Linda D. Mercer

Cholecystokinin Modulates Migration of Gonadotropin-Releasing Hormone-1 Neurons

Expression of the brain–gut peptide cholecystokinin (CCK) in the developing olfactory–gonadotropin-releasing hormone-1 (GnRH-1) neuroendocrine systems was characterized, and the function of CCK in these systems was analyzed both in vivo and in vitro. We present novel data demonstrating that CCK transcript and protein are expressed in sensory cells in the developing olfactory epithelium and vomeronasal organ, with both ligand and receptors (CCK-1R and CCK-2R) found on olfactory axons throughout prenatal development. In addition, migrating GnRH-1 neurons in nasal regions express CCK-1R but not CCK-2R receptors. The role of CCK in olfactory–GnRH-1 system development was evaluated using nasal explants, after assessing that the in vivo expression of both CCK and CCK receptors was mimicked in this in vitro model. Exogenous application of CCK (10-7 m) reduced both olfactory axon outgrowth and migration of GnRH-1 cells. This inhibition was mediated by CCK-1R receptors. Moreover, CCK-1R but not CCK-2R antagonism caused a shift in the location of GnRH-1 neurons, increasing the distance that the cells migrated. GnRH-1 neuronal migration in mice carrying a genetic deletion of either CCK-1R or CCK-2R receptor genes was also analyzed. At embryonic day 14.5, the total number of GnRH-1 cells was identical in wild-type and mutant mice; however, the number of GnRH-1 neurons within forebrain was significantly greater in CCK-1R–/– embryos, consistent with an accelerated migratory process. These results indicate that CCK provides an inhibitory influence on GnRH-1 neuronal migration, contributing to the appropriate entrance of these neuroendocrine cells into the brain, and thus represent the first report of a developmental role for CCK.

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate

SummaryThe possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [3H]L-glutamate ([3H]GLU) and [3H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [3H]Glycine always labelled a single population of sites; densities of binding sites (Bmax) in cortical, cerebellar and “granule” membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (Kd) in the same three preparations were 0.13, 0.31 and 1.9 μM, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [3H]GLU was saturable and to a single population of sites: Kd 0.5–0.9 μM and Bmax 3.2–3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [3H]GLU was always L-aspartate>L-cysteate>L-cysteinesulphinate>L-serine-O-sulphate>ibotenate>L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [3H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations. Although the binding of [3H]GLU was relatively insensitive to NMDA itself and competitive NMDA antagonists, binding may be to a recognition site for NMDA-like agonists, since they fully inhibited specific binding. This excitatory amino acid recognition for NMDA agonists was conserved in the three membrane preparations. In cortical and “granule” membranes the Bmax values for the binding of [3H]GLU and [3H]glycine had a stoichiometry of 1∶:1, whilst in cerebellar synaptic membranes this ratio was 4∶:1. Receptor autoradiography of NMDA-related [3H]GLU and [3H]glycine binding in tissue sections failed to reveal any differential labelling patterns in cerebral cortex and cerebellum. In the cerebellum, densities of silver grains found with both [3H]ligands were concentrated in the granule cell layer relative to the molecular layer, but the differences detected in membrane binding studies were not observed in cerebellum. Our findings suggest the existence of three types of heterogeneity for the glycine domain of the NMDA receptor: (1) differing affinities for glycine, (2) differing pharmacological profiles, and (3) differing stoichiometry in relation to the putative NMDA-like agonist site. Our evidence supports an hypothesis for the existence of multiple glycine domains which might differentially modulate NMDA-mediated neurotransmission.

Electrophysiological and autoradiographical evidence for cholecystokinin A receptors on rat isolated nodose ganglia

The sulphated octapeptide, cholecystokinin (CCK-8S), is believed to be a neurotransmitter of vagal sensory neurones, and here the presence of functional receptors for CCK-8S in the rat vagus nerve has been investigated by electrophysiological and autoradiographic techniques. CCK-8S caused concentration-dependent depolarizations when superfused over the rat isolated nodose ganglion at 37 degrees C as measured by a silicone grease gap technique. Concentration-response curves to CCK-8S were shifted to the right by low concentrations of the CCKA receptor antagonist, Devazepide, but not by the CCKB receptor antagonist, L-365,260, data which indicate that receptors were of the CCKA subtype. Consistent with this notion, the CCKB agonist, unsulphated CCK-8, was without effect until high concentrations (> 1 microM) were used. A synthetic analogue of CCK-8S, D-Tyr25(Nle28,31)-CCK 25-33S, which has been reported to be more stable and peptidase-resistant than CCK-8S, was equipotent with CCK-8S in depolarizing the nodose ganglion. When D-Tyr25(Nle28,31)-CCK 25-33S was labelled with 125I, it bound to tissue sections of nodose ganglion. By light microscopic autoradiography, silver grains were found to be highly localized over cell bodies of vagal sensory neurones. An excess of CCK-8S inhibited binding as did Devazepide, but not L-365,260, confirming that binding sites were CCKA subtype receptors. These results indicate the existence of functional CCKA receptors in the nodose ganglion and strengthen the case for the involvement of vagal sensory neurones in gastric emptying and satiety.

