Uncommon liver lesions with multimodality imaging and pathology correlation.

Uncommon liver lesions pose a diagnostic challenge because of unfamiliar imaging findings. For simplification, these lesions can be divided into four broad categories based on the dominant imaging feature in each: hypervascular, hypovascular, fat-containing, or cystic lesions. In this review, we profile the radiological features of uncommon liver lesions on multimodality imaging including ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), and nuclear medicine.

The Huntington's Disease health-related Quality of Life questionnaire (HDQoL): a disease-specific measure of health-related quality of life.

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by motor, cognitive and psychiatric disturbances, and yet there is no disease-specific patient-reported health-related quality of life outcome measure for patients. Our aim was to develop and validate such an instrument, i.e. the Huntington's Disease health-related Quality of Life questionnaire (HDQoL), to capture the true impact of living with this disease. Semi-structured interviews were conducted with the full spectrum of people living with HD, to form a pool of items, which were then examined in a larger sample prior to data-driven item reduction. We provide the statistical basis for the extraction of three different sets of scales from the HDQoL, and present validation and psychometric data on these scales using a sample of 152 participants living with HD. These new patient-derived scales provide promising patient-reported outcome measures for HD.

Impact of Huntington's across the entire disease spectrum: the phases and stages of disease from the patient perspective.

Although Huntington's disease (HD) is a neurodegenerative disease characterized by motor, cognitive and behavioural disturbances, there has been little empirical data examining what patients are most concerned about throughout the different stages of disease, which can span many years. Semi-structured face-to-face interviews were individually conducted with 31 people living with different stages of Huntington's, from pre-clinical gene carriers to advanced stage. We examined how often participants raised issues and concerns regarding the impact of Huntington's on everyday life. The Physical/functional theme hardly featured pre-clinically, but was strongly present from Stage 1, rose steadily and peaked at Stage 5. There were no significant changes between stages for the Emotional, Social, and Self themes that all featured across all stages, indicating that these issues were not raised more frequently over the course of the disease. Likewise, the more rarely mentioned Financial and Legal themes also remained similar across stages. However, the Cognitive theme only featured between Stages 1 and 4, and hardly at all pre-clinically and at Stage 5. These findings provide insight into patients' important and unique perspective and have implications for the management and development of interventions across the spectrum of HD stages.

The role of repressor proteins in the adrenergic induction of type II iodothyronine deiodinase in rat pinealocytes.

In this study, we investigated the transcriptional regulation of the adrenergic induction of type II iodothyronine deiodinase (Dio2) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA level of Dio2 that peaked around 2 h and declined over the next 5 h. Both beta- and alpha1-adrenergic receptors contributed to the NE induction of Dio2 expression through a cAMP/protein kinase A mechanism. In pinealocytes that had been stimulated by NE, inhibition of transcription by actinomycin had no discernible effect on Dio2 expression. In contrast, inhibition of protein synthesis by cycloheximide enhanced the NE induction of Dio2 expression, suggesting the involvement of a repressor protein. Transient transfection of pinealocytes with adenovirus expressing small interfering RNA against Fos-related antigen 2 (Fra2) enhanced the NE induction of Dio2 expression, whereas the effect of overexpression of the full-length transcript of Fra2 was inhibitory. Time-course study indicated that preventing the NE induction of Fra2 enhanced the NE induction of Dio2 after 3 h, and the enhancement persisted beyond 6 h after NE stimulation. In comparison, transient transfection of pinealocytes with small interfering RNA against inducible cAMP early repressor (Icer) had no effect on the NE induction of Dio2 expression, whereas overexpression of the full-length transcript of Icer caused a small reduction of the NE-stimulated Dio2 expression. Together, our results support Fra-2 as an important transcriptional repressor that helps shape the time profile of the adrenergic induction of Dio2 expression in the rat pineal gland.

Acetylation of histone H3 and adrenergic-regulated gene transcription in rat pinealocytes.

