-reactivity was observed either with reserpine metabolites or with other drugs commonly used in conjunction with reserpine for the treatment of hypertension. The assay was able to detect as little as 150 pg of reserpine in serum (15 ng/ml), without the need for an extraction procedure. Serum concentrations were measured in rats following the oral and intravenous administration of 1 mg/kg of reserpine.     

Subcellular distribution and chemical nature of the receptor for 5-hydroxytryptamine in the central nervous system

Abstract— In vitro binding experiments with 5-hydroxy[¹⁴C]tryptamine (3.3 × 10^?6 M) were carried out on subcellular fractions of the cat brain. The highest specific activity was observed in some fractions of nerve-ending membranes isolated from the hypothalamus, basal ganglia, and gray areas of the mesencephalon. The specificity of this high affinity binding was demonstrated by competition with reserpine, butanolamide of lysergic acid, and desmethylimipramine. With butanol-water extraction the [¹⁴C]5-HT was found in the butanol while the gangliosides were separated in the water phase. Several experiments with thin layer and column chromatography suggest that in the organic phase the [¹⁴C]5-HT is not bound to the lipids but to a special proteolipid. This proteolipid is different from that found in myelin and has similar chromatographic properties to that previously observed in the proteolipid which binds d-[¹⁴C]tubocurarine in nerve-ending membranes of the cerebral cortex.     

Effects of reserpine administration on the fine structure of the rat pars intermedia

Summary Hormone release from the pars intermedia is under inhibitory control of the hypothalamus. Control may be mediated via dopaminergic fibers which directly contact secretory cells. Administration of reserpine in vivo to adult male rats for four consecutive days results in major alternations in pars intermedia cytology. Cells show expanded areas and whorls of rough endoplasmic reticulum, as well as extensive Golgi zones with numerous dense granules. Some nerve fibers exhibit alterations in vesicle content, while others retain a more normal appearance. Freeze-fracture of glands from reserpine-treated animals provides evidence for exocytosis of granules, although such phenomena are not observed in thin sections. The ultrastructural findings suggest that reserpine alters the content of local inhibitory neurotransmitters in the pars intermedia, leading to unrestrained hormone release, followed by renewed granule synthesis.My thanks to Ms. Judi DeLongo for skillful technical assistance     

Combined reserpine and pituitary irradiation therapy for Cushing's disease patients following unsuccessful transsphenoidal microsurgery

The effectiveness of treatment with reserpine and pituitary irradiation, and with reserpine alone was evaluated in three female patients with Cushing's disease whose transsphenoidal pituitary microsurgery (TPM) had been unsuccessful. In these patients, endocrinological examination after the surgery demonstrated a recurrence of the disease although the microadenomas had apparently been curetted out from the pituitary in all patients. The first patient therefore received 1.0-2.0 mg/day of reserpine with 60 Gy x-ray irradiation, and there was complete remission within 3 months and the patient remained asymptomatic even when reserpine was reduced to 0.1 mg/day 10 years later. The second case was treated with low dose x-ray (20 Gy) and reserpine (0.5-2.0 mg/day), which were also effective. However, 2 weeks discontinuation of the drug caused urinary 17-hydroxycorticosteroids (17-OHCS) and serum cortisol to increase abnormally again, but these were finally re-normalized by an additional administration of reserpine. The third case was given reserpine alone (1.0-2.0 mg/day). She also had a remission in 3 months and the treatment was continued for one year, requiring no further treatment. These results suggest that additional treatment with reserpine and pituitary irradiation or with reserpine alone after unsuccessful TPM may be an effective alternative for patients with Cushing's disease.     

