D5 dopamine receptor regulation of phospholipase D

D(1)-like receptors have been reported to decrease oxidative stress in vascular smooth muscle cells by decreasing phospholipase D (PLD) activity. However, the PLD isoform regulated by D(1)-like receptors (D(1) or D(5)) and whether abnormal regulation of PLD by D(1)-like receptors plays a role in the pathogenesis of hypertension are unknown. The hypothesis that the D(5) receptor is the D(1)-like receptor that inhibits PLD activity and serves to regulate blood pressure was tested using D(5) receptor mutant mice (D(5)(-/-)). We found that in the mouse kidney, PLD2, like the D(5) receptor, is mainly expressed in renal brush-border membranes, whereas PLD1 is mainly expressed in renal vessels with faint staining in brush-border membranes and collecting ducts. Total renal PLD activity is increased in D(5)(-/-) mice relative to congenic D(5) wild-type (D(5)(+/+)) mice. PLD2, but not PLD1, expression is greater in D(5)(-/-) than in D(5)(+/+) mice. The D(5) receptor agonist fenoldopam decreases PLD2, but not PLD1, expression and activity in human embryonic kidney-293 cells heterologously expressing the human D(5) receptor, effects that are blocked by the D(5) receptor antagonist SCH-23390. These studies show that the D(5) receptor regulates PLD2 activity and expression. The hypertension in the D(5)(-/-) mice is associated with increased PLD expression and activity. Impaired D(5) receptor regulation of PLD2 may play a role in the pathogenesis of hypertension.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.     

Vitamin D Metabolism and Guidelines for Vitamin D Supplementation

Vitamin D is essential for bone health and is known to be involved in immunomodulation and cell proliferation. Vitamin D status remains a significant health issue worldwide. However, there has been no clear consensus on vitamin D deficiency and its measurement in serum, and clinical practice of vitamin D deficiency treatment remains inconsistent. The major circulating metabolite of vitamin D, 25-hydroxyvitamin D (25(OH)D), is widely used as a biomarker of vitamin D status. Other metabolic pathways are recognised as important to vitamin D function and measurement of other metabolites may become important in the future. The utility of free 25(OH)D rather than total 25(OH)D needs further assessment. Data used to estimate the vitamin D intake required to achieve a serum 25(OH)D concentration were drawn from individual studies which reported dose-response data. The studies differ in their choice of subjects, dose of vitamin D, frequency of dosing regimen and methods used for the measurement of 25(OH)D concentration. Baseline 25(OH)D, body mass index, ethnicity, type of vitamin D (D₂ or D₃) and genetics affect the response of serum 25(OH)D to vitamin D supplementation. The diversity of opinions that exist on this topic are reflected in the guidelines. Government and scientific societies have published their recommendations for vitamin D intake which vary from 400–1000 IU/d (10–25 μg/d) for an average adult. It was not possible to establish a range of serum 25(OH)D concentrations associated with selected non-musculoskeletal health outcomes. To recommend treatment targets, future studies need to be on infants, children, pregnant and lactating women.     

24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2: novel metabolites of vitamin D2

Understanding of the inactivation pathways of 25-hydroxyvitamin D2 and 24-hydroxyvitamin D2, the two physiologically significant monohydroxylated metabolites of vitamin D2, is of importance, especially during hypervitaminosis D2. In a recent study, it has been demonstrated that the inactivation of 24-hydroxyvitamin D2 occurs through its conversion into 24,26-dihydroxyvitamin D2 [Koszewski, N.J., Reinhardt, T.A., Napoli, J.L., Beitz, C.D., & Horst, R.L. (1988) Biochemistry 27, 5785]. At present, little information is available regarding the inactivation pathway of 25-hydroxyvitamin D2 except its further metabolism into 24,25-dihydroxyvitamin D2 [Jones, G., Rosenthal, A., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Rabinovich, D., & Shakked, Z. (1979) Biochemistry 18, 1094]. In our present study, we investigated the metabolic fate of 25-hydroxyvitamin D2 in the isolated perfused rat kidney and demonstrated its conversion not only into 24,25-dihydroxyvitamin D2 but also into two other new metabolites, namely, 24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2. The structure identification of the new metabolites was established by the techniques of ultraviolet absorption spectrophotometry and mass spectrometry and by the characteristic nature of each new metabolite's susceptibility to sodium metaperiodate oxidation. In order to demonstrate the physiological significance of the two new trihydroxy metabolites of vitamin D2, we induced hypervitaminosis D2 in a rat using [3 alpha-3H]vitamin D2 and analyzed its plasma for the various [3 alpha-3H]vitamin D2 metabolites on two different high-pressure liquid chromatography systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polypeptides of the synaptic membrane antigens D1, D2, and D3