In this study we investigated the effect of histone acetylation on the transcription of adrenergic-induced genes in rat pinealocytes. We found that treatment of pinealocytes with trichostatin A (TSA), a histone deacetylase inhibitor, caused hyperacetylation of histone H3 (H3) Lys14 at nanomolar concentrations. Hyperacetylation of H3 was also observed after treatment with scriptaid, a structurally unrelated histone deacetylase inhibitor. The effects of TSA and scriptaid were inhibitory on the adrenergic induction of arylalkylamine-n-acetyltransferase (aa-nat) mRNA, protein, and enzyme activity, and on melatonin production. TSA at higher concentrations also inhibited the adrenergic induction of mapk phosphatase-1 (mkp-1) and inducible cAMP early repressor mRNAs. In contrast, the effect of TSA on the norepinephrine induction of the c-fos mRNA was stimulatory. Moreover, the effect of TSA on adrenergic-induced gene transcription was dependent on the time of its addition; its effect was only observed during the active phase of transcription. Chromatin immunoprecipitation with antibodies against acetylated Lys14 of H3 showed an increase in DNA recovery of the promoter regions of aa-nat, mkp-1, and c-fos after treatment with TSA. Together, our results demonstrate that histone acetylation differentially influences the transcription of adrenergic-induced genes, an enhancing effect for c-fos but inhibitory for aa-nat, mkp-1, and inducible cAMP early repressor. Moreover, both inhibitory and enhancing effects appear to be mediated through specific modification of promoter-bound histones during gene transcription.

Histone H3 phosphorylation in the rat pineal gland: adrenergic regulation and diurnal variation.

In this study, we investigated phosphorylation of Ser10 in histone H3 by norepinephrine (NE) in the rat pineal gland. In whole-animal studies, we demonstrated a marked increase in histone H3 phosphorylation in the rat pineal gland during the first half of the dark period. Exposure to light during this period caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 15 min, indicating a high level of dephosphorylation activity. Corresponding studies in cultured pineal cells revealed that treatment with NE produced an increase in histone H3 phosphorylation that peaked between 2 and 3 h and declined rapidly by 4 h. The NE-induced histone H3 phosphorylation was blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor, but not by prazosin or other kinase inhibitors. Moreover, only treatment with dibutyryl cAMP but not other kinase activators mimicked the effect of NE on histone H3 phosphorylation. The NE-stimulated H3 phosphorylation was markedly increased by cotreatment with a serine/threonine phosphatase inhibitor, tautomycin or okadaic acid, supporting a high level of ongoing histone H3 dephosphorylation activity. Together, our results indicate that histone H3 phosphorylation is a naturally occurring event at night in the rat pineal gland that is driven almost exclusively by a NE-->beta-adrenergic-->cAMP/protein kinase A signaling mechanism. This transient histone H3 phosphorylation probably reflects the nocturnal activation of multiple adrenergic-regulated genes in the rat pineal gland.

The role of inducible repressor proteins in the adrenergic induction of arylalkylamine-N-acetyltransferase and mitogen-activated protein kinase phosphatase-1 in rat pinealocytes.

In this study, we investigated the role of two inducible repressor proteins, inducible cAMP early repressor (ICER) and Fos-related antigen 2 (Fra-2) in the adrenergic induction of MAPK phosphatase-1 (MKP-1) as compared with their roles in the induction of arylalkylamine-N-acetyltransferase (AA-NAT) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA and protein levels of MKP-1 and AA-NAT, as well as in the AA-NAT activity and melatonin production. NE stimulation also caused a simultaneous increase in the mRNA and protein levels of ICER and Fra-2. Transient knockdown of icer using adenovirus expressing small interfering RNA (siRNA) abolished the NE induction of icer expression but had little effect on the NE induction of mkp-1 or aa-nat expression. In contrast, pretreatment with adenovirus overexpressing icer was effective in reducing the NE induction of mkp-1 and aa-nat. The inhibitory effect of overexpressing icer was reversed by cotreatment with siRNA against icer. siRNA against fra-2 also abolished the NE-stimulated expression of fra-2 but had little effect on the NE induction of mkp-1 and aa-nat expression. Proteasomal inhibition, which reduced the NE-stimulated induction of aa-nat, caused a reduction of ICER and Fra-2. Together, these results indicate that whereas overexpression of ICER can suppress the NE induction of aa-nat and mkp-1, the amount of the repressors, ICER and Fra-2, present during NE induction appears insufficient to exert a significant effect in controlling the expression of these genes.

