Effect of the antipsychotic agent amisulpride on glucose lowering and insulin secretion.

AIMS: To investigate the effects of the second generation antipsychotic (R/S)-amisulpride, and the chirally purified enantiomers, on glucose homeostasis in diet-induced obese (DIO) mice. METHODS: Normal and DIO mice were treated with pharmacologically relevant doses of amisulpride prior to oral glucose tolerance tests (OGTTs). Blood glucose, insulin, glucagon-like peptide-1, prolactin and amisulpride drug levels were determined. RESULTS: Racemic amisulpride significantly reduced glucose excursions during OGTT in both normal and DIO mice. This potent effect was preserved with the 'off-isomer', R-amisulpride (ED(50) 1 mg/kg). Insulin secretion was significantly increased with R-amisulpride with only a minor increase in prolactin levels. CONCLUSIONS: Amisulpride has antidiabetic actions in DIO mice resulting from increased insulin secretion. This provides some explanation for why amisulpride, unlike other atypical antipsychotics, is not diabetogenic in man. Furthermore, the observation that R-amisulpride is also antidiabetic and has minimal impact on prolactin levels presents the opportunity for development of this isomer as an antidiabetic agent.

Differential effects of traumatic brain injury on vesicular acetylcholine transporter and M2 muscarinic receptor mRNA and protein in rat.

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may result from presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and the M2 muscarinic receptor subtype can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2 muscarinic receptor subtype protein and medial septal mRNA levels at 4 weeks following injury. Rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 2.5 to 2.9 mm in depth) and sham surgery. Animals were sacrificed, and coronal sections (35 microm thick) were cut through the dorsal hippocampus for VAChT and M2 immunohistochemistry. Semiquantitative measurements of VAChT and M2 protein in hippocampal homogenates from injured and sham rats were assessed with Western blot analysis. Changes in VAChT and M2 mRNA levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). At 4 weeks after injury, both immunohistochemical and Western blot methods demonstrated an increase in hippocampal VAChT protein. An increase in VAChT mRNA was also observed. Immunohistochemistry demonstrated a loss of M2; however, there was no significant change in M2 mRNA levels in comparison with sham controls. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI through differential transcriptional regulation.

Thrombin induces surface and intracellular secretion of amyloid precursor protein from human endothelial cells.

Thrombin, a major coagulant and inflammatory mediator, was shown to regulate amyloid precursor protein (APP) secretion. APP is the protein from which the amyloid beta peptide (A(beta)) is derived. A(beta) forms the core of vascular and cerebral plaques in Alzheimer's disease (AD). In this study, human umbilical vein endothelial cells (HUVEC) were used to examine the effects of thrombin on APP expression. Cell supernatants from thrombin-treated HUVEC were immunoblotted to measure secreted APP. Thrombin-induced secretion of APP peaks at approximately 30 min post-treatment. Immunohistochemical analysis found that APP is not colocalized with or secreted through the same pathway as coagulation factor VIII. The secretion of APP is thrombin receptor-mediated, since it is inhibited by the thrombin antagonist N-Acetyl-D-Phe-Pro-1-Amido-4-Guanidino-Butyl-1-Boronic Acid. It also is induced by treatment with a calcium ionophore. Moreover, APP secretion is protein kinase C (PKC)-dependent because it is blocked by the PKC inhibitor bisindolylmaleimide. APP secretion also occurs from the cell surface, possibly through direct cleavage by thrombin. Immunoreactivity on the surface of HUVEC decreased after thrombin treatment but not after treatment with a non-proteolytic thrombin receptor activator. These data suggest that thrombin induces APP secretion through a PKC-dependent mechanism, as well as from the cell surface. Our results are consistent with thrombin playing a role in AD pathology.

Chronic effects of traumatic brain injury on hippocampal vesicular acetylcholine transporter and M2 muscarinic receptor protein in rats.