Characterization of the interaction between reserpine and bovine serum albumin: spectroscopic approaches

The characteristics of the interaction between reserpine and bovine serum albumin (BSA) were studied by fluorescence, UV-vis absorption and Fourier transform infrared (FT-IR) spectroscopy. Spectroscopic analysis revealed that fluorescence quenching of BSA by reserpine was through a static quenching procedure. The binding constant K(A) of reserpine with BSA at 293, 301 and 309 K was 1.63, 1.78 and 2.35 x 10(5) moL(-1) L respectively, which indicated degree of binding force between reserpine and BSA. There was one binding site between reserpine and BSA. The entropy and enthalpy changes were positive, indicating that interaction of reserpine and BSA was driven mainly by hydrophobic forces. The average binding distance between the donor (BSA) and the acceptor (reserpine) was about 3.84 nm based on the Forster non-radiation energy transfer theory. Results of synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of BSA were changed by the binding of reserpine. The results may provide important insights into the physiological activity of reserpine.     

Effect of reserpine on the differentiation of the external granular layer of the cerebellum in the chick embryo

In this work we analyse the effect produced by reserpine on the development of thickness and cell number in the external granular layer in the cerebellum of chick embryo. A striking 48-hour histogenetic delay is observed in the treated embryos relative to controls, as show by greater thickness and cell density of this layer in the former, as well as by retarded appearance of a typical radial morphological organization of the external granular layer.     

Selective regulation of carboxypeptidase peptide hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells

Bovine adrenomedullary chromaffin cells in culture were incubated with reserpine or forskolin, two agents acting through different mechanisms, which increase cellular [Met]enkephalin levels by 2-fold after 72 h. Cells were harvested and chromaffin granules were purified on a linear sucrose gradient. After reserpine treatment, carboxypeptidase-processing enzyme specific activity in chromaffin granule fractions was stimulated 1.9-fold, and Co2+-stimulated carboxypeptidase specific activity was stimulated 3-fold. The increase in enzyme activity was dependent on the time of reserpine treatment. Forskolin, on the other hand, had no significant effect on carboxypeptidase activity. The differential effects of reserpine and forskolin suggest that the carboxypeptidase-processing enzyme may be selectively regulated during periods of elevated enkephalin formation. Kinetic studies revealed that in cells exposed to reserpine, the Km value for [Met]enkephalin-Arg6 for the Co2+-stimulated carboxypeptidase activity was lowered to 0.136 from 0.447 mM, but there was no change in the Km values of the non-Co2+-stimulated carboxypeptidase activity from reserpine and control groups. Cellular levels of immunoreactive carboxypeptidase-processing enzyme, measured by a radioimmunoassay method, were not altered after reserpine treatment. These data suggest that while the total number of carboxypeptidase enzyme molecules remained constant, there may be a conversion of existing enzyme molecules to a more active form which displays a higher affinity for [Met]enkephalin-Arg6 in the presence of Co2+.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC STUDIES OF THE CAROTID BODY FOLLOWING INJECTIONS OF LABELED BIOGENIC AMINE PRECURSORS

Adult Syrian hamsters were given a subcutaneous injection of reserpine 3 days before an intraperitoneal injection of ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan and the carotid bodies were subsequently prepared for electron microscopic radioautography. Other Syrian hamsters were given a subcutaneous injection of reserpine and the carotid bodies were subjected to a sensitive cytochemical test for the detection of unsubstituted amines. These studies were made to determine whether the labeled amine precursors were incorporated into the cells and to see whether the parenchymal cells were affected by reserpine treatment. Material from hamsters treated first with reserpine and subsequently injected with ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan exhibited reduced grains of silver over the cells which were associated mainly with the dense cores of the cytoplasmic granules. These studies offer evidence that the granules of the carotid body incorporate catecholamine and indolamine precursors. Material from hamsters incubated for the presence of unsubstituted amines gave a positive reaction (opaque cytoplasmic granules) for catecholamines but not for indolamines. The latter substances may not be present in quantities sufficient to register a positive reaction in the cytochemical test. The opaque granules, indicative of the presence of catecholamines, decreased in density after reserpine treatment. 5 days after one reserpine injection the granules had regained opacity and were comparable to those seen in the control cells.     

