Concomitant depletion of dopamine and secretory granules from cells in the ultimobranchial gland of vitamin D2-treated chicken

Summary The ultimobranchial gland (UBG) is a rich source of the polypeptide hormone calcitonin, which is present in a cell system analogous to the mammalian parafollicular cells (C cells) of the thyroid gland. Both types of cells are argyrophilic and, ultrastructurally, they are furnished with numerous electron-dense granules considered to contain the hormone. In the chicken, the main cells of the UBG contain large amounts of dopamine. The possible functional relationship between this amine and the hormone has been studied by a combination of fluorescence and electron microscopy of the UBG from chickens treated with vitamin D₂. This stimulus produced a depletion of dopamine and a pronounced degranulation of the UBG cells, concomitant with a loss in their argyrophilia. Administration of l-3,4-dihydroxyphenylalanine (l-DOPA) to vitamin D₂-treated animals was followed by a reappearance of dopamine in the cytoplasm of the UBG cells, whereas electron-dense granules or argyrophilia were not restored. It is suggested that this concomitant depletion of dopamine and the secretory granules from the UBG cells reflects a participation of the amine in the secretion of the polypeptide hormone.     

Monoamines in rat thyroid parafollicular cells and the effect of vitamin D2-induced degranulation

Summary Thyroid parafollicular cells of normocalcemic and vitamin D₂-treated rats were investigated by electron microscopy and with the histochemical fluorescence technique of Hillarp and Falck.Administration of high doses of vitamin D₂ caused hypercalcemia and an extensive degranulation of the parafollicular cells.The formation and storage of monoamines in granulated and degranulated parafollicular cells was investigated by fluorescence microscopy after injection of monoamine precursors (DOPA, 5-HTP), alone or in combination with Ro 4-4602, nialamide or reserpine.No fluorescence was observed in parafollicular cells of untreated rats. l-DOPA and l-5-HTP (but not the corresponding D-amino acids) were taken up by a process closely linked to the decarboxylation of the amino acids to the corresponding amines (dopamine and 5-hydroxytryptamine). Treatment with vitamin D₂ did not seem to affect the formation of amines in the parafollicular cells or the formation and storage of amines in other cell systems investigated. The amine itself (dopamine) was not taken up by the parafollicular cells.In normocalcemic rats, the amine formed was retained in the cytoplasm of the parafollicular cells by a partially reserpine-resistant mechanism. The storage of amines is concluded to occur in association with the calcitonin-containing granules.In parafollicular cells of vitamin D₂-treated rats, a certain amount of amine was bound in the cytoplasm in the absence of typical granules. As a considerable amount of calcitonin is known to remain in the thyroid of vitamin D₂-treated rats, the present observations may indicate an association between the amine and the polypeptide hormone calcitonin, whether the latter is confined to typical granules or not.The present study was supported by grants B72-12X-3352-02 and B72-14X-2207-06B from the Swedish Medical Research Council and by grants from Magnus Bergwall's Foundation, Gustav and Majen Lundgren's Foundation, Wilhelm and Martina Lundgren's Foundation and from the Faculty of Medicine, University of Göteborg, Sweden. For skilful technical assistance we are indebted to Mrs. Kirsten Collin and Mr. Pär-Anders Larsson.     

Depletion of secretory granules,calcitonin, and formaldehyde-ozone-induced fluorescence from cat thyroid C cells by vitamin D2 treatment

Summary Using a combination of electron microscopy, fluorometry, and bioassay, the C cells of the cat thyroid were investigated with respect to their content of secretory granules, and calcitonin, and to their formaldehyde-ozone-induced fluorescence. This fluorescence is assumed to reflect the presence of a peptide with NH₂-terminal tryptophan. In cats injected with large doses of vitamin D₂ daily for 5 days, the C cells were degranulated, their fluorescence intensity was lowered and the calcitonin content of the thyroid was markedly reduced. It is suggested that the proposed tryptophyl peptide in the C cells is stored in the secretory granules and that it is engaged in the storage and/or release of the hormone.     