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may be due to presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and M2 receptors can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2 muscarinic subtype receptor protein levels at four time points: 1 day, 1 week, 2 weeks, and 4 weeks following injury. Rats were anesthetized and surgically prepared for controlled cortical impact injury (4 m/s, 2.5- to 2.9-mm depth) and sham surgery. Animals were sacrificed and coronal sections (35 micro(m) thick) were cut through the dorsal hippocampus for VAChT and M2 immunohistochemistry. Semiquantitative measurements of VAChT and M2 protein in hippocampal homogenates from injured and sham rats were assessed using Western blot analysis. Immunohistochemistry showed no obvious changes in VAChT and M2 immunoreactivity at 1 day and 1 week postinjury. At 2 and 4 weeks postinjury, an increase in hippocampal VAChT protein and a corresponding loss of hippocampal M2 protein was observed compared to sham controls. Consistent with these results, Western blot analyses at 4 weeks postinjury demonstrated a 40-50% increase in VAChT and a 25-30% decrease in M2. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI, by upregulating the storage capacity and subsequent release of ACh and downregulating presynaptic inhibitory receptors.

CD5-mediated negative regulation of antigen receptor-induced growth signals in B-1 B cells.

A subset of B lymphocytes present primarily in the peritoneal and pleural cavities is defined by the expression of CD5 and is elevated in autoimmune diseases. Upon signaling through membrane immunoglobulin M (mIgM), splenic B lymphocytes (B-2) proliferate, whereas peritoneal B cells (B-1) undergo apoptosis. However, in CD5-deficient mice, B-1 cells responded to mIgM crosslinking by developing a resistance to apoptosis and entering the cell cycle. In wild-type B-1 cells, prevention of association between CD5 and mIgM rescued their growth response to mIgM crosslinking. Thus the B cell receptor-mediated signaling is negatively regulated by CD5 in normal B-1 cells.

A role for calcitonin gene related peptide (CGRP) in the regulation of early B lymphocyte differentiation.

In previous studies we identified high affinity adenylyl cyclase linked receptors for calcitonin gene related peptide (CGRP) on rat T and B cells, on lymphocyte cell lines including the mouse pre-B cell line 70Z/3, and on cells in mouse bone marrow. The effect of CGRP on early B cell differentiation has been examined using the 70Z/3, and on cells in mouse bone marrow. The effect of CGRP on early B cell differentiation has been examined using the 70Z/3 cell line. CGRP inhibits the lipopolysaccharide (LPS) induction of surface immunoglobulin (sIg) protein expression in 70Z/3 cells, an effect that is associated with a decrease in the steady-state levels of Ig heavy (mu) and light (kappa) chain mRNA. In this report, experiments are described that provide further information on the mechanism by which CGRP inhibits sIg expression. The kinetics of CGRP inhibition of LPS-induced sIg expression was examined in 70Z/3 cells. An optimal window for the inhibitory effect of CGRP on SIg induction occurs at least 24 h after the cells are treated with LPS. To determine whether the inhibitory effects of CGRP on sIg expression are mediated by an inhibition of NF kappa-B translocation to the nucleus, electrophoretic mobility shift assays were performed using nuclear proteins from 70Z/3 cells. There was no difference in NF kappa-B binding activity in cells that had been treated with LPS or LPS + CGRP, suggesting that the inhibitory effect of CGRP is not mediated by an inhibition of NF kappa-B activity. These studies provide further evidence that CGRP plays an inhibitory role in early B cell differentiation. Finally, a model is proposed that describes an integrated role for CGRP in the homeostatic regulation of early B cell differentiation.

Heat shock alters Alzheimer's beta amyloid precursor protein expression in human endothelial cells.

