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The amplitude and duration of Ca2+ signaling is crucial for B-cell development and self-tolerance; however, the mechanisms for terminating Ca2+ signals in B cells have not been determined. In lymphocytes, plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 (PMCA1 and PMCA4, aka ATP2B1 and ATP2B4) are the main candidates for expelling Ca2+ from the cell through the plasma membrane. We report here that Pmca4 (Atp2b4) KO mice had normal B-cell development, while mice with a conditional KO of Pmca1 (Atp2b1) had greatly reduced numbers of B cells, particularly splenic follicular B cells, marginal zone B cells, and peritoneal B-1a cells. Mouse and naive human B cells showed only PMCA1 expression and no PMCA4 by western blot, in contrast to T cells, which did express PMCA4. Calcium handling was normal in Pmca4-/- B cells, but Pmca1 KO B cells had elevated basal levels of Ca2+ , elevated levels in ER stores, and reduced Ca2+ clearance. These findings show that the PMCA1 isoform alone is required to ensure normal B-cell Ca2+ signaling and development, which may have implications for therapeutic targeting of PMCAs and Ca2+ in B cells.

The outcome of T cell activation is determined by mechanisms that balance Ca2+ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn−/−), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn−/− T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn−/− T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn−/− T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.

Adaptation to stress is a basic phenomenon in mammalian life that is mandatorily associated with the activity of the hypothalamic–pituitary–adrenal (HPA) axis. An increased resting activity of the HPA axis can be measured during pregnancy and lactation, suggesting that these reproductive states lead to chronic load in females. In this study, we examined the consequences of the congenital lack of vasopressin on the activity of the HPA axis during lactation using vasopressin-deficient Brattleboro rats. Virgin and lactating, homozygous vasopressin-deficient rats were compared with control, heterozygous rats. In control dams compared with virgins, physiological changes similar to those observed in a chronic stress state (thymus involution, adrenal gland hyperplasia, elevation of proopiomelanocortin mRNA levels in the adenohypophysis, and resting plasma corticosterone levels) were observed. In vasopressin-deficient dams, adrenal gland hyperplasia and resting corticosterone level elevations were not observed. Corticotropin-releasing hormone (Crh) mRNA levels in the hypothalamic paraventricular nucleus were elevated in only the control dams, while oxytocin (OT) mRNA levels were higher in vasopressin-deficient virgins and lactation induced a further increase in both the genotypes. Suckling-induced ACTH and corticosterone level elevations were blunted in vasopressin-deficient dams. Anaphylactoid reaction (i.v. egg white) and insulin-induced hypoglycemia stimulated the HPA axis, which were blunted in lactating rats compared with the virgins and in vasopressin-deficient rats compared with the controls without interaction of the two factors. Vasopressin seems to contribute to the physiological changes observed during lactation mimicking a chronic stress state, but its role in acute HPA axis regulation during lactation seems to be similar to that observed in virgins. If vasopressin is congenitally absent, OT, but not the CRH, compensates for the missing vasopressin; however, the functional restitution remains incomplete.

Human Molecular Genetics, 21 (2), ISSN:0964-6906, ISSN:1460-2083

This study was undertaken to examine the importance of nitric oxide (NO) generated by the neural isoform of the nitric oxide synthase (nNOS) on the activity of the hypothalamic neurohypophyseal system in neural nitric oxide synthase knock-out (KO) and wild-type (WT) mice under basal conditions and in response to forced swimming. The intensity of the hybridisation signal for vasopressin (AVP) in the hypothalamic supraoptic nucleus (SON) was significantly higher in KO mice when compared with WT, whereas oxytocin (OXT) basal mRNA levels were similar in both groups. Although the basal peripheral release of AVP and OXT was equivalent in both genotypes, we observed in KO mice a significant drop of AVP and OXT plasma values 15 min after stressor onset and a robust increase in the OXT plasma concentration at 60 min. These findings suggest that in the male mouse, NO inhibits AVP gene transcription in magnocellular neurones of the SON and collaborates in maintaining constant AVP and OXT plasma levels following acute stressor exposure, exerting a bimodal regulatory action on OXT secretion. We conclude that NO is involved in the regulation of magnocellular neurones of the SON, and it is preferentially implicated in the attenuation of the peripheral release of OXT induced by acute stressor exposure.