The role of protein turnover in regulating MKP-1 levels in rat pinealocytes.

We have previously shown that mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is induced at night under the control of a photoneural system in the rat pineal gland. Because of the established roles of MAPKs, glucocorticoids and proteasome activity in regulating MKP-1 expression in other cell types, their relative contributions to MKP-1 regulation were investigated in rat pinealocytes. We found that neither inhibition of MAPKs nor treatment with dexamethasone affected norepinephrine-stimulated MKP-1 expression. In contrast, treatment with proteasome inhibitors increased norepinephrine-stimulated MKP-1 protein levels and abolished the decline in norepinephrine-stimulated MKP-1 protein levels caused by inhibition of transcription or translation, or blockade of alpha-adrenergic receptors. Taken together, our results indicate that in rat pinealocytes, the continuous and rapid turnover of MKP-1 protein allows for its rapid induction but is not sufficient to generate the sustained increase in MKP-1 expression post-adrenergic stimulation.

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p.

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

The integral membrane protein snl1p is genetically linked to yeast nuclear pore complex function.

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23 degrees C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2-1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland.

Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE-->cGMP activation of p42/44MAPK is rapid and transient, the NE-->cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland.

Opposite effects of proteasome inhibitors in the adrenergic induction of arylalkylamine N-acetyltransferase in rat pinealocytes.

In the rat pineal gland, the steady-state level of arylalkylamine N-acetyltransferase (AANAT) protein is controlled by transcriptional and translational mechanisms as well as by proteasome-mediated degradation. Studies with proteasome inhibitors, MG132 and clasto-lactacystin beta-lactone (c-lact), show two opposite effects of proteasomal inhibition on norepinephrine (NE)-induction of Aanat. Addition of MG132 or c-lact following NE stimulation causes an increase in AANAT protein level and enzyme activity without affecting the level of Aanat mRNA. In contrast, addition of inhibitors prior to NE stimulation reduces the NE-stimulated Aanat mRNA, AANAT protein, and enzyme activity. The inhibitory effect of proteasomal inhibition on adrenergic-induced Aanat transcription appears specific for Aanat because it has no effect on the adrenergic induction of mitogen-activated protein kinase phosphatase-1 (mkp-1). The effects of the proteasome inhibitors on NE-stimulated Aanat induction appear to be mediated by accumulation of a protein repressor.

Role of protein turnover in the activation of p38 mitogen-activated protein kinase in rat pinealocytes.

Differences in the time profiles of activation between p38MAPK and p42/44MAPK by norepinephrine (NE) in rat pinealocytes suggest involvement of mechanisms other than the phosphorylation cascades in their activation. In the present study we investigated whether protein turnover played a role in regulating p38MAPK activation in the rat pineal gland. NE stimulation caused an increase in MAPK kinase3/6 (MKK 3/6) and p38MAPK phosphorylation that occurred in the absence of changes in the mRNA or protein levels of p38MAPK or MKK3/6. The stimulatory effect of NE on phosphorylated MKK3/6 and p38MAPK, but not phosphorylated p42/44MAPK, was blocked by treatment with actinomycin or cycloheximide, indicating a requirement of transcription and translation in activation of the p38MAPK but not the p42/44MAPK pathway. Moreover, inhibition of proteasomes by clasto-lactacystin beta-lactone or Z-Leu-Leu-Leu-CHO (MG132) selectively increased basal and NE-stimulated phosphorylated MKK3/6 and p38MAPK levels without affecting the mRNA or protein levels of MKK3 or p38MAPK. In contrast, the effect of proteasomal inhibition on NE-stimulated p42/44MAPK phosphorylation was inhibitory. Treatment with MG132 also reduced the decline in the phosphorylated levels of NE-stimulated MKK3/6 and p38MAPK that normally follows beta-adrenergic blockade. Together, our results indicate that p38MAPK but not p42/44MAPK activation in the rat pineal gland is tightly coupled to protein synthesis and degradation. The synthesis of an activator upstream of MKK3/6 is required for the NE-activation of p38MAPK.

Mechanistic studies on the alkyltransferase activity of serotonin N-acetyltransferase.

