MQN-Mapplet: Visualization of Chemical Space with Interactive Maps of DrugBank, ChEMBL, PubChem, GDB-11, and GDB-13

The MQN-mapplet is a Java application giving access to the structure of small molecules in large databases via color-coded maps of their chemical space. These maps are projections from a 42-dimensional property space defined by 42 integer value descriptors called molecular quantum numbers (MQN), which count different categories of atoms, bonds, polar groups, and topological features and categorize molecules by size, rigidity, and polarity. Despite its simplicity, MQN-space is relevant to biological activities. The MQN-mapplet allows localization of any molecule on the color-coded images, visualization of the molecules, and identification of analogs as neighbors on the MQN-map or in the original 42-dimensional MQN-space. No query molecule is necessary to start the exploration, which may be particularly attractive for nonchemists. To our knowledge, this type of interactive exploration tool is unprecedented for very large databases such as PubChem and GDB-13 (almost one billion molecules). The application is freely available for download at www.gdb.unibe.ch.

Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice

High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50 = 290 nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure–activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport.

The SLC1 high-affinity glutamate and neutral amino acid transporter family

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-β-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Illuminating somatostatin analog action at neuroendocrine tumor receptors

Somatostatin analogs for the diagnosis and therapy of neuroendocrine tumors (NETs) have been used in clinical applications for more than two decades. Five somatostatin receptor subtypes have been identified and molecular mechanisms of somatostatin receptor signaling and regulation have been elucidated. These advances increased understanding of the biological role of each somatostatin receptor subtype, their distribution in NETs, as well as agonist-specific regulation of receptor signaling, internalization, and phosphorylation, particularly for the sst2 receptor subtype, which is the primary target of current somatostatin analog therapy for NETs. Various hypotheses exist to explain differences in patient responsiveness to somatostatin analog inhibition of tumor secretion and growth as well as differences in the development of tumor resistance to therapy. In addition, we now have a better understanding of the action of both first generation (octreotide, lanreotide, Octreoscan) and second generation (pasireotide) FDA-approved somatostatin analogs, including the biased agonistic character of some agonists. The increased understanding of somatostatin receptor pharmacology provides new opportunities to design more sophisticated assays to aid the future development of somatostatin analogs with increased efficacy.

Syntheses, receptor bindings, in vitro and in vivo stabilities and biodistributions of DOTA-neurotensin(8-13) derivatives containing β-amino acid residues - a lesson about the importance of animal experiments

Neurotensin(8-13) (NTS(8-13)) analogs with C- and/or N-terminal β-amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1-6). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, 6a, into a crystallographically identified receptor NTSR1 (Fig.1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell-membrane homogenates, while, with NTSR1-exhibiting cancer tissues, affinities in the single-digit nanomolar range can be observed (Table 2). Most of the β-amino acid-containing NTS(8-13) analogs (Table 1 and Fig.2), including the (68) Ga complexes of the DOTA-substituted ones (6; Figs.2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two (68) Ga complexes (of 6a and 6b) in HT29 tumor-bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET-imaging experiments with the tumor-bearing mice (Fig.6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10-15 min) of the two (68) Ga complexes shows that they are rapidly cleaved in the animals (Fig.5).

Peptide receptor radionuclide therapy in a patient with disabling non-functioning pituitary adenoma

Non-functioning pituitary adenoma (NFPA) with higher proliferation index (WHO II) are often a therapeutical challenge. Low somatostatin receptor expression in these tumors usually prevents a treatment with somatostatin analogs. In 1996, a 55-year-old patient was referred due to right-sided headache. A pituitary macroadenoma with infiltration into the right cavernous sinus was diagnosed. There was no visual field deficit and the clinical and biochemical work up was consistent with a NFPA. The patient underwent transsphenoidal surgery. Residual adenoma remained in the right cavernous sinus. Histologically, a null-cell adenoma with a high proliferation index was documented (MIB-1: 11.6 %, WHO II). Somatostatin receptor autoradiography was performed in the surgical specimen showing a homogenous expression of sst2 receptors. Radiosurgery was completed with stable disease for 8 years. In 2004, the patient was diagnosed with an incomplete palsy of the right oculomotorius nerve and a significant increase in the volume of the adenoma in the right cavernous sinus. After a positive Octreoscan(®) the patient consented to an experimental therapy approach using Lutetium DOTATOC (3 × 200 mCi). The palsy of the oculomotorius nerve improved and remained stable until today (March 2013), the follow-up MRI scans demonstrated stable disease. This is the first case of a patient with a NFPA (WHO II) in whom PRRT successfully improved the local complications of the tumor for more than 8 years after ineffective surgery and gamma knife therapy. The determination of sst2 in vitro using autoradiography and in vivo by Octreoscan was instrumental to administer this therapy in a challenging situation.

