Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of reserpine treated guinea pigs

Summary The individual granules of enterochromaffin cells of normal and reserpine treated guinea pigs have been studied by staining slides of the duodenum first by an argentaffin method and subsequently by an argyrophile method. Some argentaffin cells can be shown to contain not only argentaffin granules, but also granules that are purely argyrophile. The relative number of such argentaffin cells is greatly increased following administration of reserpine, as depletion of their 5-hydroxytryptamine content converts argentaffin granules into purely argyrophile ones. On the basis of this finding it is confirmed that the argyrophile granule is merely an argentaffin granule depleted of its 5-HT content, and that the argyrophile (nonargentaffin) and the argentaffin cells represent different phases of a secretory cycle.This investigation was supported by a grant from the Indian Council of Medical Research. I am grateful to Ms. Ciba of India Ltd. for making available reserpine (Serpasil) and solvent for reserpine. It is a pleasure to thank Mr. Anand Parkash for technical assistance and Mr. M. L. Sharma for the photographs.     

The relative proportion of pre-enterochromaffin (argyrophile) and enterochromaffin (argentaffin) cells in the gastro-intestinal tract of human foetuses

Summary The proportion of argyrophile cells that are argentaffin varies considerably over different parts of the gastro-intestinal tract of the human foetus. In the small intestine plots of the number of argentaffin cells against the number of argyrophile cells, at various cranio-caudal levels, reveal no correlation. The percentage of argyrophile cells that are argentaffin decreases in a caudal direction in the proximal part of the small intestine, but markedly increases in the distal part. In the large intestine there is a positive correlation between the numbers of argyrophile and argentaffin cells, but the percentage of argyrophile cells that are argentaffin also varies with cranio-caudal level. The pattern of this variation is not consistent in the various foetuses studied.     

Amine-producing endocrine-like cells in the epithelium of urethra and prostate of the guinea-pig a chemical,fluorescence histochemical,and electron microscopic study

Summary The urethra and prostate of the guinea-pig contain at least two types of endocrine-like cells in the epithelium. The predominant type is argentaffin and stores 5-hydroxytryptamine. Treatment with reserpine or a dopa decarboxylase inhibitor markedly reduces the 5-hydroxytryptamine content of this cell type. The other less numerous cell type, which is argyrophil but not argentaffin, is devoid of 5-hydroxytryptamine but can be induced to store dopamine if supplied with dopa. Both cell types occur disseminated in the urethral epithelium, whilst only the argyrophyl, non-argentaffin cell type devoid of 5-hydroxytryptamine is found in the prostate. At the ultrastructural level the argentaffin cell type contains numerous electron-dense cytoplasmic 800–1000 Å granules. These granules are argentaffin, suggesting that they are the storage site for 5-hydroxytryptamine. The cells sometimes reach the urethral lumen via a narrow neck, the apex being endowed with microvilli. This arrangement suggests that the cells are capable of responding to stimuli in the urethral lumen. Preliminary attempts to test the effect of depriving or loading guinea-pigs with water failed to induced changes in the 5-hydroxytryptamine content of the urethral endocrine-like cells.     

Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of the guinea pig

Summary Successive staining of cells of the enterochromaffin system of the guinea pig by an argentaffin and an argyrophile method shows that, as in man and rabbit, they can be divided into (a) those in which all granules are apparently argentaffin, (b) those in which all granules are argyrophile but none are argentaffin, and (c) those in which some granules are argentaffin (as well as argyrophile) while others are purely argyrophile. The presence of the third type makes it evident that no hard and fast line of distinction can be drawn between the so called argentaffin and argyrophile cells.I am grateful to Mr. Mohan Lal Sharma for help with the photographs and to Mr. Anand Parkash for technical assistance.     

Enteroglucagon and substance P-like immunoreactivity in argentaffin and argyrophil rectal carcinoids.

A specific immunofluorescence for enteroglucagon or substance P or for both hormones was demonstrated in nine out of 12 examined rectal carcinoids. One tumor was argentaffin, contained ultrastructurally pleomorphic granules of the entero-chromaffin cell type, and showed immunofluorescence for substance P. The rest were non-argentaffin but were argyrophil with the Grimelius technique and contained round granules. The argyrophil carcinoids were immunoreactive to one or both hormones in eight cases and not fluorescent in three cases. In two of the non-argentaffin carcinoids a small number of argyrophil cells was found with the method of Sevier-Munger.     