Axoplasmic transport of dopamine in nigro-striatal neurons

The possibility that dopamine is transported in the nigro-striatal system was investigated by the stereotaxic injection of labelled tyrosine or l -DOPA into the substantia nigra of tranylcypromine-pretreated rats. At various intervals thereafter (2-48 h), significant quantities of labelled material were recovered from the ipsilateral substantia nigra, globus pallidus and caudate-putamen, The activity in the substantia nigra consisted of DOPA, dopamine, methoxytyramine, acid metabolites and other unidentified metabolites. In the caudate-putamen, however, nearly all of the activity (85 per cent) was recovered in the dopamine fraction, the remainder being distributed among some of the metabolites. No DOPA was recovered from the caudate-putamen. On the basis of time-course studies after the injection of [¹⁴C]DOPA into the substantia nigra, we calculated the transport rate of dopamine in the nigro-striatal bundle to be 0.8 mm/h. Electrolytic lesions of the nigrostriatal bundle at the level of the lateral hypothalamus, pretreatment with 6-hydroxydopamine, or injections of [¹⁴C]DOPA dorsal to the substantia nigra each produced profound reductions in the amount of activity subsequently recovered from the caudate-putamen. These data suggest that the activity recovered from the caudate-putamen after injections of [¹⁴C]DOPA into the or substantia nigra reflected axonal transport rather than other processes such as diffusion or transport via the circulation. Pretreatment with the DOPA decarboxy-lase inhibitor, R_o 4-4602, significantly reduced the amount of activity recovered in the caudate-putamen, an indication that decarboxylation of DOPA to dopamine was a prerequisite for transport. Pretreatment with reserpine also severely reduced the transport of dopamine in the nigro-striatal bundle, an observation suggesting that dopamine was transported by binding to the amine storage granules. There was no evidence of retrograde transport of dopamine in the nigrostriatal bundle. Injections of larger than tracer quantities of labelled tyrosine into the substantia nigra did not produce the degree of transport of dopamine that was obtained after injections of DOPA, a result suggesting that the amine storage granules may not normally be filled during axonal transport.     

Different effects of hypercalcemic state induced by walker tumor (HWCS 256) and 1,25 (OH)D3 intoxication on rat thyroid C cells

Summary Two different models of chronic C cell stimulation by the hypercalcemic state were compared with respect to their morphology, immunocytochemistry, and biochemistry. In the chronic hypercalcemic state due to the HWCS 256 strain of the Walker tumor C cells show signs of degeneration such as vacuolation, on day 7 after tumor implantation. On day 10 tumor induced hypercalcemia leads to irreversible cell damage with karyopyknosis and karyorhexis. These morphological changes are accompanied by a decline in radioimmunologically measurable calcitonin content of the thyroid and by the loss of response to acute stimulation of C cells. In contrast, in the hypercalcemic state due to 1,25(OH)₂D₃ intoxication we find an almost complete degranulation of C cells but no signs of degeneration or cell damage, although the thyroid calcitonin content and the calcitonin secretion capacity is greatly reduced. Tumor induced cachexia as a reason for C cell damage in tumor bearing rats could be excluded. Other possible reasons, such as acute overstimulation and tumor factors acting directly on C cells are discussed.     

Studies of calcitonin cells and parathyroid glands of the Indian palm squirrel, Funambulus pennanti in response to experimental hypercalcaemia.

Long term hypercalcaemia was induced in F. pennanti by alternate day intramuscular injections of 50,000 IU of vitamin D2 and by giving them 1% CaCl2 solution prepared in tap water to drink. The controls were not injected with vitamin D2 and were given tap water. The serum calcium levels at various stages of the experiment (1-29 days) show increased values as compared with those of control animals. The calcitonin cells in the treated animals generally exhibit an increase in their number up to the 15th day. Mitotic figures are also encountered between the 7th and the 15th day of treatment. This exhibits the increase in the number of C cells. Constant calcium challenge results in increased quantities of secretory granules among these cells up to the 15th day and in degranulation from the 17th day onwards. It also causes degenerative changes in a certain number of C cells. The parathyroids exhibit atrophic changes (25 days onwards) due to chronic hypercalcaemia. For short term hypercalcaemia, animals were injected intravenously with 1 ml of 10% solution of calcium gluconate. The calcitonin cells do not exhibit any change during the first half hour but thereafter they exhibit progressive degranulation, resulting in marked degranulation after 5 hours of the injection. The parathyroids remain unaffected throughout the experiment and show no histological change.     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.     