One of the pathological lesions in Alzheimer's disease (AD) is the amyloid or senile plaque. The plaque core is predominantly made up of amyloid beta peptide (A beta), a 42-43 amino acid peptide derived from amyloid precursor protein (APP). APP is a membrane bound glycoprotein which is expressed ubiquitously in many cells. Although normal or pathological functions for APP are not well understood, several observations suggest that APP may play a role in cellular stress and inflammation at the endothelial cell/vascular barrier. APP is found in platelets and endothelial cells, it can inhibit a blood coagulation factor, and secreted APP can be neuroprotective. Changes in expression of APP during cellular stress or inflammation may contribute to pathological deposition of A beta. In the present studies, expression of APP in human endothelial cells was examined following heat shock. In human umbilical vein endothelial cells (HUVECs) exposed to 42 degrees C for 30 min, there was a five- to eight-fold increase in APP mRNA levels which peaked at 4 hr. The increase in APP mRNA was followed by an increase in APP protein immunoreactivity in the cytoplasm in a perinuclear Golgi-like region, and in discrete granular cytoplasmic structures. Immunoblot analysis of APP in the cell media found a transient increase in APP which peaked at 1 hr after heat shock. These results suggest that cellular stress induces the secretion of APP from endothelial cells followed by a subsequent increase in APP mRNA and protein synthesis. The upregulation of APP mRNA and protein supports a cellular stress role for APP.

Characterization of a calcitonin gene-related peptide (CGRP) receptor on mouse bone marrow cells.

Calcitonin gene-related peptide, (CGRP), a vasoactive neuropeptide, is found throughout the peripheral nervous system, and CGRP receptors are present on mature lymphocytes. The current studies describe a CGRP receptor on isolated mouse bone marrow cells. The affinity, distribution and specificity of CGRP receptors were analyzed using radioligand binding assays. [125I]CGRP binding in mouse bone marrow cells was dependent on cell concentration and was stable from 5 to 60 min at room temperature. The average Kd is 3.29 +/- 1.24 nM and the average receptor density is 2796 +/- 365 sites/cell. Competition binding analysis found rat alpha and beta CGRP to be the most inhibitory, (Ki values 0.899 and 0.711 nM, respectively), followed by human alpha CGRP and the antagonist human CGRP8-37. The neuropeptides human and salmon calcitonin did not inhibit [125I]CGRP binding to bone marrow cells. The presence of CGRP receptors on mouse bone marrow cells provides further evidence for a direct role for CGRP in modulating the function and differentiation of cellular components of the immune and inflammatory systems.

Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). I. Characterization of high-affinity CGRP receptors on murine 70Z/3 cells.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide found in both peripheral and central neurons and in certain endocrine tissues. Previous studies have identified specific high-affinity CGRP receptors on normal T and B cells and have described modulatory effects of CGRP on freshly isolated lymphocytes. In these studies several lymphocyte cell lines were screened for 125I-CGRP binding. One cell line, the murine 70Z/3 pre-B cell line, was found to exhibit a high level of 125I-CGRP binding and was used for further characterization of the lymphocyte CGRP receptor. Several protease inhibitors were evaluated for their ability to stabilize 125I-CGRP binding to 70Z/3 cells. Bacitracin, a relatively nonspecific protease inhibitor, and chymostatin, a cysteine protease inhibitor, enhanced 125I-CGRP binding, suggesting that 70Z/3 cells express a protease capable of inactivating CGRP. 125I-CGRP binding had a linear dependence on cell concentration with binding being detectable with as few as 200,000 cells/ml and was rapid, with equilibrium binding being reached by 30 min. Saturation binding studies demonstrated that the 70Z/3 CGRP receptor has both low- and high-affinity binding states, with Kds of approximately 0.2 and 3.3 nM, respectively. The density of the low-affinity binding site was approximately 20,000 sites/cell and was unchanged following LPS treatment. In contrast, LPS treatment for 48 hr induced a fourfold increase in the density of the high-affinity site, from 105 to 401 sites/cell. In competition binding studies, only CGRP and the CGRP antagonist CGRP8-37 inhibited 125I-CGRP binding, whereas the unrelated neuropeptides calcitonin, SP, and CRH had no effect. Inhibition of 125I-CGRP binding by rat CGRP was best described by a two-site model, with Kis of 44.1 pM and 0.6 nM. Inhibition of binding by human CGRP and CGRP8-37 was best described by a one-site model with Kis of 3.92 and 6.50 nM, respectively. The 70Z/3 CGRP receptor protein was biochemically characterized by affinity labeling the receptor protein with 125I-CGRP and the covalent cross-linking reagents DSS or BS. SDS-PAGE analysis revealed a major band of 103,000 MW. In addition a high-molecular-weight complex which did not penetrate the gel was also observed. The presence of CGRP receptors on 70Z/3 cells provides further evidence for the immunomodulatory role for CGRP and provides a model system for studying the cellular mechanisms of action of CGRP in modulating lymphocyte differentiation and function.

Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). II. Inhibition of LPS-induced kappa light chain expression by CGRP.

The presence of calcitonin gene-related peptide (CGRP) in nerve endings in lymphoid organs, around blood vessels, and in bone marrow suggests that it might influence the function and differentiation of lymphoid cells. Previous studies identified specific CGRP receptors on mature T and B lymphocytes and on 70Z/3 pre-B cells. In these studies, it was found that CGRP stimulated a rapid, prolonged elevation of cAMP with in 70Z/3 cells with an ED50 of approximately 20 fM. Following CGRP treatment, cAMP levels peaked within 5 min and were still elevated after 60 min. The effect of CGRP on surface immunoglobulin (sIg) expression was examined by treating 70Z/3 cells with CGRP, or combinations of CGRP and LPS, and then measuring sIg expression by FACS. When 70Z/3 cells were treated with LPS, CGRP, or calcitonin for 48 hr, only LPS induced the expression of sIg, increasing the percentage of cells expressing sIg from less than 10% positive in untreated cells to 80-98% positive. Subsequent experiments examined the effect of CGRP on LPS-induced sIg. CGRP inhibited LPS-induced sIg expression at concentrations ranging from 10(-15) to 10(-7) M. The maximal inhibition was observed at CGRP concentrations ranging from 10(-10) to 10(-8) M, and the maximal reduction of sIg expression was about 40%. The inhibitory effect of CGRP was specific in that it could not be mimicked by calcitonin and could be blocked by the CGRP receptor antagonist CGRP8-37. A similar dose-dependent inhibitory effect on LPS induction was observed in 70Z/3 cells treated with LPS and dibutyryl cAMP, suggesting that the inhibitory effect of CGRP on sIg expression is mediated by stimulation of intracellular cAMP. The inhibitory effect of CGRP on LPS induction of sIg appears to be mediated by a reduction in the expression of both mu and kappa mRNA. When mu and kappa expression were examined by Northern blot analysis, it was found that CGRP caused a 50% reduction in the amount of mu and kappa mRNA induced by LPS. The ability of CGRP to inhibit differentiation of 70Z/3 cells and sIg expression provides evidence that CGRP can influence the differentiation of lymphopoietic precursors.

Boron neutron capture therapy of a murine melanoma with p-boronophenylalanine: dose-response analysis using a morbidity index.

Boron-10 concentrations of 20 or 40 micrograms/g were attained in mouse B16 melanomas following one or two intragastric doses of p-boronophenylalanine (750 mg/kg body weight per dose), respectively. Tumor-to-normal-tissue (blood, muscle) boron concentration ratios were 4:1-6:1. The efficacy of boron neutron capture irradiation was monitored using the Wilcoxon two-sample test in conjunction with a system of ranking outcomes of different therapies that compared living mice and mice sacrificed because of excessive tumor growth concomitantly. Median survivals were extended progressively as radiation doses were increased up to 38.7 gray-equivalent (gray X relative biological effectiveness), with one of five and one of six tumors cured in each of the two highest dose groups, respectively. When comparable tumor inhibitory doses of 250-kVp X rays were used to treat these tumors, instead of the transient erythema and edema that resulted from boron neutron capture therapy, there resulted irreversible muscle necrosis in the irradiated zone and atrophy of the foot distal to the irradiated zone. The improvement in treatment outcome with boron neutron capture therapy is attributable to unprecedented tumor-to-normal-tissue radiation dose ratios of approximately 2.8 to 3.6.