Thin layer compared to direct smear in thyroid fine needle aspiration

The efficacy of preparing thyroid fine needle aspirations (FNAs) by the thin layer as opposed to the direct smear method has not been evaluated sufficiently in a regional laboratory setting. At the Foothills Hospital (Calgary, Canada), the method of processing thyroid FNAs was changed from direct smear to thin layer in January 1996. The results of 327 patients who had direct smear from 1994 to 1995 were compared to 401 who had thin layer between 1996 and 1997. While there were no significant differences across a broad range of quality indicators, thin layer showed a trend towards a higher proportion of true benign diagnoses (31% vs 24%), a lower proportion of inadequate specimens (41% vs 50%) and, most importantly, a lower false negative rate (3% vs 9%). In conclusion, the changeover to thin layer did not compromise the interpretation of thyroid FNAs.     

Monoaminergic nerves in the skin of plaice Pleuronectes platessa (L.)

1. The fluorescent histochemical technique of Falck and Hillarp was applied to plaice skin. The presence of monoaminergic nerve terminals, containing predominantly stores of adrenaline, forming a plexus in and around the melanophore layer was demonstrated. 2. Such stores were enriched by noradrenaline in the presence of monoamine oxidase inhibitor, unaffected by spinal section, depleted by spinal nerve section or ligatures and abolished by reserpine. 3. The observations support the view that teleost sympathetic melanophore aggregating nerves are truly adrenergic.     

An assessment of the models of thin filament of the sarcomere using small-angle X-ray data on rabbit relaxed muscle

Models of a thin filament based on various G-actin atomic structures and modes of their packing into helical structures were considered. The calculated intensities of actin layer lines were compared with the X-ray data for thin fiber bundles of a relaxed rabbit skeletal muscle. The effect of the main components of thin filament on the intensity of actin layer lines was also analyzed. The F-actin PDB 3BYH model gave the best fit to the experimental data.     

Reserpine-induced central effects: pharmacological evidence for the lack of central effects of reserpine methiodide

Reserpine, an alkaloid from Rauwolfia serpentina, was widely used for its antihypertensive action. However, its use has been reduced because of its sedative and extra pyramidal symptoms. In the present investigation, reserpine methiodide (RMI), a quaternary analogue of reserpine, was synthesized and pharmacologically evaluated in rats and mice for its central (barbiturate hypnosis, spontaneous motor activity, body temperature, and avoidance of conditioned response) and peripheral actions (blood pressure) in comparison with reserpine. The results indicate that reserpine produced a dose-dependent depression of the central nervous system. RMI at doses equal to and double the equimolar doses of reserpine did not produce any behavioural changes compared with control animals. Nevertheless, both reserpine and RMI were found to produce dose-dependent reduction in the blood pressure of anaesthetized rats, although only at higher doses of RMI, indicating that quaternization of reserpine not only attenuated the entry of RMI into the central nervous system, but also reduced its access to the target tissue in the periphery. It is speculated that the hypotensive actions of RMI may also be due to peripheral depletion of catecholamines.     

Monoamine oxidase inhibitors and the depletion of 5-hydroxytryptamine from enterochromaffin cells by reserpine

Summary The influence of Nialamide (a monoamine oxidase inhibitor) on the depletion of 5-hydroxytryptamine from enterochromaffin cells, has been studied in rabbits. Experimental animals were given varying doses of Nialamide in N/10 HCl by single or repeated injections, followed by injections of reserpine. Control animals were given N/10 HCl followed by reserpine or by reserpine solvent. Nialamide did not prevent the depletion of enterochromaffin cell granules by reserpine in any of the experimental animals.     

