The effect of ergosterol, ergocalciferol and cholecalciferol on calcium-controlled peroxidase secretion by sugarbeet cells

Provitamin D₂ (ergosterol), vitamins D₂ and D₃ reduced the calcium-mediated peroxidase secretion in three types of sugarbeet cells in suspension cultures. Vitamin D₂ was the most effective in habituated non-organogenic cells which were the less sensitive to calcium; provitamin D₂ was the most effective in habituated organogenic cells, while normal non-organogenic cells were equally sensitive to the three types of vitamin D. The calcium ionophore A23187 slightly restricted peroxidase release in all cases, except in the habituated organogenic cells in the presence of calcium where it exerted a promotive effect. The inhibiting effect of vitamin D₂ was not counteracted by the ionophore except in this habituated organogenic cell line.     

Adventitious root formation in green cuttings of Populus tremula: Characterisation of the effect of vitamin D3 and indolylbutyric acid

Indolyl-3-butyric acid and vitamin D₃ enhance adventitious root formation in green cuttings of Populus tremula L. A significant synergistic effect is observed between these two substances. The number of roots formed on application of the individual substances and on simultaneous application depends on the growth substance concentration, the timing of application, the age of the cuttings and the number of leaves. Of the vitamin D₃ animal metabolites tested, only 1,25-dihydroxyvitamin D₃ markedly promoted adventitious rooting, and this to a lesser extent than vitamin D₃ itself. The 3-O-glucopyranosides of vitamin D₃ and the vitamin D₃ animal metabolites, promoted rooting to the same extent as the parent compounds.     

Calcaemic responses in the freshwater mud eel, Amphipnous cuchia, to vitamin D3 administration

Vitamin D₃ (1000 iu 100 g⁻¹ body wt) was administered to the freshwater mud eel, Amphipnous cuchia , which was maintained either in tap water or a calcium-rich environment. Elevation of serum calcium levels was observed after the Vitamin D₃ administration. This response was faster in the fishes which were maintained in a calcium rich environment after the vitamin D₃ administration.     

Genetic polymorphism of the vitamin D binding protein (Gc) in the musk shrew (Suncus murinus)

In the musk shrew ( Suncus murinus ), the electrophoretic bands in the post-albumin region were identified as vitamin D binding protein (Gc) by the [³HI vitamin D₃ binding method. Three Gc phenotypes were distinguished from each other: a single faster band (Gc-A), a single slower band (Gc-B) and the double bands (Gc-AB). Results of mating experiments indicated that the Gc-A and Gc-B are controlled by two codominant alleles, Cc ^a and Gc ^b at an autosomal locus ( Cc ), respectively. It was noticed that, in the Gc-AB phenotypes, the Gc-B band was constantly more intense than the Gc-A band in the protein staining. The same tendency was also observed btween the homozygous Gc-A and Gc-B bands, and further, radioactivity of the Gc-B bound with [³H] vitamin D₃ was about twofold higher than that of the Gc-A. These results suggest that the Gc^b yields its protein product twofold more than the Gc ^a. No cross-reaction between the shrew proteins and a rabbit anti-human Gc protein was observed.     

RNA and protein metabolism during adventitious root formation in stem cuttings of Phaseolus aureus

Indole-3-butyric acid (IBA, 10⁻⁴ M ), spermine (7 × 10⁻⁵ M ) and vitamin D₂ (6.3 × 10⁻⁵ M ), all of which enhance rooting in mung bean cuttings ( Phaseolus aureus Roxb. cv. Berkin), influence RNA metabolism. Total and poly (A)⁺-RNA synthesis within the hypocotyl is inhibited by each of these chemicals within 24 h. These changes precede induced cell division and are therefore associated with the so-called inductive period of regeneration during which some cells in the hypocotyl undergo dedifferentiation. However, following subsequent transfer of cuttings to borate, which is an essential prerequisite for development of root primordia in these cuttings, RNA synthesis is enhanced by pretreatments with IBA, spermine or vitamin D₂. Furthermore, IBA inhibits synthesis and turnover of protein within the hypocotyl.     

Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

A dual block to cell cycle progression in HL60 cells exposed to analogues of vitamin D,

Abstract. The physiologically active form of vitamin D₃, 1,25-dihydroxy-vitamin D₃, (1,25(OH)₂, D₃), induces differentiation of several types of myeloid leukaemia cells. The acquisition of monocyte-like phenotype is accompanied by slower progression through the cell cycle, and G₁, block has been reported to be the basis of this effect. It is shown here that human promyelocytic leukaemia HL60 cells treated with analogues of vitamin D₃, which are potent inducers of monocytic differentiation, have an additional cell cycle block. Exposure to 10-⁷m 1,25(OH)₂, D₃, or 1,25-(OH)₂,-16-ene-D₃ resulted in monocytic differentiation and the expected G₁, block evident at approximately 48 h in a rapidly differentiating variant of HL60 cells (HL60-G), and at 96 h in the more slowly differentiating HL60-240 cells. In addition, a G₂,+M block was noted at approximately 72 h in HL60-G and HL60-240 cells. Exposure to vitamin D₃, analogues also markedly increased the number of dikaryons, suggesting that cytokinesis was impaired more than karyokinesis. Treatment with a third analogue 25-hydroxy-16,23-diene-D₃, produced little differentiation and had minimal effects on the cell cycle parameters. These findings indicate that vitamin D₃, analogues regulate cell proliferation by control of the transition of G₁, and G₂,+M phases, reminiscent of the cdc2/CDK2 type of cell cycle control.     

Effect of excess vitamin D3 on calcium metabolism in rainbow trout Salmo gairdneri Richardson

The effect of excess vitamin D₃ on calcium metabolism in rainbow trout was investigated. Trout were reared for 24 weeks on diets containing 4000, 104 000 or 1 004 000 iu of supplemental vitamin D₃ kg^-1 dry diet. No effect of excess vitamin D₃ was detected in the weight gain, feed efficiency or total mortalities of trout in the different groups. None of the fish showed any signs of hypercalcaemia and no significant difference was detected in the calcium, magnesium, phosphorus and sodium content of the trout vertebrae or total carcass. However, there was a significant increase in the calcium, magnesium and phosphorus content of the skin in trout reared on the highest supplemental level of vitamin D₃ (1 004 000 iu kg^-1 diet). No overt signs of renal calcinosis were detected in any of the trout. This study indicates that trout are resistant to excess vitamin D₃, and that excess vitamin D₃ does not appear to be related to the increased incidence of renal calcinosis in rainbow trout.     

Chimeric D2/D3 Dopamine Receptors Efficiently Inhibit Adenylyl Cyclase in HEK 293 Cells

Abstract: Despite a high degree of sequence homology, the dopamine D₂ and D₃ receptors have substantially different second messenger coupling properties. We have used chimeric D₂/D₃ receptors to investigate the contribution of the intracellular loops to the signaling properties of these receptors. In HEK 293 cells, D₂ receptors inhibit prostaglandin E₁-stimulated cyclic AMP levels by >90%, whereas D₃ receptors inhibit cyclic AMP accumulation by only 20%. In chimeras that have the second or third intracellular loop, or both loops simultaneously, switched between the D₂ and D₃ receptors, the maximal inhibition of adenylyl cyclase is 60–90%. In addition, the potency of quinpirole to inhibit adenylyl cyclase activity at some of the chimeras is altered compared with the wild-type receptors. It appears that the intracellular loops of the D₃ receptor are capable of interacting with G proteins, as when these loops are expressed in the D₂ receptor, the chimeras inhibit adenylyl cyclase similarly to the wild-type D₂ receptor. Our data suggest that the overall conformation of the D₃ receptor may be such that it interacts with G proteins only weakly, but when the intracellular loops are expressed in another context or the D₃ receptor structure is altered by the introduction of D₂ receptor sequence, this constraint may be lifted.     

Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum

Cattle and water buffalo sera labelled with vitamin D₃[¹⁴C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin.
Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.     