TEMOZOLOMIDE AND BEVACIZUMAB THERAPY IN ADVANCED HEMANGIOPERICYTOMA/ SOLITARY FIBROUS TUMOR

Tumors comprising the spectrum of hemangiopericytoma/ malignant solitary fibrous tumor (HPC/SFT) are thought to arise from fibroblasts and represent a small subset of soft tissue sarcomas. Surgery is typically the treatment of choice for localized disease, with reported 10-year overall survival rates of 54-89% after complete surgical resection. However, for the approximately 20% of HPC/SFT patients who eventually develop local recurrences and/or distant metastases, options for effective treatment are limited and are poorly defined. Alternative therapeutic options are therefore needed for improved palliation and disease control. We hypothesize that HPC/SFT are a spectrum of soft tissue tumors with unique clinical, pathological, and molecular makeup and clinical behavior. HPC/SFT respond to unique therapeutic agents that specifically target aberrations specific to these tumors. We retrospectively reviewed the characteristics and the clinical outcomes for all HPC/SFT patients whose tumor specimens have been reviewed at the MD Anderson Cancer Center from January 1993 to June 2007 by a MD Anderson pathologist and were treated at the institution with available electronic medical records. We identified 128 patients, 79 with primary localized disease and 49 with recurrent and/or metastatic disease. For the 23 patients with advanced HPC/SFT who received adriamycin-based, gemcitabine based, or paclitaxel chemotherapy as first- or second-line therapy, the overall RECIST response rate was 0%. Most patients achieved a brief duration of disease stabilization on chemotherapy, with median progression-free survival (PFS) period of 4.6 months. For the 14 patients with advanced HPC/SFT who received temozolomide and bevacizumab systemic therapy, the overall RECIST response rate was 14%, with the overall Choi response rate of 79%. The median PFS for the cohort was 9.7 months with a median 6-month progression free rate of 78.6%. The most frequently observed toxic effect of temzolomide-bevacizumab therapy was myelosuppression. We have designed a phase II study to evaluate the safety and efficacy of temozolomide-bevaciumab in locally advanced, recurrent, and metastatic HPC/SFT in a prospective manner. Combination therapy with temozolomide and bevacizumab may be a potentially clinically beneficial regimen for advanced HPC/SFT patients.

Differential uptake of (68)Ga-DOTATOC and (68)Ga-DOTATATE in PET/CT of gastroenteropancreatic neuroendocrine tumors

PURPOSE Abundant expression of somatostatin receptors (sst) is a characteristic of neuroendocrine tumors (NET). Thus, radiolabeled somatostatin analogs have emerged as important tools for both in vivo diagnosis and therapy of NET. The two compounds most often used in functional imaging with positron emission tomography (PET) are (68)Ga-DOTATATE and (68)Ga-DOTATOC. Both analogs share a quite similar sst binding profile. However, the in vitro affinity of (68)Ga-DOTATATE in binding the sst subtype 2 (sst2) is approximately tenfold higher than that of (68)Ga-DOTATOC. This difference may affect their efficiency in detection of NET lesions, as sst2 is the predominant receptor subtype on gastroenteropancreatic NET. We thus compared the diagnostic value of PET/CT with both radiolabeled somatostatin analogs ((68)Ga-DOTATATE and (68)Ga-DOTATOC) in the same patients with gastroenteropancreatic NET. PATIENTS AND METHODS Twenty-seven patients with metastatic gastroenteropancreatic NET underwent (68)Ga-DOTATOC and (68)Ga-DOTATATE PET/CT as part of the workup before prospective peptide receptor radionuclide therapy (PRRT). The performance of both imaging methods was analyzed and compared for detection of individual lesions per patient and for eight defined body regions. A region was regarded as positive if at least one lesion was detected in that region. In addition, radiopeptide uptake in terms of the maximal standardized uptake value (SUV(max)) was compared for concordant lesions and renal parenchyma. RESULTS Fifty-one regions were found positive with both (68)Ga-DOTATATE and (68)Ga-DOTATOC. Overall, however, significantly fewer lesions were detected with (68)Ga-DOTATATE in comparison with (68)Ga-DOTATOC (174 versus 179, p < 0.05). Mean (68)Ga-DOTATATE SUV(max) across all lesions was significantly lower compared with (68)Ga-DOTATOC (16.9 ± 6.8 versus 22.1 ± 12.0, p < 0.01). Mean SUV(max) for renal parenchyma was not significantly different between (68)Ga-DOTATATE and (68)Ga-DOTATOC (12.6 ± 2.6 versus 12.6 ± 2.7). CONCLUSIONS (68)Ga-DOTATOC and (68)Ga-DOTATATE possess similar diagnostic accuracy for detection of gastroenteropancreatic NET lesions (with a potential advantage of (68)Ga-DOTATOC) despite their evident difference in affinity for sst2. Quite unexpectedly, maximal uptake of (68)Ga-DOTATOC tended to be higher than its (68)Ga-DOTATATE counterpart. However, tumor uptake shows high inter- and intraindividual variance with unpredictable preference of one radiopeptide. Thus, our data encourage the application of different sst ligands to enable personalized imaging and therapy of gastroenteropancreatic NET with optimal targeting of tumor receptors.