Studies of calcitonin cells and parathyroid gland of house shrew, Suncus murinus in response to experimental hypercalcemia

1. Hypercalcemia was induced in S. murinus by alternate day intramuscular injections of vitamin D (25 000 IU) and by providing them 1% CaCl2 solution (prepared in tap water) for drinking. 2. After such a treatment the serum calcium values recorded a rise as compared to those of the control specimens. 3. The histological picture of the thyroid of the treated specimens reveals increased number of calcitonin cells. This observation is supported by the occurrence of mitotic figures among them. 4. Perpetual calcium challenge results in degranulation of the secretory material (calcitonin) among these cells (at 26th and 30th day of treatment). 5. It also results in degenerative changes in certain number of C cells. 6. The blood capillaries around these cells get dilated and secretory granules of C cells tend to gather at the periphery of cytoplasm and towards vascular pole. 7. The parathyroid shows atrophic changes.     

In vitro metabolism of vitamin D3 by isolated liver cells

Summary Liver cells were prepared from rats fed a rachitogenic diet to investigate the hepatic metabolism of [ — 1,2 —³H₂] vitamin D₃. Rat hepatocytes suspended in Hanks medium rapidly took up labeled vitamin D₃ from the incubation medium and converted this sterol to various metabolites, including 25-hydroxy vitamin D₃ (25-OH-D₃). There was a steady increment in the cellular production of 25-OH-D₃ and of the more polar metabolites of vitamin D₃ over 3 hr of incubation as determined by thin layer chromatography. Neither the addition of cyclic nucleotides or dexamethasone to, nor the removal of calcium or phosphate from the medium resulted in changes in the rate of conversion of vitamin D₃ to its products. Rats pretreated with sodium diphenylhydantoin converted labeled vitamin D₃ to its metabolites at the same rate as control rats. These data indicate that isolated liver cells retain the capacity for vitamin D₃ hydroxylation, but suggest that the rate of this process does not undergo rapid changes in response to metabolic stimulation.Recipient of Research Career Development Award 1 K04 HL-00089.     

Effects of Dopamine D2 Receptor Partial Agonist Antipsychotic Aripiprazole on Dopamine Synthesis in Human Brain Measured by PET with L-[β-11C]DOPA

Dopamine D₂ receptor partial agonist antipsychotic drugs can modulate dopaminergic neurotransmission as functional agonists or functional antagonists. The effects of antipsychotics on presynaptic dopaminergic functions, such as dopamine synthesis capacity, might also be related to their therapeutic efficacy. Positron emission tomography (PET) was used to examine the effects of the partial agonist antipsychotic drug aripiprazole on presynaptic dopamine synthesis in relation to dopamine D₂ receptor occupancy and the resulting changes in dopamine synthesis capacity in healthy men. On separate days, PET studies with [¹¹C]raclopride and L-[β-¹¹C]DOPA were performed under resting condition and with single doses of aripiprazole given orally. Occupancy of dopamine D₂ receptors corresponded to the doses of aripiprazole, but the changes in dopamine synthesis capacity were not significant, nor was the relation between dopamine D₂ receptor occupancy and these changes. A significant negative correlation was observed between baseline dopamine synthesis capacity and changes in dopamine synthesis capacity by aripiprazole, indicating that this antipsychotic appears to stabilize dopamine synthesis capacity. The therapeutic effects of aripiprazole in schizophrenia might be related to such stabilizing effects on dopaminergic neurotransmission responsivity.     

Topographical and developmental studies on target sites of 1,25 (OH2) vitamin D3 in skin

Summary Tritium-labeled 1,25 (OH₂) vitamin D₃, when injected into vitamin D-deficient adult and pregnant rats is concentrated and retained strongest in nuclei of cells in the outer root sheath of the hair, followed by the stratum granulosum, spinosum, and basale of the epidermis. In the hair follicle, in addition to the most heavily labeled outer root sheath, nuclear labeling exists also in cells of the hair bulb and of the inner root sheath, as well as in basal cells of the sebaceous gland. In contrast, cells of the dermal papilla and the connective tissue of the dermis are generally unlabeled, except for labeled cells in the outer connective tissue sheath at the infundibulum of vibrissae of 20-day fetal rats and a few scattered labeled cells in the dermis, probably macrophages. In the developing hair, in 18- and 20-day fetal rats, a distinct topographic pattern of labeled cells can be seen, which is characteristic of the different stages of hair follicle development. In the hair germ, heavily labeled cells appear first in the stratum spinosum. In the hair peg, they remain in this position in its juxtaepidermal portion; however, when a dermal papilla develops, heavily labeled cells assume a marginal position. This suggests a sequential epidermal-epidermal and mesenchymal-epidermal receptor induction. Injection of tritium labeled 25 (OH) vitamin D₃ did not show nuclear concentration in these tissues and excess unlabeled 25 (OH) vitamin D₃ — unlike excess 1,25 (OH₂) vitamin D₃ — did not prevent nuclear uptake of tritium labeled 1,25 (OH₂) vitamin D₃. The results indicate differential effects of 1,25 (OH₂) vitamin D₃ on different structures in the epidermis and dermis.Supported by US PHS grant PCM8200569     