Further observations on the alleged presence of non-argyrophile argentaffin cells in the human gastro-intestinal tract

Summary The relationship between the argentaffin and argyrophile cells of the human gastro-intestinal tract has been studied by staining paraffin sections first by the Masson-Hamperl method and then by the Bodian method. The investigations confirms the author's earlier observation (Singh 1963) that all argentaffin cells of the human gastro-intestinal tract are also argyrophile and that there is no evidence to support the claim of Hellweg (1952) and of Hamperl (1952) regarding the presence of non-argyrophile argentaffin cells.     

The distribution of enterochromaffin cells in the human small intestine

Summary The distribution of argyrophile and argentaffin cells has been studied in the small intestine of five human adults. In proceeding cranio-caudally the characteristic feature of their distribution is the presence of eight to ten waves of rising and falling density. A progressive decrease in density of cells from duodenum to terminal ileum (described by previous workers) is not present.Re-examination of findings reported earlier in the small intestines of human foetuses shows that a predominant U shaped pattern of distribution is present in younger foetuses. This changes to the adult pattern by full term. The appearance of the adult pattern occurs earlier for argyrophile cells than for argentaffin cells.     

On the alleged presence of non-argyrophile argentaffin cells in the human gastro-intestinal tract

Summary The relationship between the argentaffin and argyrophile cells of the human gastrointestinal tract has been studied, in foetal and adult material, by a technique involving the staining of sections first by an argentaffin method (Gomori-hexamine silver, Schmorl, Diazonium) and subsequently by an argyrophile method (Bodian). A comparison of the cells staining by the two methods shows that all argentaffin cells of the human gastrointestinal tract are also argyrophile and that there is no evidence to support the claim of Hellweg (1952) and of HamPerl (1952) regarding the presence of non-argyrophile argentaffin cells.W. H. O. fellow from the Department of Anatomy, Medical College, Rohtak, India. — I am very grateful to Professor J. D. Boyd and to the World Health Organisation for having made it possible for me to carry out this research at the Anatomy School, Cambridge. I am indebted to Dr. G. A. Gresham and his staff for their very willing cooperation in providing material from surgical resections. My thanks are also due to Mr. J. F. Crane for the photographs and to Mr. J. W. Cash and Mr. R. Smith for helpful discussions on staining techniques.     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

Differences in the effects of amphetamine and methylphenidate on brain dopamine turnover and serum prolactin concentration in reserpine-treated rats.

In the present study we compared the ability of amphetamine and methylphenidate to antagonize the elevation of serum prolactin produced by reserpine because of the differences in the actions of amphetamine and methylphenidate on brain dopamine turnover. Groups of male rats were treated with either methylphenidate (10 mg/kg) or amphetamine (5 mg/kg) alone or in combination with reserpine (5 mg/kg). The reserpine treatment was given 4 hours before methylphenidate or amphetamine, and the rats were killed 5 hours after reserpine. Neither amphetamine nor methylphenidate alone was able to suppress serum prolactin. Amphetamine but not methylphenidate was able to block the increase of serum prolactin in response to reserpine. Amphetamine lowered brain DOPAC in control and reserpine-treated rats, but methylphenidate elevated brain DOPAC in control rats and had no effect in reserpine-treated rats. These results indicate that the methylphenidate group of CNS stimulants can be differentiated on the basis of their neuroendocrine effects from the amphetamine group of stimulants.     

The reserpine effects on the gonadotrophic cells of the male common carp Cyprinus carpio (Osteichtyes: Cyprinidae)

The secretion of gonadotropins (GtH) in goldfish and carp, is stimulated by GtH-releasing hormone (GnRH) and is inhibited by dopamine. Studies with antidopaminergics have demonstrated to be effective in order to stimulate the spermiation and the ovulation in different species of teleosts. The reserpine, a drug that deplets the dopamine, has shown to stimulate the spermiation in the common carp. We report here, the effects of reserpine on the number and volume of gonadotrophic cells of the common carp. Eight injections of reserpine alone, at doses of 0.5, 1.0 or 1.5 mg/ml/kg of body weight and at intervals of 48 hours, caused an increase in the number and volume of gonadotrophic cells. The dose 0.5 mg/ml/kg, presented an increase in the number and volume of gonadotrophic cells of 382% and 123%, respectively, above the control group. The dose 1.0 mg/ml/kg, showed an enhanced number and volume of gonadotrophic cells of 704% and 152%, respectively. With the dose 1.5 mg/ml/kg increase in number (171%) and volume (106%) of gonadotrophic cells was lower. The gonads of the experimental groups had an abundance of advanced states of spermatogenesis. Our results show that eight intraperitoneal injections of reserpine were responsible for an increase in gonadodrophic cell, number and volume.     