ATM Signaling to TSC2: Mechanisms and Implications for Cancer Therapy

Ataxia telangiectasia mutated (ATM) is a critical component of the cellular response to DNA damage, where it acts as a damage sensor, and signals to a large network of proteins which execute the important tasks involved in responding to the damage, namely inducing cell cycle checkpoints, inducing DNA repair, modulating transcriptional responses, and regulating cell death pathways if the damage cannot be repaired faithfully. We have now discovered that an additional novel component of this ATM-dependent damage response involves induction of autophagy in response to oxidative stress. In contrast to DNA damage-induced ATM activation however, oxidative stress induced ATM, occurs in the cytoplasm, and does not require nuclear-to-cytoplasmic shuttling of ATM. Using several cell culture systems including MCF7 breast carcinoma cells, SKOV3 ovarian cancer cells, and various lineages of mouse embryonic fibroblasts, we showed that once activated by reactive oxygen species (ROS), ATM signals to mTORC1 to induce autophagy via the LKB1-AMPK-TSC2 pathway. Targeting dysregulation of mTORC1 in Atm-deficient mice, which succumb to lymphomagenesis within 3-4 months of age with daily administration of rapamycin, could significantly extend survival and cause regression of tumors, suggesting that pharmacologically targeting this pathway has therapeutic implications in cancer. We also identified a second contrasting pathway for DNA damage-induced mTORC1 repression which does not require AMPK activation, but does require ATM and TSC2. Several potential mechanisms including mTOR localization and p53-mediated pathways were ruled out however we identified that TSC2 may be an additional cytoplasmic direct ATM substrate that is engaged in response to DNA damage specifically. Lastly, a study was performed to examine whether autophagy induced by ovarian cancer therapeutics (focusing on cisplatin, since paclitaxel does not induce autophagy in the SKOV3 cell line model we used) plays a role in resistance to therapy since autophagy can play both pro-survival mechanisms or be a mechanism of cell death. Using a genetic approach to knock-down Atg5 expression with shRNA in SKOV3 ovarian carcinoma cells, we compared the cytotoxicity of cisplatin in vector or Atg5 knock-down cells, and demonstrated that autophagy does not play any significant role in the response to cisplatin in this cell line.

Structure-function relation in retinoid regulated gene expression

Retinoids are known to inhibit proliferation of and induce terminal differentiation of many normal and transformed cells. It has been postulated that retinoids exert their effect by altering gene expression. HL-60 cells and macrophages both respond to retinoic acid action by the rapid induction of the enzyme tissue transglutaminase. The induction has been shown to be due to increased transcription of the transglutaminase gene. The first part of the dissertation studied the structure-function relationship of retinoid-regulated transglutaminase induction, differentiation and proliferation in HL-60 cells using retinoid analogs. The results indicated strict structural constraints and a strong structure-function correlation between transglutaminase induction and differentiation; those retinoids that induced transglutaminase also induced differentiation, those analogs that did not induce transglutaminase could not induce differentiation. The ability of the retinoids to induce transglutaminase in HL-60 cells was paralleled in macrophages. However, the antiproliferative effect of the retinoids displayed less stringent structural constraints than their differentiation- and transglutaminase-inducing properties. Specifically all the retinoids were able to inhibit proliferation to varying extents. It is concluded that the induction of transglutaminase and of differentiation by retinoids is mediated by receptors. While receptor mediation cannot be entirely ruled out, with the current data no definitive statement can be made about the antiproliferative activity of retinoids. Also, the concordance in the ability of the retinoids to induce transglutaminase and the ability to induce differentiation of HL-60 cells suggests that the former is an early response of the cells to retinoids and differentiation a later consequence on the same pathway. Using the induction of transglutaminase as an index of the direct, or primary, effect of retinoids on gene expression, the second part of the dissertation investigates, by 2D gel electrophoresis, the alteration in the rates of synthesis of other proteins in macrophages and HL-60 cells in response to short incubations with retinoic acid. Any changes in parallel with transglutaminase were taken to indicate proteins directly under the control of retinoic acid. It is concluded that retinoic acid regulates the expression of a circumscribed set of genes in a cell-specific manner. The results support the hypothesis that retinoids exert their multiple effects on myeloid cells, in part, by receptor-mediated alternations in gene expression. ^