The rat brain synaptic membrane antigens D1, D2, and D3 were labelled by 125I and precipitated by antibodies in a crossed immunoelectrophoresis. The precipitates were stained, scraped off, reduced, and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The D1 antigen was composed of two polypeptide chains, apparent molecular weights 50 300 and 116 000 D2 of only one polypeptide chain, apparent molecular weight 139 000, and D3 of three polypeptides, apparent molecular weights 14 100, 23 500, and 34 400. Higher apparent molecular weight polypeptides were present in variable amounts in the D3 precipitate, except when the synaptic membrane extracts had been pre-treated with phospholipase D.     

PSS-3D1D: an improved 3D1D profile method of protein fold recognition for the annotation of twilight zone sequences

Annotation of any newly determined protein sequence depends on the pairwise sequence identity with known sequences. However, for the twilight zone sequences which have only 15–25% identity, the pair-wise comparison methods are inadequate and the annotation becomes a challenging task. Such sequences can be annotated by using methods that recognize their fold. Bowie et al. described a 3D1D profile method in which the amino acid sequences that fold into a known 3D structure are identified by their compatibility to that known 3D structure. We have improved the above method by using the predicted secondary structure information and employ it for fold recognition from the twilight zone sequences. In our Protein Secondary Structure 3D1D (PSS-3D1D) method, a score (w) for the predicted secondary structure of the query sequence is included in finding the compatibility of the query sequence to the known fold 3D structures. In the benchmarks, the PSS-3D1D method shows a maximum of 21% improvement in predicting correctly the α + β class of folds from the sequences with twilight zone level of identity, when compared with the 3D1D profile method. Hence, the PSS-3D1D method could offer more clues than the 3D1D method for the annotation of twilight zone sequences. The web based PSS-3D1D method is freely available in the PredictFold server at .     

Localization of the antigens D1, D2 and D3 in the rat brain synaptic membrane

The inside-outside localization of the nervous system specific membrane proteins D1, D2 and D3 was investigated by a immunoabsorption technique. It was found that D1 was located at least partly on the outside of the synaptic membrane in contrast to D3 which was inside on the membrane, facing the cytoplasm. The protein D2 was outside on the synaptic membrane, and it was found very accessible to the antiserum. It is speculated that D2 might be involved in the axonal-dendritic recognition process during synaptogenesis.     

Hepatic accumulation of vitamin D3 and 25-hydroxyvitamin D3.

Concomitant intravenous administration of 25-hydroxycholecalciferol and [3H] vitamin D3 to vitamin D-depleted rats did not affect the conversion of [3H] vitamin D3 to 25-OH-[3H] vitamin D3 as indicated by a serum 25-OH-[3H] vitamin D3 to content at 3 and 24 h identical to those observed in animals receiving [3H] vitamin D3 alone. Similarly, pre-dosing with 25-OH vitamin D3 24 h earlier did not affect the conversion. Co-administration to vitamin D depleted rats of vitamin D2 or D3, at 200-fold higher doses than a control group receiving tracer [3H] vitamin D3 alone, resulted in serum 25-OH vitamin D levels that were 15-20 fold higher than the control, indicating a similar metabolic fate for synthetic and natural vitamin D in rats and the ability of increased substrate to overwhelm hepatic constraints on 25-OH vitamin D production. Following intravenous administration of 25-OH-[3H] vitamin D3 to vitamin D depleted rats, hepatic 3H content decreased in parallel with serum radioactivity. Hepatic accumulation of intravenously administered vitamin D3 ([14C] vitamin D3) alone or with 25-OH-[3H] vitamin D3, by vitamin D-depleted rats revealed a marked preference for vitamin D3; the hepatic accumulation of [14C] vitamin D3 increased to 35% of the dose by 45 min, at which time 25-OH-[3H] vitamin D3 hepatic content was 7-fold less, and decreasing. Chromatography of extracts of hepatic subcellular fractions revealed more [14C] vitamin D3 than 25-OH-[3H] vitamin D3 in the microsomes, the reported site of calciferol 25-hydroxylase. Circulating 25-OH vitamin D, therefore, has comparatively minimal potential for hepatic accumulation. Product inhibition of the calciferol 25-hydroxylase must, therefore, result from recently synthesized hepatic 25-OH vitamin D, and is not affected by exogenous 25-OH vitamin D3.     