Chromogranin A,B and C immunoreactivities of mammalian endocrine cells

Summary Antibodies specific for chromogranin A, B or C have been used to detect immunohistochemically these three anionic proteins. Pancreatic A, B and PP cells, gut argentaffin EC, argyrophil ECL and gastrin G cells, thyroid C cells, parathyroid cells, adrenal medullary cells, pituitary TSH, FSH and LH cells as well as some axons of visceral nerves have been found to react with chromogranin A antibodies. Pancreatic A, gut EC and G, adrenal medullary and pituitary cells as well as some gut nerve fibers showed chromogranin B immunoreactivity. Chromogranin C immunoreactivity has been detected in pancreatic A, pyloric D₁, intestinal L, thyroid C, adrenal medullary and pituitary cells, as well as in some gut neurons and nerve fibers. No crossreactivity has been found in immunohistochemical tests between chromogranins A, B or C and costored monoamines or peptide hormones/prohormones, from which chromogranins can be separated by selective extraction during fixation. On both morphological and chemical grounds a relationship seems to exist between chromogranin A and Grimelius' argyrophilia. Sialooligosaccharide chains of chromogranin A and, possibly, chromogranins' phosphoserine/phosphothreonine groups, seem to interact with guanidyl, amino, and/or imidazole groups of non-chromogranin components to form silver complexing sites accounting for granules' argyrophilia, which can be removed or blocked without affecting chromogranin immunoreactivities. The abundant anionic groups of the three proteins should contribute substantially to granules' basophilia, the partly masked pattern of which supports the existence of a close interaction of such groups with other components of secretory granules, including monoamines and peptide hormones or prohormones. Chromogranins could play a role in hormone postranslational biosynthesis and intragranular packaging.     

DOPAMINERGIC PROPERTIES OF A SOMATIC CELL HYBRID LINE OF MOUSE NEUROBLASTOMA X SYMPATHETIC GANGLION CELLS

Abstract— Studies were made on the regulation of dopamine metabolism in a cell line derived by hydridization of a non-tyrosine-hydroxylase-containing line of murine neuroblastoma cells with a neur-onally-enriched population of murine embryonic sympathetic ganglion cells. Hybrid subclones with tyrosine hydroxylase activity were selected by exposure to tyrosine-free medium. The cells also exhibited DOPA decarboxylase activity and the subclone (named T28) with the highest specific activities of both enzymes was further characterized. The hybrid T28 line did not contain dopamine-β-hydroxylase activity. The specific activity of tyrosine hydroxylase as well as of DOPA decarboxylase increased significantly in T28 cultures when the cells entered the stationary phase of growth. Both of these enzymes were also induced after several days of exposure to 1 m m -dibutyryl cyclic AMP in culture medium containing either 5% or 0.8% serum. However, maintenance in medium containing 0.8% serum alone, which inhibited cell multiplication, did not induce either enzyme. The dopamine content of T28 cells was also regulated as a function of cell density. High density (stationary phase) cultures of T28 cells contained about 300 pmol dopamine per mg protein and at least half of this endogenous amine appeared to he stored in vesicles or granules (as judged by depletion with reserpine or α-methyl- m -tyramine). The T28 and other neuronal hybrid lines appear to be useful model systems for neuro-chemical studies.     