BACKGROUND: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) catalyzes the first, rate-limiting step in the biosynthesis of the circadian hormone melatonin (5-methoxy-N-acetyltryptamine) from serotonin. Our recent discovery that, in addition to catalyzing the acetyl transfer from acetyl-coenzyme A (acetyl-CoASH) to serotonin, AANAT is also a robust catalyst for the alkyl transfer reaction between CoASH and N-bromoacetyltryptamine has not only opened up a new way to develop cell-permeable AANAT acetyltransferase inhibitors that are valuable in vivo tools in helping elucidate melatonin's (patho)physiological roles, but has also raised a question - how does AANAT accelerate the alkyl transfer reaction? In this study, mechanistic aspects of the AANAT-catalyzed alkyl transfer reaction were explored by employing CoASH and a series of N-haloacetyltryptamines that were also evaluated for their AANAT acetyltransferase inhibitory activities. RESULTS: Investigation of various N-haloacetyltryptamine analogs showed a similar leaving group effect on the enzymatic and non-enzymatic reaction rates. Steady-state kinetic analyses demonstrated that AANAT alkyltransferase obeys a sequential, ternary complex mechanism, with random substrate binding. Rate versus pH profiles revealed the catalytic importance of an ionizable group with pK(a) of approximately 7. All those N-haloacetyltryptamines that serve as substrates of AANAT alkyltransferase are also potent (low micromolar) in vitro inhibitors against AANAT acetyltransferase activity. In particular, N-chloroacetyltryptamine was also shown to be a potent inhibitor of intracellular melatonin production in a pineal cell culture assay. CONCLUSIONS: This is the first detailed investigation of the alkyltransferase activity associated with an acetyltransferase. Our results indicate that AANAT does not accelerate the alkyl transfer reaction by simple approximation effect as previously proposed for the similar alkyl transfer reaction catalyzed by other acyltransferases. This study has general implications for developing novel inhibitors by taking advantage of the promiscuous alkyltransferase activity associated with several acyltransferases.

Evidence for the simultaneous translocation of muscarinic acetylcholine receptor and G protein by carbachol.

Interactions between guanine nucleotide regulatory proteins (G proteins) and muscarinic acetylcholine receptors (mAChRs) were studied in vivo following carbachol treatment. Rat brain homogenates were separated by high speed ultracentrifugation into heavy and light membrane and 300,000 g supernate fractions. The G proteins were partially purified by Sephadex-G200 and heptylamine-Sepharose and the mAChRs by (3,2'-aminobenzhydryloxy)-tropane-(ABT)-affinity chromatographies. Radioligand binding assays showed that acute carbachol induced a biphasic translocation of the mAChRs and G proteins into the light membrane fraction with an initial release at 5-10 min and a second phase at 60 min. Portions of the released mAChRs and the G proteins, were found in the 300,000 g supernates and light membranes and were eluted in the same peak fractions from a Sephadex G-200 column. This dually labelled peak dissociated in the presence of digitonin, suggesting close association between the mAChR and G protein. ABT-affinity chromatography yielded dually labelled mAChR-G protein fractions which eluted as a single radioactive peak on a second ABT column. The partially purified G proteins from these fractions were photoaffinity labelled with 8-azidoguanosine-5'-triphosphate, [gamma-32P]. SDS-PAGE autoradiography revealed the presence of Go alpha and Gi alpha which may be released simultaneously with the mAChRs from the plasma membrane. In addition, 110,000 molecular weight polypeptide was dually labelled by [3H]-PrBCM and [gamma-32P]-8-azido-GTP suggesting the presence of a "mAChR-G protein complex." These findings provide direct evidence for the release of mAChRs and G proteins and a mAChR-G protein complex by agonist occupation of the mAChRs.

Phosphorylation-dephosphorylation of muscarinic acetylcholine receptors: evidence for the in vivo and in vitro release of receptors from rat brain plasma membrane.