Using antibodies against synaptic protein preparations, we cloned the cDNA of a new Ca2+-binding protein. Its C-terminal portion displays significant similarity with calmodulin and contains two EF-hand motifs. The corresponding mRNA is highly expressed in rat brain, primarily in cerebral cortex, hippocampus, and cerebellum; its expression appears to be restricted to neurons. Transcript levels increase during postnatal development. A recombinant C-terminal protein fragment binds Ca2+ as indicated by a Ca2+-induced mobility shift in SDS-polyacrylamide gel electrophoresis. Antisera generated against the bacterial fusion protein recognize a brain-specific protein doublet with apparent molecular masses of 33 and 36 kDa. These data are confirmed by in vitro translation, which generates a single 36-kDa polypeptide, and by the heterologous expression in 293 cells, which yields a 33/36-kDa doublet comparable to that found in brain. On two-dimensional gels, the 33-kDa band separates into a chain of spots plausibly due to differential phosphorylation. This view is supported by in situ phosphorylation studies in hippocampal slices. Most of the immunoreactivity is detectable in cytoskeletal preparations with a further enrichment in the synapse-associated cytomatrix. These biochemical data, together with the ultra-structural localization in dendrites and the postsynaptic density, strongly suggest an association with the somato-dendritic cytoskeleton. Therefore, this novel Ca2+-binding protein was named caldendrin.

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals.

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.

The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopyin siturevealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.

Glycoproteins gp65 and gp55 are major components of synaptic membranes prepared from rat forebrain. Both are recognized by the monoclonal antibody SMgp65. We have used SMgp65 to screen a rat brain cDNA expression library. Two sets of overlapping cDNAs that contain open reading frames of 397 and 281 amino acids were isolated. The deduced proteins are members of the immunoglobulin (Ig) superfamily containing three and two Ig domains, respectively. The common part has approximately 40% sequence identity with neurothelin/basigin. The identity of the proteins as gp65 and gp55 was confirmed by production of new antisera against a common recombinant protein fragment. These antisera immunoprecipitate gp65 and gp55. Furthermore, expression of gp65 and gp55 cDNAs in human 293 cells treated with tunicamycin results in the production of unglycosylated core proteins of identical size to deglycosylated gp65 and gp55. Northern analysis revealed that gp65 transcripts are brain-specific, whereas gp55 is expressed in most tissues and cell lines examined. The tissue distribution was confirmed at the protein level though the pattern of glycosylation of gp55 varies between tissues. In situ hybridization experiments with a common and a gp65-specific probe suggest differential expression of gp65 and gp55 transcripts in the rat brain.

To investigate the expression of isoforms of the cyclic AMP (cAMP)-dependent protein kinase (cAK) in the transition zone of the human prostate and the functional significance of the enzyme in the control of prostate smooth muscle.Using Western blot analysis and immunohistochemistry, the expression and distribution in the prostate of cAKIalpha, cAKIbeta, cAKIIalpha, and cAKIIbeta in relation to alpha-actin and the phosphodiesterase PDE4 (types A and B) were investigated. The effects of the cAK inhibitor Rp-8-CPT-cAMPS on the reversion of the adrenergic tension of isolated prostate tissue induced by forskolin, rolipram, sodium nitroprusside (SNP), and tadalafil were examined by means of the organ bath technique.Immunosignals specific for cAKIalpha, cAKIIalpha, and cAKIIbeta were observed in the smooth musculature and glandular structures of the prostate. Double stainings revealed the colocalization of alpha-actin and PDE4 with the cAK isoforms. The expression of the cAK isoforms was confirmed by Western blot analysis. The relaxation of the tension induced by norepinephrine brought about by forskolin, rolipram, SNP, and tadalafil was significantly attenuated by Rp-8-CPT-cAMPS.The colocalization of smooth muscle alpha-actin and PDE4 with cAK, as well as the results from the organ bath experiments, provide further evidence for a pivotal role of the cAMP-dependent signaling in the regulation of prostate smooth muscle contractility. Compounds interacting with the cAMP/cAK pathway might represent a new therapeutic avenue to treat symptoms of benign prostatic hyperplasia and lower urinary tract symptomatology.