UVB Exposure of Farm Animals: Study on a Food-Based Strategy to Bridge the Gap between Current Vitamin D Intakes and Dietary Targets

Vitamin D deficiency is a global health problem. This study aimed to investigate the efficacy of ultraviolet (UV) B radiation for improving vitamin D₃ content of eggs and meat. In a two-factorial design hens that received diets with 0 (-D₃) or 3,000 IU (+D₃) vitamin D₃/kg were non-exposed (-UVB) or exposed to UVB radiation (+UVB) for 3 h daily over 4 weeks. Data show that UVB radiation was very effective in raising the vitamin D₃ content of egg yolk and meat. Egg yolk from +UVB/−D₃ hens had a higher vitamin D₃ content (17.5±7.2 µg/100 g dry matter (DM)) than those from the –UVB/+D₃ group (5.2±2.4 µg/100 g DM, p<0.01). Vitamin D₃ content in egg yolk of vitamin D₃-supplemented hens could be further increased by UVB radiation (32.4±10.9 µg/100 g DM). The content of 25-hydroxyvitamin D₃ (25(OH)D₃) in the egg yolk also increased in response to UVB, although less pronounced than vitamin D₃. Meat revealed about 4-fold higher vitamin D₃ contents in response to UVB than to dietary vitamin D₃ (p<0.001). In conclusion, exposure of hens to UVB is an efficient approach to provide consumers with vitamin D₃-enriched foods from animal sources.     

Effect of vitamin D3 supplementation on hepatic lipid dysregulation associated with autophagy regulatory AMPK/Akt-mTOR signaling in type 2 diabetic mice

Vitamin D₃ has been reported to protect liver against non-alcoholic fatty liver disease (NAFLD) by attenuating hepatic lipid dysregulation in type 2 diabetes mellitus (T2DM). However, the mechanism of vitamin D₃ on hepatic lipid metabolism-associated autophagy in hyperglycemia-induced NAFLD remains yet to be exactly elucidated. C57BL/6J mice were intraperitoneally injected with 30 mg/kg of streptozotocin and fed a high-fat diet for induction of diabetes. All mice were administered with vehicle or vitamin D₃ (300 ng/kg or 600 ng/kg) by oral gavage for 12 weeks. Histological demonstrations of the hepatic tissues were obtained by H&E staining and the protein levels related to lipid metabolism and autophagy signaling were analyzed by Western blot. Treatment with vitamin D₃ improved insulin resistance, liver damage, and plasma lipid profiles, and decreased hepatic lipid content in the diabetic mice. Moreover, vitamin D₃ administration ameliorated hepatic lipid dysregulation by downregulating lipogenesis and upregulating lipid oxidation under diabetic condition. Importantly, vitamin D₃ treatment induced autophagy by activating AMP-activated protein kinase (AMPK), inactivating Akt and ultimately blocking mammalian target of rapamycin (mTOR) activation in the T2DM mice. Additionally, vitamin D₃ was found to be effective in anti-apoptosis and anti-fibrosis in the liver of diabetic mice. The results suggested that vitamin D₃ may ameliorate hepatic lipid dysregulation by activating autophagy regulatory AMPK/Akt-mTOR signaling in T2DM, providing insights into its beneficial effects on NAFLD in type 2 diabetic patients.     

Lack of effect of chronic desipramine treatment on dopaminergic activity in the nucleus accumbens of the rat

The binding of [³H]SCH 23390 to dopamine (DA) D₁-receptors was measured in the nucleus accumbens of rats treated chronically with desipramine for 14 days. DA D₁ — and D₂-receptor binding using [³H]SCH 23390 and [³H]spiperone, respectively as ligands, was determined in rats treated for 28 days. NeitherB _max norK _d values were influenced by chronic desipramine treatment. In addition, chronic desipramine treatment (28 days) did not influence the dose dependent, quinpirole (10–1000 nM)-mediated inhibition of the electrically stimulated release of [³H]DA and [¹⁴C]ACh from nucleus accumbens slices or the dose dependent increase in [³H]DA release and decrease in [¹⁴C]ACh release in the presence of 1 and 10 M nomifensine. Therefore, our results suggest that the effect of chronic antidepressant treatment cannot be attributed to changes in either DA D₁₁-or D₂-receptor binding or DA D₂-receptor function in the nucleus accumbens.     

Acute Erythemal Ultraviolet Radiation Causes Systemic Immunosuppression in the Absence of Increased 25-Hydroxyvitamin D3 Levels in Male Mice