Molecular structure of antibodies against small ligands

Antibodies which bind bioactive ligands can serve as a template for the generation of a second antibody which may react with the physiological receptor. This phenomenon of molecular mimicry by antibodies has been described in a variety of systems. In order to understand the chemical and molecular mechanisms involved in these interactions, monoclonal antibodies directed against two pharmacologically active alkaloids, morphine and nicotine, were carefully studied using experimental and theoretical molecular modeling techniques. The molecular characterization of these antibodies involved binding studies with ligand analogs and determination of the variable region amino acid sequence. A three-dimensional model of the anti-morphine binding site was constructed using computational and graphics display techniques. The antibody response in BALB/c mice to morphine appears relatively restricted, in that all of the antibodies examined in this study contained a $\lambda$ light chain, which is normally found in only 5% of mouse immunoglobulins. This study represents the first use of theoretical and experimental modeling techniques to describe the antigen binding site of a mouse Fv region containing a $\lambda$ light chain. The binding site model indicates that a charged glutamic acid residue and aromatic side chains are key features in ionic and hydrophobic interactions with the ligand morphine. A glutamic acid residue is found in the identical position in the anti-nicotine antibody and may play a role in binding nicotine. ^

Characterization of neurotransmitter inputs to cholinergic amacrine cells of the rabbit retina: Analysis of ACh release

The cholinergic amacrine cells of the rabbit retinal are the only neurons which accumulate choline and also synthesize acetylcholine (ACh). It is widely accepted that the physiologically evoked release of acetylcholine can be taken as a measure of the activity of the entire cholinergic population. Initially, we examined the possibility that these cells receive excitatory input via glutamate receptors from glutamatergic neurons. Glutamate analogs were found to cause massive ACh release from the rabbit retina. Glutamate was found to activate several different receptor subtypes. Selective glutamate antagonists were used to separate the responses evoked by the different glutamate receptor subtypes. The kainate receptor was determined pharmacologically to be the subtype activated physiologically. Since bipolar cells make direct contact with cholinergic amacrine cells, our results support the hypothesis the bipolar cell neurotransmitter is glutamate. Although NMDA receptors can be activated by NMDA analogs, they are not activated during the physiologically evoked release of ACh. A separate study examined the possibility that L-homocysteate could be the bipolar cell neurotransmitter and the results placed serious constraints on this possibility.^ GABA$\sb{\rm A}$ agonists and antagonists are known to have powerful effects on ACh release from the rabbit retina. By pharmacologically blocking the excitatory input from bipolar cells, we attempted to determine the site of GABA$\sb{\rm A}$ input. Our results suggest that the predominant site of GABA$\sb{\rm A}$ input is onto the bipolar cells presynaptic to cholinergic amacrine cells. In a separate study, we found SR-95531 to be a potent and selective GABA$\sb{\rm A}$ receptor antagonist. In addition, GABA$\sb{\rm B}$ agonists and antagonists were found to have minor or no effects on ACh release. Glycine was also examined, its inhibitory effects were found to be very similar to GABA$\sb{\rm A}$ agonists. In contrast, strychnine was found to increase basal but inhibit light evoked ACh release. Additional results indicated that the predominant site of glycinergic input is onto the presynaptic bipolar cells. Our results suggest a different role for glycine compared to GABA in shaping the light evoked release of ACh from the rabbit retina. ^

Analysis of endogenous retinoic acid receptor expression and function in murine melanoma cells

