Recently several groups have reported the existence of D-serine in serum (2), urine (3), and brain (4, 5) of mammals, including humans (6). Hashimoto et at (7) showed with biochemical techniques that the distribution of D-serine in rat brain regions parallels the distribution of N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors. This is of interest because stimulation of NMDA receptors by glutamate requires the binding of glycine (8,9), and D-serine mimics glycine at these sites (10).To ascertain whether endogenous D-serine plays a physiologic role in neurotransmission, we have developed polyclonal antibodies that are selective for D-serine. We now report a localization of D-serine throughout rat brain that closely parallels NMDA receptors and is concentrated in gray-matter astrocytes of the telencephalon. We also demonstrate that D-serine is released from type 2 astrocyte cultures stimulated with agonists of non-NMDA glutamate receptors. MATERIALS AND METHODSMaterials. D-[3H]Serine (22 Ci/mmol; 1 Ci = 37 GBq) was provided by Steven Hurt (New England Nuclear). Antibody to glial fibrillary acidic protein (GFAP) was from Dako, and antibody to glutamine synthetase was from Chemicon. Glutaraldehyde was from EM sciences. Gold chloride was from Aldrich. Alkaline phosphatase-coupled anti-rabbit antibody was from The Jackson Laboratories. The peroxidase Elite staining kit was from Vector Laboratories. Glutamate receptor drugs were from Research Biochemicals (Natick, MA). CellThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. culture reagents were from GIBCO/BRL. All other reagents were from Sigma.Preparation of Polyclonal Antiserum to D-Serine. D-Serine was coupled to bovine serum albumin (BSA) with glutaraldehyde and then reduced with NaBH4 (11). After extensive dialysis against water, the conjugate was adsorbed to freshly prepared 45-nm colloidal gold particles (12). A rabbit was immunized intradermally every 3 weeks with the BSA conjugate alone and intravenously with the gold particles. Before use, all D-serine antiserum used in this study was incubated for 2 hr at room temperature with Sepharose beads coupled to glutaraldehyde-treated BSA, to eliminate antibodies not selective for D-serine (13). Liquid-phase conjugates of various amino acids to glutaraldehyde were prepared identically to the original immunogen, except that BSA was omitted and free aldehyde groups were blocked with excess Tris. For dot blot screens, various amino acids were coupled to dialyzed rat brain cytosol with glutaraldehyde as described above for D-serine/ BSA conjugates and then spotted on nitrocellulose. After overnight incubation with the primary antiserum, blots were visualized with an alkaline phosphatase-coupled anti-rabbit secondary antibody. High-affinity stereoselective antibodies appeared after 5 months of immunization.Immunohistochemistry. Anesthetized rats (age, 45-...

Following the discovery of inositol-1,4,5-trisphosphate as a second messenger, many other inositol phosphates were discovered in quick succession, with some understanding of their synthesis pathways and a few guesses at their possible functions. But then it all seemed to go comparatively quiet, with an explosion of interest in the inositol lipids. Now the water-soluble phase is once again becoming a focus of interest. Old and new data point to a new vista of inositol phosphates, with functions in many diverse aspects of cell biology, such as ion-channel physiology, membrane dynamics and nuclear signalling.