Exocrine pancreas under experimental conditions

Summary At least four types of endocrine-like cells have been detected histochemically in the mucosa of the human colon and rectum, i.e. argentaffin cells storing 5-hydroxytryptamine (5 HT) and non-argentaffin cells reacting with glucagon, somatostatin and bovine pancreatic peptide (BPP) antibodies. Ultrastructurally, four main types and three rare types of endocrine-like cells have been identified. Among the former cells were: (1) argentaffin EC₁ cells, known to store 5 HT and substance P, (2) poorly argyrophil L cells, corresponding to the glucagon-immunoreactive cells storing enteroglucagon or glucagon-like immunoreactivity (GL1), (3) inconstantly argyrophil F-like cells, possibly corresponding to BPP-immunoreactive cells, and (4) fairly argyrophil H cells of unknown function. Rare D cells, corresponding to somatostatin cells, N cells, corresponding to neurotensin cells, and P cells, of unknown function, have been also found.Supported in part by the Italian National Research Council (Grant N. 76.01558.04)     

Retrograde ejaculation occurs in the dog, but can be prevented by pre-treatment with phenylpropanolamine: a urodynamic study

Retrograde ejaculation is partial or total propulsion of semen from the posterior urethra into the urinary bladder; it is well characterized (and relatively common) in humans, with only a few reports in animals. Our objectives were to determine whether retrograde flow of semen occurred during ejaculation in mature dogs with normal fertility, and to determine the effects of phenylpropanolamine on this phenomenon (dose-titration, switch-back study). Retrograde ejaculation and urethral pressure profile measurements were evaluated (double-blind) in six dogs after 5 days of oral treatment with phenylpropanolamine (0, 2, 4, or 8mg/kg); all dogs received all treatments (at 2-week intervals). The number of sperm in the urine was determined before and after each manual sperm collection. Urethral pressure profiles were obtained three times during each procedure. In the absence of phenylpropanolamine, sperm were present in the bladder after semen collection in all dogs (number varied significantly among individuals). The mean (+/-S.D.) number of sperm in the bladder was 17.0+/-5.0, 18.5+/-1.2, 5.1+/-5.0, and 4.8+/-0.1x10(6) sperm for 0, 2, 4, and 8mg/kg, respectively (no significant difference between dogs given 4 or 8mg/kg, but both were significantly lower than those given 0 or 2mg/kg). This reduction was significantly correlated to the increase in mean urethral pressure at the level of the sphincter (39cm versus 59cm H(2)O in placebo-treated dogs versus those given 8mg/kg). In conclusion, we confirmed that retrograde ejaculation occurred during the ejaculatory process in normal dogs, and we demonstrated that phenylpropanolamine (4 or 8mg/kg once daily for 5 days before collection) increased urethral pressure and reduced the number of sperm voided into the bladder during ejaculation.     

Reserpine pretreatment prevents increases in extracellular striatal dopamine following L-DOPA administration in rats with nigrostriatal denervation

The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p<0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.     

Trace dosages of the neurotoxins MPTP and MPP+ may affect brain dopamine in vivo.