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.     

D1-like dopamine receptor-mediated function in congenic mutants with D1 vs. D5 receptor "knockout"

Current understanding of the functional roles of individual dopamine D1-like [D1, D5] and D2-like [D2L/s, D3, D4] receptor subtypes remains incomplete. In particular, the lack of pharmacological agonists and antagonists able to distinguish between D1 and D5 receptors means that any differential roles in the regulation of behavior are poorly understood. Mutant mice with targeted gene deletion ("knockout") of individual dopamine receptor subtypes offer an important alternative approach to resolving these functional roles. In congenic D1 mutants examined ethologically, progressive increases in specific topographies of behavior over wildtypes were considerably greater than those in D1 mutants on a mixed genetic background; D1 knockout appears to influence the neuronal substrate(s) of habituation to disrupt sculpture of the changing topography of behavior from initial exploration through to quiescence. Similarly, the D1 receptor appears to regulate specific topographies of orofacial movement in the mouse as these are "sculpted" in a time-dependent manner. Although the well-recognized role of the D1-like family in regulating several aspects of behavioral topography has been assumed to involve primarily D1 receptors, this presumption may require modification to accommodate a subtle but not negligible role for their D5 counterparts as evidenced in the phenotype of congenic D5 mutants.     

D2/D3 dopamine receptor heterodimers exhibit unique functional properties

Evidence for heterodimerization has recently been provided for dopamine D(1) and adenosine A(1) receptors as well as for dopamine D(2) and somatostatin SSTR(5) receptors. In this paper, we have studied the possibility that D(2) and D(3) receptors interact functionally by forming receptor heterodimers. Initially, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D(2trunk) or D(3trunk), and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D(2tail) or D(3tail). Then we defined the pharmacological profiles of the homologous (D(2trunk)/D(2tail) and D(3trunk)/D(3tail)) as well as of the heterologous (D(2trunk)/D(3tail) and D(3trunk)/D(2tail)) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D(2) or D(3) receptors. In most cases, the D(3trunk)/D(2tail) was the one with the highest affinity for most agonists and antagonists. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D(2) and D(3) receptors, suggesting that D(2) and D(3) receptors can form complexes with these fragments and that these complexes bind [(3)H]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the ability of the split and the wild type receptors to inhibit adenylyl cyclase (AC) types V and VI. All of the native and split receptors inhibited AC-V and AC-VI, with the exception of D(3), which was unable to inhibit AC-VI. We therefore studied the ability of D(2) and D(3) to interact functionally with one another to inhibit AC-VI. We found that with D(2) alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC(50) of 2.05 +/- 0.15 nm, while in the presence of D(2) and D(3) it inhibited AC-VI with an IC(50) of 0.083 +/- 0.011 nm. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D(2) and D(3) receptors are capable of physical interaction.     

Individual quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in human milk

Extraction, lipid-reduction, and chromatographic methods suitable for the resolution and subsequent quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxy-vitamin D3 from human milk are described. This procedure utilizes a methanol:methylene chloride extraction, precipitation of unwanted lipids with cold methanol and ether, backwash with alkaline buffer, silica Sep-Pak preparative chromatography, normal- and reverse-phase high-performance liquid chromatography with final quantitation of the antirachitic sterols by competitive protein binding assay. The described assay was used to determine these antirachitic sterols in milk from women receiving various supplements of vitamin D or undergoing ultraviolet phototherapy.     

Low digit ratio 2D:4D in alcohol dependent patients

The ratio of the lengths of the second and fourth finger (2D∶4D) has been described as reflecting the degree of prenatal androgen exposure in humans. 2D∶4D is smaller for males than females and is associated with traits such as left-handedness, physical aggression, attention-deficit-hyperactivity disorder and a genetic polymorphism of the androgen receptor. All of these traits are known to be correlated to the vulnerability for alcohol dependency. We therefore hypothesized low 2D∶4D in patients with alcohol dependency. In the present study on 131 patients suffering from alcohol dependency and 185 healthy volunteers, we found that alcohol dependent patients had smaller 2D∶4D ratios compared to controls with preserved sexual dimorphism but with reduced right-left differences. The detection of alcohol dependency based on 2D∶4D ratios was most accurate using the right hand of males (ROC-analysis: AUC 0.725, sensitivity 0.667, specificity 0.723). These findings provide novel insights into the role of prenatal androgen exposure in the development of alcohol dependency and for the use of 2D∶4D as a possible trait marker in identifying patients with alcohol dependency.     