Activation of D1 and D2 Dopamine Receptors Inhibits Protein Kinase C Activity in Striatal Synaptoneurosomes

Abstract: The effects of D₁ and D₂ dopamine ligands on protein kinase C (PKC) activity were examined in synaptoneurosomes. Incubation with D₁ agonists (SKF 38393, fenodopam), in the presence of calcium, decreased the soluble and increased the particulate PKC activity. These effects were reversed by SCH 23390, which by itself had the opposite effect of increasing the soluble and decreasing the particulate PKC activity. In contrast, incubation with the D₂ agonists [LY 171555, (+)-3-(3-hydroxyphenyl)- N - n -propylpiperidine, RU 24213] increased the soluble and decreased the particulate PKC activity. These effects were reversed by sulpiride. (−)-3-(3-Hydroxyphenyl)- N - n -propylpiperidine had a D₂ antagonist profile. Apomorphine showed a biphasic dose-response change; i.e., it decreased particulate PKC activity at the D₂ receptor at low concentrations (0.1 µ M ) and increased it at the D₁ receptor at higher concentrations (10 µ M ). Pretreatment with tetrodotoxin or omission of calcium in the incubation medium did not alter the responses of the D₂ agonists, but it reversed the changes in PKC activity induced by the D₁ agonists and converted the biphasic response of apomorphine to a monophasic inhibition. These results indicate that (1) D₁ and D₂ dopamine receptors are negatively coupled to PKC and (2) the increase in particulate PKC activity seen with the D₁ drugs in the presence of calcium is mediated indirectly via a transneuronal effect.     

Structural Organization of the Murine D3 Dopamine Receptor Gene

Abstract: We have cloned the gene encoding the murine D₃ dopamine receptor and have analyzed its intron-exon structural organization, to gain a better understanding of the detailed architecture of the D₂ dopamine receptor genes. Restriction and sequence analysis reveal the presence of six introns, in contrast to the five introns previously reported for the rat D₃ receptor. The extra intron is located in the receptor's putative third cytoplasmic loop and generates an intron-exon organization directly analogous to that found in the D₂ receptor gene. In addition, we have sequenced the 5' and 3' nontranslated sequences flanking the coding region and have identified a putative poly(A) adenylation signal. These sequences are found to have a far lower homology with the corresponding rat nontranslated sequences than is found for the D₂ receptor, suggesting that the control of D₃ receptor expression may vary more between species than the control of D₂ receptor expression.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Opposing Actions of D1 and D2-Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D₁-and D₂-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D₁ or D₂ receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [³H]AA, D₂-receptor stimulation enhanced release of [³H]AA produced by application of the Ca²⁺ ionophore A23187 or of the purinergic agonist ATP. By contrast, D₁-receptor stimulation inhibited [³H]AA release. This inhibitory effect of D₁ receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D₂ and D₁ receptors in striatum, potentiation and inhibition, respectively, of Ca²⁺-evoked AA release.     

Polymorphic post-albumin of cattle and horse plasma identified as vitamin D binding protein (Gc protein)

Cattle and horse plasma samples of known post-albumin types were radiolabelled with ¹⁴C-vitamin D₃. These samples were then analysed by polyacrylamide gel electrophoresis, followed by autoradiography. The patterns observed were identical to those of post-albumin variants. The polymorphic post-albumin protein of cattle and horse was thus identified as the vitamin D binding protein and homologous to the Gc protein of human plasma.     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

Are Striatal Dopamine D4 Receptors Increased in Schizophrenia?

Abstract: The density of dopamine D₂-like receptors was determined using [³H]emonapride binding in putamen tissue taken postmortem from schizophrenic subjects and matched controls. A 72% increase in number of these receptors was identified in the schizophrenics, although three patients not receiving antipsychotic drug treatment before death exhibited receptor densities in the control range. Displacement of 1 n M [³H]emonapride binding by raclopride was used to define the contribution of the D₄ subtype of dopamine receptors to total [³H]emonapride binding. No evidence was obtained for the presence of D₄ receptors in putamen tissue from either control or schizophrenic subjects, indicating that the increase in D₂-like receptor density in schizophrenia is due not to an increase in number of D₄ sites in the disease, but to an up-regulation of D₂ or D₃ receptors probably induced by chronic treatment with antipsychotic drugs.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.