D-Serine is localized in mammalian brain to a discrete population of glial cells near NMDA receptors, suggesting that D-serine is an endogenous agonist of the receptor-associated glycine site. To explore this possibility, we have compared the immunohistochemical localizations of D-serine, glycine, and NMDA receptors in rat brain. In the telencephalon, D-serine is concentrated in protoplasmic astrocytes, which are abundant in neuropil in close vicinity to NMDA receptor 2A/B subunits. Ultrastructural examination of the CA1 region of hippocampus reveals D-serine in the cytosolic matrix of astrocytes that ensheath neurons and blood vessels, whereas NR2A/B is concentrated in dendritic spines. By contrast, glycine immunoreactivity in telencephalon is the lowest in brain. During postnatal week 2, D-serine levels in cerebellum are comparable to those in adult cerebral cortex but fall to undetectable levels by day 26.During week 2, we observe parallel ontogeny of D-serine in Bergmann glia and NR2A/B in Purkinje cells, suggesting a role for astrocytic D-serine in NMDA receptor-mediated synaptogenesis. D-Serine in the radial processes of Bergmann glia is also well positioned to regulate NMDA receptor-dependent granule cell migration. In the inner granule layer, D-serine is found transiently in protoplasmic astrocytes surrounding glomeruli, where it could regulate development of the mossy fiber/granule cell synapse. D-Serine seems to be the endogenous ligand of glycine sites in the telencephalon and developing cerebellum, whereas glycine predominates in the adult cerebellum, olfactory bulb, and hindbrain. Key words: D-serine; glycine; NMDA receptor; glia; D-amino acid; cerebellumActivation of NMDA receptor channels requires both glutamate and stimulation of a "glycine site" (Johnson and Ascher, 1987;Reynolds et al., 1987;Kemp and Leeson, 1993). Neurophysiological studies of expressed NMDA receptors indicate that with certain combinations of NR1 and NR2 subunits, D-serine is up to three times more potent than glycine at the glycine site (Matsui et al., 1995;Priestley et al., 1995). Although D-amino acids have long been known to exist in bacteria, worms, and insects (Corrigan, 1969), only very recently have high levels of D-serine been demonstrated in mammalian tissues, especially in the brain (Hashimoto et al., 1992a(Hashimoto et al., , 1993aNagata, 1992;Chouinard et al., 1993;Nagata et al., 1994).We have mapped D-serine immunohistochemically in rat brain and observed a pattern that parallels the localization of D-serine binding sites associated with NMDA receptors in the forebrain (Schell et al., 1995). D-Serine is concentrated in gray matter regions enriched in NMDA receptors and is selectively localized to protoplasmic astrocytes. We have also demonstrated that agonists of non-NMDA receptors enhance the efflux of preloaded D-serine from cultures of cortical type 2 astrocytes. These data suggest that D-serine is an endogenous ligand at the glycine site of many NMDA receptors and that glutamate releases D-serine from glial ...

Purpose: Prostate cancer eventually recurs during androgen deprivation therapy despite castrate levels of serum androgens. Expression of androgen receptor and androgen receptor^regulated proteins suggests androgen receptor activation in recurrent prostate cancer. Many groups have pursued mechanisms of ligand-independent androgen receptor activation but we found high levels of testicular androgens in recurrent prostate cancer tissue using RIA. Experimental Designs: Prostate specimens from 36 men were procured preserving blood flow to prevent ischemia and cyropreserved immediately. Recurrent prostate cancer specimens from18 men whose cancer recurred locally during androgen deprivation therapy and androgen-stimulated benign prostate specimens from 18 men receiving no hormonal treatments were studied. Tissue levels of testosterone and dihydrotestosterone were measured in each specimen using liquid chromatography/electrospray tandem mass spectrometry.Testosterone and dihydrotestosterone levels were compared with clinical variables and treatment received. Results: Testosterone levels were similar in recurrent prostate cancer (3.75 pmol/g tissue) and androgen-stimulated benign prostate (2.75 pmol/g tissue,Wilcoxon two-sided, P = 0.30). Dihydrotestosterone levels decreased 91% in recurrent prostate cancer (1.25 pmol/g tissue) compared with androgen-stimulated benign prostate (13.7 pmol/g tissue; Wilcoxon two-sided, P < 0.0001) although dihydrotestosterone levels in most specimens of recurrent prostate cancer were sufficient for androgen receptor activation.Testosterone or dihydrotestosterone levels were not related to metastatic status, antiandrogen treatment, or survival (Wilcoxon rank sum, all P > 0.2).Conclusions: Recurrent prostate cancer may develop the capacity to biosynthesize testicular androgens from adrenal androgens or cholesterol. This surprising finding suggests intracrine production of dihydrotestosterone and should be exploited for novel treatment of recurrent prostate cancer.Prostate cancer recurs in almost all men receiving androgen deprivation therapy (ADT) and is the main cause of death in prostate cancer. Recurrent prostate cancer retains androgen receptor protein expression, with androgen receptor remaining active in growth signaling despite castrate levels of circulating androgens (1). Androgen receptor protein and androgen receptor -regulated proteins are expressed in prostate cancer that recurs during ADT in both the primary (2 -5) and bone metastases (6, 7). Androgen receptor activation in recurrent prostate cancer may occur by a variety of mechanisms that alter the sensitivity (8 -10) or specificity (11) of androgen receptor (reviewed in refs. 12 -14). Recent studies using androgenindependent prostate cancer cell lines (8, 9) and xenografts (9) showed that androgen receptor overexpression allowed recurrent prostate cancer growth in the presence of castrate levels of circulating androgens. However, androgen receptor mutations that prevented ligand-binding prevented recurrent growth; ove...