There have been numerous reports over the past several years on the ability of vitamin A analogs (retinoids) to modulate cell proliferation, malignant transformation, morphogenesis, and differentiation in a wide variety of cell types and organisms. Two families of nuclear retinoid-inducible, trans-acting, transcription-enhancing receptors that bear strong DNA sequence homology to thyroid and steroid hormone receptors have recently been discovered. The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) each have at least three types designated $\alpha,$ $\beta,$ and $\gamma,$ which are encoded by separate genes and expressed in a tissue and cell type-specific manner. We have been interested in the mechanism by which retinoids inhibit tumor cell proliferation and induce differentiation. As a model system we have employed several murine melanoma cell lines (S91-C2, K1735P, and B16-F1), which are sensitive to the growth-inhibitory and differentiation-inducing effects of RA, as well as a RA-resistant subclone of one of the cell lines (S91-C154), in order to study the role of the nuclear RARs in these effects. The initial phase of this project consisted of the characterization of the expression pattern of the three known RAR and RXR types in the murine melanoma cell lines in order to determine whether any differences exist which may elucidate a role for any of the receptors in RA-induced growth inhibition and differentiation. The novel finding was made that the RAR-$\beta$ gene is rapidly induced from undetectable levels by RA treatment at the mRNA and protein level, and that the induction of RAR-$\beta$ by other biologically active retinoids correlated with their ability to inhibit the growth of the highly RA-sensitive S91-C2 cell line. This suggests a role for RAR-$\beta$ in the growth inhibiting effect of retinoids. The second phase of this project involves the stable expression of RAR-$\beta$ in the S91-C2 cells and the RAR-$\beta$ receptor-null cell line, K1735P. These studies have indicated an inverse correlation between RAR-$\beta$ expression and proliferation rate. ^

Characterization of calcium signaling mechanisms in osteoblasts

Bone remodeling is controlled by the osteoclast, which resorbs bone, and the osteoblast, which synthesizes and secretes proteins that are eventually mineralized into bone. Ca$\sp{2+}$ homeostasis and signaling contribute to the function of nearly all cell types, and understanding both in the osteoblast is of importance given its secretory properties and interaction with osteoclasts. This study was undertaken to identify and investigate the physiology of the Ca$\sp{2+}$ signaling mechanisms present in osteoblasts. The Ca$\sp{2+}$ pumps, stores and channels present in osteoblasts were studied. RT-PCR cloning revealed that osteoblast-like cells express PMCA1b, an alternatively spliced transcript of the plasma membrane Ca$\sp{2+}$-ATPase. The PMCA1b isoform contains a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. The regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca$\sp{2+}$-ATPase.^ Calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, and the compartmentalization and regulated release of calcium is cell-type specific. Fura-2 was employed to monitor intracellular Ca$\sp{2+}$. Thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum Ca$\sp{2+}$-ATPase activity, both emptied a single intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The Ca$\sp{2+}$ storage system present in osteoblasts is typical of a non-excitable cell type, despite these cells sharing characteristics of excitable cells such as voltage-sensitive Ca$\sp{2+}$ channels (VSCCs).^ VSCCs are important cell surface regulators of membrane permeability to Ca$\sp{2+}$. In non-excitable cells VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. Functional VSCCs have been shown to exist in osteoblasts, however, no molecular cloning has been reported. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Sequencing of the products indicated that osteoblasts express at least two isoforms of the L-type VSCC, $\alpha 1\sb{\rm C-a}$ and the $\alpha 1\sb{\rm C-d}$, which share regions of identity to the $\alpha \sb{\rm 1C}$ isoform first identified in cardiac myocytes. The ability of $1,25(\rm OH)\sb2D\sb3$ and structural analogs to modulate expression of Ca$\sp{2+}$ channel mRNA was then investigated. Cells were cultured for 48 hr in the presence of $1,25(\rm OH)\sb2D\sb3$ or vitamin D analogs, and the levels of mRNA encoding VSCC $\alpha \sb{\rm 1C}$ were quantitated using a competitive RT-PCR assay. It was found that $1,25(\rm OH)\sb2D\sb3$ and analog BT reduced steady state levels of $\alpha \sb{\rm 1C}$ mRNA. Conversely, analog AT did not alter steady state levels of Ca$\sp{2+}$ channel mRNA. Since it has been shown previously that analog BT, but not AT, binds and activates the nuclear vitamin D receptor, these findings suggest that the down regulation of channel mRNA involves the nuclear receptor for $1,25(\rm OH)\sb2D\sb3$. ^