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.     

Fluorescence microscopy of the 5-HTP turnover in the exocrine pancreas of mice and rats

Summary The turnover ofl-5-HTP,d-5-HTP and 5-HT in the exocrine pancreas have been studied by means of the fluorescence method ofFalck andHillarp. l- andd-5-HTP are easily taken up by the acinar cells, whereas 5-HT seems to pass into the cells only to a minor extent. After the administration ofl-5-HTP (and in some cases after 5-HT administration), specific fluorescence is seen in the form of apically located granules (probably identical with the zymogen granules) for a short period, which is prolonged, if the animals are pretreated with a MAO inhibitor. Decarboxylase inhibition prevents the appearance of these fluorescent granules. Administration ofd-5-HTP does not give rise to this granular fluorescence but to a diffuse fluorescence throughout the cells. Thus, there are reasons to assume that the granular fluorescence derives from 5-HT. The results obtained in this work correspond well with those from a similar study withl-DOPA and some of its analogues.abbreviations DOPA 3,4-dihydroxyphenylalanine - DA dopamine - NA noradrenaline - A adrenaline - 5-HTP 5-hydroxytryptophan - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase This work was supported by grants from the Swedish Medical Research Council (B68-12X-712-03B and B68-14X-56-04B), the United States Public Health Service (06701-02) and the Faculty of Medicine, University of Lund, Lund, Sweden.     

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.     

Regulation of expression of antioxidant enzymes by vitamin E and curcumin in <Emphasis Type="SmallCaps">l</Emphasis>-thyroxine-induced oxidative stress in rat renal cortex

The present study investigates the antioxidative effects of vitamin E and curcumin against l-thyroxine (T₄)-induced oxidative stress in renal cortex of adult male rats. Rats were made hyperthyroid by administration of l-thyroxine (0.0012%) in their drinking water for 30 days. Vitamin E (200 mg/kg body weight/day) and curcumin (30 mg/kg body weight/day) were supplemented singly or in combination orally for 30 days along with l-thyroxine treatment. The elevated level of oxidative stress parameters (lipid peroxidation and protein carbonylation) and decline level of small antioxidant molecules (reduced glutathione and ascorbic acid) in renal cortex of T₄-treated rats were restored back by supplementation of vitamin E or/and curcumin. Increased superoxide dismutase and catalase activities in kidney cortex of T₄-treated rats were ameliorated in response to vitamin E or/and curcumin treatment. The elevated translated product of Cu/Zn-SOD, Mn-SOD and catalase in T₄-treated rats were differentially reduced by the administration of vitamin E and curcumin independently or in combination. Cu/Zn-SOD expression was ameliorated by both vitamin E and curcumin independently or in combination, whereas Mn-SOD expression was ameliorated by the supplementation of vitamin E or curcumin independently. However, the expression of catalase was alleviated by only supplementation of vitamin E to T₄-treated rats. The results suggest that both vitamin E and curcumin may play an important role in protecting T₄-induced oxidative stress in rat renal cortex by differentially modulating the activities of antioxidant enzymes and oxidative stress parameters.     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

Effect of vitamin D2 stimulation on amine stores and secretory granules in the calcitonin cells of the mouse

Summary The calcitonin-producing cells (C cells) of the thyroid gland are characterized by numerous suhmicroscopic granules, believed to contain the hormone, by a strong argyrophilia, and by the ability to synthesize and store certain arylethylamines (such as dopamine) from corresponding immediate precursors (such as DOPA).In mice, vitamin D₂ treatment—known to increase calcitonin secretion—evoked a degranulation and a loss of argyrophilia of the C cells, and a reduction in their content of dopamine (induced by exogenous DOPA). Additional treatment with a monoamine oxidase inhibitor restored the dopamine content of the C cells, although they were still degranulated. Treatment with reserpine, which inhibits the amine-storing mechanism, prevented the degranulation following vitamin D₂ treatment.The findings support the assumption that a functional rather than only a structural relationship exists between the intracellular amine and the secretory granules in cells elaborating calcitonin, and that the amine is involved in the secretion of the polypeptide hormone.     

MORPHOLOGICAL AND PHARMACOLOGICAL EFFECTS OF RESERPINE, GIVEN ALONE OR AFTER IPRONIAZID, ON THE CATECHOL AMINES OF THE ADRENAL GLANDS OF THE RAT

Adrenomedullary cells, after fixation with OsO₄, are filled with well formed granules which are considered to represent their catechol amine content. The submicroscopic appearance of these cells was studied in reserpine-treated rats during the late phase of catechol amine depletion and during the period of its restoration. At 3 days after the beginning of reserpine treatment, the granules appeared to be emptied of their content and small vesicles containing scattered, dense deposits of, presumably, catechol amines began to be seen. At 9 days after the beginning of treatment, these deposits had already become granules and the cells had attained a completely normal appearance. The submicroscopic structure of the adrenomedullary cells of rats pretreated with iproniazid (before reserpine), in which a complete inhibition of monoamine oxidase activity had thus been obtained, was similar to that seen in non-treated animals. In numerous cases, however, some characteristic features were noted: the sacs which usually contained a dense granule of catechol amines appeared swollen and many fine granules could be seen around them; the latter were dispersed in a way suggesting that they may represent a partial breakdown of the large granules which, under the inhibitory action of iproniazid, do not release the catechol amines contained within them.     

Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

Summary Thein vivo andin vitro effects of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on calcium uptake by isolated chick duodenal cells were studied.In vivo, 1,25-(OH)₂D₃ given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion.In vitro, pre-incubation of 1,25-(OH)₂D₃ with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10^–15 m, was maximal at 10^–13 m, and was diminished at higher (10^–11 m) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D₃ analogs was 1,25-(OH)₂D₃=1-(OH)D₃>25-(OH)D₃>1,24,25-(OH)₃D₃>24,25-(OH)₂D₃>D₃. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)₂D₃ was five orders of magnitude greater than D₃. Kinetically, 1,25-(OH)₂D₃ increased theV _max of calcium uptake; the affinity for calcium (K _m=0.54mm) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)₂D₃,in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)₂D₃. These findings suggest that thein vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)₂D₃ induction of calcium transport and specific biochemical correlates.     

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.     

Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats

Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D₃ in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D₃ treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D₃ supplementation improved the behavioural deficit. In conclusion, vitamin D₃ shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D₃ modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D₃ supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D₃ and muscarinic M3 receptors in regulating insulin secretion from pancreas.     

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     

Target cells for 1,25 dihydroxyvitamin D3 in the pancreas

Summary Mature rats raised on a vitamin D deficient diet were injected with tritium labeled 1,25 dihydroxyvitamin D₃. Concentration of radioactivity, which is prevented by pretreatment with unlabeled 1,25 dihydroxyvitamin D₃, is found in nuclei of cells that are centrally located in pancreatic islets. The central location of these cells and supportive evidence from the literature suggest that they are B-cells, and that 1,25 dihydroxyvitamin D₃ has a direct and genomic action on B-cell functions including insulin secretion.Supported by US Public Health Service Grants NS 09914 and AM 14881     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

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     

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.     

The effect of toxic doses of cholecalciferol (Vitamin D3) on the serum zinc levels in rats

Zinc is a trace element important to bone mineralization as well as, in general, nutrition. It is known that cholecalciferol (vitamin D₃) affects bone metabolism. In this study toxic doses of vitamin D₃ were injected subcutaneously (25 μg/d) to rats for 5 wk. It caused a significant increase in serum zinc levels (p<0.02). On the other hand, no significant increase was detected in the other groups. Excessive amounts of vitamin D₃ caused bone breakdown and increased the levels of zinc in blood.     

Impact of vitamin D3 on cardiovascular responses to glucocorticoid excess

Although the cardiovascular system is not a classical target for 1,25-dihydroxyvitamin D₃, both cardiac myocytes and vascular smooth muscle cells respond to this hormone. The present study aimed to elucidate the effect of active vitamin D₃ on cardiovascular functions in rats exposed to glucocorticoid excess. Adult male Wistar rats were allocated into three groups: control group, dexamethasone (Dex)-treated group receiving Dex (200 μg/kg) subcutaneously for 12 days, and vitamin D₃-Dex-treated group receiving 1,25-(OH)₂D₃ (100 ng/kg) and Dex (200 μg/kg) subcutaneously for 12 days. Rats were subjected to measurement of systolic (SBP), diastolic (DBP), and mean arterial (MAP) blood pressures and heart rate. Rate pressure product (RPP) was calculated. Rats’ isolated hearts were perfused in Langendorff preparation and studied for basal activities (heart rate, peaked developed tension, time to peak tension, half relaxation time, and myocardial flow rate) and their responses to isoproterenol infusion. Blood samples were collected for determination of plasma level of nitrite, nitric oxide surrogate. Dex-treated group showed significant increase in SBP, DBP, MAP, and RPP, as well as cardiac hypertrophy and enhancement of basal cardiac performance evidenced by increased heart rate, rapid and increased contractility, and accelerated lusitropy, together with impaired contractile and myocardial flow rate responsiveness to beta-adrenergic activation and depressed inotropic and coronary vascular reserves. Such alterations were accompanied by low plasma nitrite. These changes were markedly improved by vitamin D₃ treatment. In conclusion, vitamin D₃ is an efficacious modulator of the deleterious cardiovascular responses induced by glucocorticoid excess, probably via accentuation of nitric oxide.     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Summary Certain disseminated endocrine-like cells have reviously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (paraxons). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.This work was reported in part at the 29th Congress of the International Union of Physiological Sciences, Sydney, Australia, August 28–September 3, 1983     

Effects of BMP-2 and vitamin D3 on the osteogenic differentiation of adipose stem cells

We studied the effect of bone morphogenetic protein-2 (BMP-2) and vitamin D₃ on the osteogenic differentiation of adipose stem cells (ASCs). ASCs were treated with 10, 50, and 100 ng/ml of BMP-2, and 10⁻⁸, 10⁻⁷, 10⁻⁶ M vitamin D₃. Then, to investigate the effects of combined treatment, ASCs were treated with BMP-2 and vitamin D₃ dose-dependently and time-dependently. The osteogenic differentiation was assessed by alkaline phosphatase (ALP) activities/staining and the mineralization was evaluated by Alizarin red S staining. ALP activity and mineralization dose-dependently increased in early stages (ALP on 7th day and mineralization on the 14th day) while all three doses of BMP-2 or vitamin D₃ showed comparable effects in late stages (ALP on the 14th day and mineralization on the 21st day) in ASCs. BMP-2 and vitamin D₃ had synergistic effect on the osteogenic differentiation of ASCs. While all three doses of BMP-2 acted similarly in reinforcing the effect of vitamin D₃, vitamin D₃ dose-dependently augmented the osteogenic effect of BMP-2. When BMP-2 was constantly treated, vitamin D₃ effect did not differ depending on the period of vitamin D₃ treatment. However, when vitamin D₃ was constantly treated, the BMP was more effective when treated for the last 7 days than when treated for the first 7 days. In conclusion, BMP-2 and vitamin D₃ promote osteogenic differentiation of ASCs, and can work synergistically. These results can be used to induce effective and economical osteogenic induction of ASCs for bone tissue engineering.