Vitamin D is synthesised by ultraviolet (UV) irradiation of skin and is hypothesized to be a direct mediator of the immunosuppression that occurs following UV radiation (UVR) exposure. Both UVR and vitamin D drive immune responses towards tolerance by ultimately increasing the suppressive activities of regulatory T cells. To examine a role for UVR-induced vitamin D, vitamin D₃-deficient mice were established by dietary vitamin D₃ restriction. In comparison to vitamin D₃-replete mice, vitamin D₃-deficient mice had significantly reduced serum levels of 25-hydroxyvitamin D₃ (25(OH)D₃, <20 nmol.L⁻¹) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃, <20 pmol.L⁻¹). Following either acute erythemal UVR, or chronic sub-erythemal UVR (8 exposures over 4 weeks) treatment, serum 25(OH)D₃ levels significantly increased in vitamin D₃-deficient female but not male mice. To determine if UVR-induced vitamin D was a mediator of UVR-induced systemic immunosuppression, responses were measured in mice that were able (female) or unable (male) to increase systemic levels of 25(OH)D₃ after UVR. Erythemal UVR (≥4 kJ/m²) suppressed contact hypersensitivity responses (T helper type-1 or -17), aspects of allergic airway disease (T helper type-2) and also the in vivo priming capacity of bone marrow-derived dendritic cells to a similar degree in female and male vitamin D₃-deficient mice. Thus, in male mice, UVR-induced 25(OH)D₃ is not essential for mediating the immunosuppressive effects of erythemal UVR.     

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse

Summary The tracheo-bronchial mucosa of the mouse has been found to contain an extensive system of argyrophilic epithelial cells. In the trachea the cells morphologically resemble enterochromaffin cells. Normally, these enterochromaffin-like cells contain no fluorogenic amine, as revealed by the Falck-Hillarp formaldehyde technique. On the other hand the cells have the capacity to take up and decarboxylate 3,4-dihydroxyphenylalanine (DOPA) or 5-hydroxytryptophan (5-HTP); the amine formed is stored in the cytoplasm in a reserpine-sensitive store. This capacity to produce and store amines under experimental conditions may reflect the presence in the tracheal enterochromaffin-like cells of an amine which can not be demonstrated with available fluorescence histochemical techniques. In the electron microscope the tracheal enterochromaffin-like cells were identified by a positive argyrophil reaction and by their capacity to accumulate radioactivity after administration of ³H-DOPA or ³H-5-HTP as revealed by autoradiography. The radioactive labelling was associated with cytoplasmic electron-dense granules (800–1000 Å), suggesting that the amine formed was stored in these granules. Accordingly, the granules stained argentaffin after DOPA-pre-treatment of the animal. It is suggested that, like similar cells in the gastric mucosa, these argyrophilic enterochromaffin-like cells constitute an endocrine system in which amines are of cytophysiological importance.     

Autoradiographic studies with 3H 1,25 dihydroxyvitamin D3 in thyroid and associated tissues of the neck region

Summary Rats and mice fed a vitamin D-deficient or vitamin D-complete diet were injected with ³H 1,25 (OH)₂ vitamin D₃. Autoradiograms prepared from cross sections through the neck region revealed nuclear concentration of radioactivity strongest in parathyroid chief cells, occasionally in thyroid follicular epithelial and interfollicular cells, in the epithelium of tubular remnants of the ultimobranchial body, in epithelium of the esophagus, in chondrocytes of tracheal cartilage, and in myoepithelial cells of tracheal glands. In the thyroid, most of the follicle epithelial cells did not show nuclear concentration of radioactivity which occurred only occasionally and predominantly in follicles located in marginal positions. Thyroglobulin in lumina of thyroid follicles contained varying amounts of radioactivity that correspond to the diameter of the follicles, with relatively high amounts in large follicles and little or no radioactivity in small follicles. Competition with excess of unlabeled 1,25 (OH)₂ vitamin D₃ abolished nuclear radioactivity, but not the radioactivity in the colloid, while 25 (OH) vitamin D₃ did not affect either. When a combination of autoradiography and immunohistochemistry was applied, follicular and parafollicular C-cells positive for calcitonin antibodies, did not show nuclear concentration of radioactivity. Tubular remnants of ultimobranchial bodies, however, showed distinct nuclear labeling, but did not stain, or only weakly stain, with antibodies to calcitonin. When ³H 25 (OH) vitamin D₃ was injected, no nuclear concentration of radioactivity was noted in any of the tissues.The results from these histochemical studies suggest the existence of nuclear receptors and direct genomic effects of 1,25 (OH)₂ vitamin D₃ in heterogeneous tissues of the neck region, which include parathyroid chief cells, myoepithelial cells of tracheal glands, chondrocytes of tracheal cartilage, epithelial cells of esophagus, and certain thyroid follicle epithelial cells. No evidence could be obtained for nuclear receptors in C-cells and cells of striated muscle.