Isolation and identification of vitamin D3, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 in Solanum malacoxylon incubated with ruminal fluid.

It has been shown that Solanum malacoxylon contains 1 alpha,25-dihydroxyvitamin D3-glycoside. The presence of vitamin D3 and 25-hydroxyvitamin D3 has also been suggested. In the present study vitamin D3 and three of its metabolites, including 1 alpha,25-dihydroxyvitamin D3, were detected in plant leaf extracts preincubated with ruminal fluid (SMRF). Extraction of SMRF with non-polar organic solvents and purification of the lipid extract by TLC followed by HPLC yielded nine ultraviolet-absorbing (264 nm) peaks. Four of them comigrated on a Zorbax-Sil HPLC column with synthetic standards of vitamin D3, 25-hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3, respectively. These compounds were unequivocally identified by means of mass spectrometry. The results confirm that Solanum malacoxylon contains, in addition to 1 alpha,25-dihydroxyvitamin D3, vitamin D3, 25-hydroxyvitamin D3 and possibly other as yet unidentified derivatives. As 1,24,25-trihydroxyvitamin D3 is absent in plant extracts not incubated with ruminal fluid, the data also indicate that rumen microbes may convert 1 alpha,25-dihydroxyvitamin D3 into 1,24,25-trihydroxyvitamin D3.     

Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth

Regulation of adult beta-cell mass in pancreatic islets is essential to preserve sufficient insulin secretion in order to appropriately regulate glucose homeostasis. In many tissues mitogens influence development by stimulating D-type cyclins (D1, D2, or D3) and activating cyclin-dependent kinases (CDK4 or CDK6), which results in progression through the G(1) phase of the cell cycle. Here we show that cyclins D2 and D1 are essential for normal postnatal islet growth. In adult murine islets basal cyclin D2 mRNA expression was easily detected, while cyclin D1 was expressed at lower levels and cyclin D3 was nearly undetectable. Prenatal islet development occurred normally in cyclin D2(-/-) or cyclin D1(+/-) D2(-/-) mice. However, beta-cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin D2(-/-) mice, causing glucose intolerance that progressed to diabetes by 12 months of age. Although cyclin D1(+/-) mice never developed diabetes, life-threatening diabetes developed in 3-month-old cyclin D1(-/+) D2(-/-) mice as beta-cell mass decreased after birth. Thus, cyclins D2 and D1 were essential for beta-cell expansion in adult mice. Strategies to tightly regulate D-type cyclin activity in beta cells could prevent or cure diabetes.     

Altered Vitamin D receptor-coactivator interactions reflect superagonism of Vitamin D analogs

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], has potent antiproliferative actions on various normal and malignant cells. Calcemic effects, however, hamper therapeutic application of 1,25-(OH)(2)D(3) in hyperproliferative diseases. Two 14-epi-analogs of 1,25-(OH)(2)D(3) namely 19-nor-14-epi-23-yne-1,25-(OH)(2)D(3) (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)(2)D(3) (TX527), display reduced calcemic effects coupled to an (at least 10-fold) increased antiproliferative potency when compared with 1,25-(OH)(2)D(3). Altered cofactor recruitment by the Vitamin D receptor (VDR) might underlie the superagonism of these 14-epi-analogs. Therefore, this study aims to evaluate their effects at the level of VDR-coactivator interactions. Mammalian two-hybrid assays with VDR and the coactivators TIF2 and DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)(2)D(3). TX522 and TX527 require 30- and 40-fold lower doses to obtain the VDR-DRIP205 interaction induced by 1,25-(OH)(2)D(3) at 10(-8)M. Evaluation of additional 1,25-(OH)(2)D(3)-analogs and their impact on VDR-coactivator interactions revealed a strong correlation between the antiproliferative potency of an analog and its ability to induce VDR-coactivator interactions. In conclusion, these data show that altered coactivator binding by the VDR is one possible explanation for the superagonistic action of the two 14-epi-analogs TX522 and TX527.