The present study has examined the effects of systemically administered MPTP and MPP+ upon striatal DA and Dopac of C57 mice, also treated concurrently with either saline or reserpine. MPTP followed by saline did not affect DA level but decreased that of Dopac only at 5.0 mg/kg and higher dosages. The potency of MPTP affecting DA increased greatly when the neurotoxicant was followed by either 5.0 or 10.0 mg/kg reserpine; MPTP at 0.10 mg/kg and higher dosages significantly reversed the DA depleting effects of reserpine. MPP+ (1.0 or 10.0 mg/kg) with saline did not affect either DA or Dopac. In contrast, MPP+ at 0.10 mg/kg and higher dosages, when followed by 10.0 mg/kg reserpine, dose-dependently enhanced the DA depleting effects of reserpine. In agreement with the earlier results obtained in vitro, the present study indicates that MPTP administration at trace level dosages may lead to an inhibition of MAO in vivo. The effect of systemically given MPP+ on DA, however, appears to be more complex in nature, conceivably comprised of actions at the striatal neurones including the intraneuronal vesicles and, possibly, at the substantia nigra which may affect striatum in turn. That MPP+ may have reached brain areas in these experiments is also indicated by the observation of a significant striatal level of 3H-MPP+ after its systemic administration. In conclusion, irrespective of MPTP and MPP+ action mechanisms, trace levels of these neurotoxicants appear to affect brain dopamine neurons.     

SGC (small granule chromaffin) cells in the mouse adrenal medulla: light and electron microscopic identification using semi-thin and ultra-thin sections.

Adrenal glands of the mouse, fixed either in glutaraldehyde followed by osmium tetroxide or in a mixture of potassium dichromate and glutaraldehyde, and embedded in Epon 812, were investigated by light and electron microscopy. An argentaffin reaction was applied to semi-thin sections for light microscopy and to ultra-thin sections for electron microscopy. Since the mature secretory granules in the Small Granule Chromaffin (SGC) cell were argentaffin and were mainly located along the cell membrane, this cell was clearly distinguishable under the light microscope both from the A (adrenaline) cell whose secretory granules were non-argentaffin and from the NA (noradrenaline) cell whose cytoplasm was rich and was filled with large, strongly argentaffin granules. Chromaffinity of the SGC cell was demonstrated under the light microscope. The SGC cell was intensively stained with toluidine blue without revealing metachromasia. It was demonstrated at the EM level that not only the secretory granules but also the synaptic-like vesicles in the SGC cell contained argentaffin substances. Possible functional relationship between the secretory granules and the synaptic-like vesicles was discussed.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

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.     

A comparative study on immunosuppressive effects of cyclosporin A and FK 506 on peripheral blood lymphocytes in dogs

Immunosuppressive effects of cyclosporin A (CsA) and FK 506 (FK) on peripheral blood lymphocytes were studied in dogs in respect to mixed lymphocyte reaction, proliferative responses to recombinant interleukin-2 (rIL-2), phytohemagglutinin (PHA) and concanavalin-A (Con-A); phenotypes of OKIa1, CD3, CD8 and surface IgM; cytotoxic activity against xenogeneic tumor cells. CsA (2.0 or 5.0 mg/kg, intravenously) or FK (0.16 mg/kg, intramuscularly) was given to mongrel dogs every morning for serial 21 days. The blood concentrations of CsA, measured as trough levels by fluorescence polarization method, ranged from 37 to 350 ng/ml in dogs administered at 2.0 mg/kg and from 170 to 894 ng/ml in dogs administered at 5.0 mg/kg during treatment, respectively. In dogs treated with FK at a dose of 0.16 mg/kg, the drug concentrations in the plasma during treatment ranged from 0.16 to 1.8 ng/ml. Mixed lymphocyte reaction and proliferative responses to rIL-2, PHA and Con-A, which were declined by CsA, were not affected by FK. In contrast, the proportion of OKIa1+ cells was not affected by CsA, whereas FK decreased the proportion of OKIa1+ cells progressively during the course of treatment. Cytotoxic activity was suppressed by both CsA and FK. These results possibly indicate that CsA and FK exert their immunosuppressive effects via different mechanisms.     

Monthly variations in drug-induced gastric ulcers in rats

Clinical evidence suggests that there may be some relationship between the occurrence of peptic ulcers and the season of the year. As little experimental work has been carried out on this subject, three drugs commonly used to induce experimental ulcers in rats (acetylsalicylic acid, 300 mg/kg; phenylbutazone, 200 mg/kg; reserpine, 10 mg/kg) were tested every month for one year under standardised experimental conditions (T_a, RH, LD 12:12). In rats given phenylbutazone the maximum area of ulceration was found in October and December, and in rats given acetylsalicylic acid in February and March. In rats given reserpine, there were no significant monthly variations. An influence of climatic factors on ulcer induction cannot be completely excluded. Endogenous conditions might also account for the monthly differences in ulceration.