Changes in in vivo binding of 3H-Ro 15-1788 in mouse brain by reserpine

The effects of reserpine on the in vivo binding of ³H-Ro 15-1788, (Ro 15-1788: ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) a selective benzodiazepine antagonist, in the mouse brain were investigated. The biodistributions of tracer amounts of ³H-Ro 15-1788 in mice were significantly altered by pretreatment with reserpine (2.5 or 5.0 mg/kg, 24 h before the tracer administration). The time courses of radioactivity in the brain and the blood following i.v. injection of ³H-Ro 15-1788 with carrier Ro 15-1788 were not changed by pretreatment with reserpine, which suggested that the specific binding process might be altered by reserpine. The degree of alteration in the in vivo binding of ³H-Ro 15-1788 seemed to be dependent upon the dose of reserpine and the duration after the treatment of reserpine. The maximum changes in the biodistribution of ³H-Ro 15-1788 were observed at 1 day after injection of reserpine. The body temperature and the brain monoamine contents (dopamine, norepinephrine and 5-hydroxytryptamine) in mice were measured as indicators of pharmacological effects of reserpine, and good relationships to the degree of changes in the biodistribution of ³H-Ro 15-1788 and either the body temperature or brain monoamine contents, were observed. Furthermore, the changes in the biodistribution of ³H-Ro 15-1788 in the reserpinized mice were significantly suppressed by antidepressant imipramine treatment. These results suggest that it would be possible to detect the in vivo drug interaction with brain benzodiazepine receptors in the living human brain using ¹¹C-Ro 15-1788 and positron emission tomography (PET).     

Absorption of infrared radiation by a thin water layer]

The unhomogeneous distribution of infrared absorption in a thin water layer and its time instability were explained. The parameters of interribbon interaction in the case of thin water layers with bound influence were evaluated.     

The legume Rhizosphere

Summary Examination of the root surfaces of Medicago tribuloides Desr. with phase contrast microscopy or electron microscopy using thin sections revealed the presence of a layer of material outside the root surface. In thin sections of KMnO₄ fixed roots this layer was composed of a thin electron dense layer, an electron dense granular matrix of varying width and an enclosing electron dense membrane. After inoculation with an effective Rhizobium strain, rhizobia were found aggregated in a definite zone adjacent to the root surface when either living roots were examined by phase microscopy or thin sections by electron microscopy. This layer was also found in inoculated and uninoculated roots of Trifolium fragiferum and T. pratense. The bacteria were packed with inclusion granules and lay enclosed by a membrane layer adjacent to the granular matrix seen in uninoculated roots. The ultrastructural organisation of root hairs is essentially similar to that of other differentiated root cells. The replicated surface of the uninoculated root hair wall is largely amorphous with a few sculptured portions resembling a cuticle layer. The inoculated root hair wall often shows areas of exposed, open microfibrillar meshwork with rhizobia sitting on the microfibrils. The rhizobia resemble a flagellated, coccoid swarmer form of Rhizobium which is found in the barrel medic rhizosphere.     

An increase in putative voltage dependent calcium channel number following reserpine treatment

Rats were treated with reserpine (0.2 mg/kg) on days 1, 3, and 5. On day 6, binding parameters for alpha-1 adrenergic receptors (3H-prazosin) and putative voltage dependent calcium channels, VDCC (3H-nitrendipine), were determined. There was an increase in both the number (2.1 fold) and affinity (1.8 fold) of alpha-1 adrenergic receptors following reserpine treatment. In addition, there was a 2.7 fold increase in the number of VDCCs, but no change in VDCC binding affinity, following reserpine treatment. These data are consistant with the development of smooth muscle supersensitivity following reserpine treatment in a variety of tissues, and suggest that VDCC number may be modulated by the cell in response to tonic levels of catecholamines. Changes in the number of VDCCs may be an important regulatory mechanism for cell function in physiologic and pathologic states.     

Ultrastructure of hyphae and ascospores in the genus Eremascus eidam

Hyphae and ascospores of Eremascus fertilis and E. albus were studied in ultrathin sections. The lateral wall of the hyphae had a thick electron-light inner layer and a thin dark outer layer. The septa had a simple central pore with or without a plug, and there were Woronin bodies in the vicinity. The wall of the ascospores of E. fertilis showed a thick light inner layer and a thin dark outer layer. In the wall of the spores of E. albus a dark fibrillar layer was present between the light inner layer and the dark outer layer. The spores of this species germinated with a tube the wall of which was continuous with a newly formed layer inside the spore wall.This investigation was supported by the Netherlands Organization for the Advancement of Pure Research (Z. W. O.)