The effects of phosphorylation on muscarinic acetylcholine receptors (mAChRs) were studied in vitro and in vivo using rat brain plasma membrane and receptors partially purified at least 2500-fold. Purified mAChRs were phosphorylated in vitro by cAMP-dependent protein kinase and dephosphorylated by calcineurin. Phosphorylation of purified mAChRs was enhanced by carbachol and blocked by atropine. The filtrate which passed through glass fiber filters and high speed supernates were assayed for mAChRs by an ammonium sulfate precipitation method. Following incubation of the plasma membrane under phosphorylating conditions and ultracentrifugation at 300,000 g, the mAChRs appeared in the high speed supernate. This release was stimulated by adding carbachol to the incubation medium. In rats treated with carbachol, brain mAChRs redistributed from the heavy into the light membrane fractions. Ultrastructural examination of the light membrane fractions and the 300,000 g supernatant fractions after in vivo and in vitro carbachol treatment calcineurin increased the reincorporation of added partially purified receptors into the plasma membrane. The release and reincorporation of mAChRs strongly imply that there is a translocation and recycling of mAChRs between plasma membrane and cytosol in vivo. The significance and the function of the translocation of mAChRs remain to be investigated.

The adrenergic-regulated CRTC1 and CRTC2 phosphorylation and cellular distribution is independent of endogenous SIK1 in the male rat pinealocyte.

Salt inducible kinase 1 (SIK1) has been reported to repress cAMP-response element binding protein (CREB)-mediated gene transcription by causing the nuclear export of CREB-regulated transcription coactivators (CRTCs) through phosphorylation. Although the repressor role of SIK1 in suppressing the expression of arylalkylamine N-acetyltransferase, the enzyme that controls the daily rhythm in melatonin production in the rat pineal gland, has been established, whether SIK1 regulates the phosphorylation and localization of CRTC1 and CRTC2 in this tissue remains unclear. The present study found that overexpressing SIK1 in NE-stimulated rat pinealocytes could increase the phosphorylation of CRTC1 and CRTC2, reduced selectively the nuclear level of CRTC2 (but not that of CRTC1), and elevated the cytosolic levels of both CRTC1 and CRTC2. In contrast, transient knockdown of endogenous SIK1 had no effect on the phosphorylation or distribution of CRTC1 and CRTC2 in norepinephrine (NE)-stimulated pinealocytes. Our results also showed that adrenergic blockade during NE stimulation led to a rapid rephosphorylation and decline in the nucleus levels of CRTC1 and CRTC2; however SIK1 knockdown had no effect on this rapid rephosphorylation. Moreover, studies with kinase inhibitors revealed that kinase(s) sensitive to KT5823 appeared to be involved in this rapid rephosphorylation. Together, these results indicate that although overexpressing SIK1 can phosphorylate CRTC1 and CRTC2 in the NE-stimulated pinealocyte, the endogenous SIK1, in spite of its induction by NE, does not appear to be the main regulator of the phosphorylation and intracellular localization of these two coactivators.

24,25(OH)2 vitamin D3 modulates the L-type Ca2+ channel current in UMR 106 cells: involvement of protein kinase A and protein kinase C.

In this study, the effect of 24,25(OH)2 vitamin D3 (24,25D3), on the L-type Ca2+ channel current (L-channel current) in UMR 106 cells was investigated using the whole cell version of the patch clamp technique. It was found that 24,25D3 had a dual effect on the L-channel current: a low concentration of 24,25D3 (1 x 10(-8) M) increased the amplitude of the L-channel current by 49 +/- 11%, whereas a high concentration of 24,25D3 (1 x 10(-5) M) reduced the amplitude of the current by 55 +/- 7%. The effect of a low concentration of 24,25D3 was mimicked by 8-bromo-cAMP and inhibited by Rp-cAMPs, indicating the involvement of the cAMP/protein kinase A pathway. In contrast, the effect of a high concentration of 24,25D3 was mimicked by 4 beta-phorbol 12-myristate 13-acetate and inhibited by calphostin C, indicating the involvement of protein kinase C. In comparison, a high concentration of 1,25(OH)2 vitamin D3 (1,25D3) (1 x 10(-6) M) increased the L-channel current in UMR 106 cells. Therefore, 24,25D3 appears to have an action on the L-channel current that is distinct from that of 1,25D3. This demonstration of a non-genomic effect of 24,25D3 on calcium channels suggests that 24,25D3 is an active metabolite of vitamin D3 and may play an important role in regulating the function of bone cells.