Differential effects of human neuromelanin and synthetic dopamine melanin on neuronal and glial cells

We investigated the effects of neuromelanin (NM) isolated from the human substantia nigra and synthetic dopamine melanin (DAM) on neuronal and glial cell lines and on primary rat mesencephalic cultures. Lactate dehydrogenase (LDH) activity and lipid peroxidation were significantly increased in SK‐N‐SH cells by DAM but not by NM. In contrast, iron‐saturated NM significantly increased LDH activity in SK‐N‐SH cells, compared with 100 mg/mL ETDA‐treated NM containing a low concentration of bound iron. DAM, but not NM, stimulated hydroxyl radical production and increased SK‐N‐SH cell death via apoptotic‐like mechanisms. Neither DAM nor NM induced any changes in the glial cell line U373. 3H‐Dopamine uptake in primary rat mesencephalic cultures was significantly reduced in DAM‐ compared with NM‐treated cultures, accompanied by increased cell death via an apoptosis‐like mechanism. Interestingly, Fenton‐induced cell death was significantly decreased in cultures treated with both Fenton reagent and NM, an effect not seen in cultures treated with Fenton reagent plus DAM. These data are suggestive of a protective role for neuromelanin under conditions of high oxidative load. Our findings provide new evidence for a physiological role for neuromelanin in vivo and highlights the caution with which data based upon model systems should be interpreted.

[125I]Ifenprodil : a convenient radioligand for binding and autoradiographic studies of the polyamine-sensitive site of the NMDA receptor

Iodination of ifenprodil, a non-competitive NMDA antagonist, with Na125I/Chloramin-T gave a radioligand which bound rapidly and saturably to a single population of sites (dissociation constant 145 nM) in membranes of rat cerebral cortex. In competition studies, specific binding of [125I]-ifenprodil was inhibited by analogues of ifenprodil, as well as by spermine and spermidine. Binding was sensitive to Ca2+, Mg2+ and Zn2+. [125I]-Ifenprodil labelled a population of binding sites, which was topographically distributed in rat forebrain, as shown by autoradiography. [125I]Ifenprodil is a useful radioligand for the investigation of the polyamine site of the N-methyl-D-aspartate (NMDA) receptor-complex.

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk.

Excitotoxicity mediated via the (S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) subtype of receptor for l‐glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA‐induced neuronal injury and staurosporine (STS)‐mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up‐ and downstream of mitochondrial activation. AMPA‐mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (ΔΨm), early release of cytochrome c (cyt c), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis‐inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS‐induced apoptosis was characterized by a number of differences, a more rapid time course, non‐involvement of ΔΨm, and relatively early recruitment of SMAC and caspase 3. The AMPA‐induced rise in intracellular calcium appeared insufficient to evoke ΔΨm as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD‐fmk reduced the AMPA‐induced release of cyt c, SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS‐treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA‐mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase‐dependent AIF redistribution arguing for a model with cross‐talk between caspase‐dependent/independent apoptosis.

125I-ifenprodil : synthesis and characterization of binding to a polyamine-sensitive site in cerebral cortical membranes

Abstract: The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by 125I‐ifenprodil, a noncompetitive NMDA receptor antagonist, are described. 125I‐ifenprodil was synthesized using Na125I in the presence of chloramine‐T and purified by paper chromatography. Binding of the 125I‐ligand was optimal at pH 7.7 in 5 mM Tris · HCl buffer. Equilibrium binding of 125I‐ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 μM). Zn2+, Ca2+, and Mg2+ inhibited specific binding of 125I‐ifenprodil in a concentration‐dependent manner, with IC50 values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (KD) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single‐binding‐site model (Hill coefficient = 0.95), with a density of binding sites (Bmax) of 141 pmol/mg of protein. Binding of 125I‐ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3‐diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine‐sensitive binding of 125I‐ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that σ/antitussive agents partially inhibited specific binding, but inclusion of the σ drug GBR 12909 had little effect on the binding of 125I‐ifenprodil, suggesting this site was not involved. The binding site labeled by 125I‐ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the σ site.

Immunolocalization of CCK1R in rat brain using a new anti-peptide antibody.

An antibody directed at the carboxy tail of the cholecystokinin-1 receptor (CCK1R) was characterized by ELISA and Western blotting. Immunohistochemistry established that CCK1R-like immunoreactivity (CCK1R-LI) was widely and topographically distributed through the neuroaxis, appearing relatively higher in rhi- and diencephalon, and intense in both neuronal somata (cytoplasmic) and processes. CCK1R-LI was found in new loci, but also in areas previously identified by receptor autoradiography, electrophysiology and in situ hybridization of CCK1R mRNA. The widespread distribution of CCK1R has implications for the functional roles of these receptors in brain. The high titre and low background seen with this new antiserum makes it of great value for cell and tissue research.

A novel role for cholecystokinin: regulation of mesenteric vascular resistance

The aim of this work was to characterize the vasoactive effect of cholecystokinin on mesenteric vasculature. The mesenteric vascular bed of 3-month-old Sprague-Dawley rats was isolated and perfused at constant flow and changes in perfusion pressure monitored. CCK peptides lacked any direct contractile or relaxing effect on the mesenteric smooth muscle. Transmural nerve stimulation (TNS, 200 mA, 0.2 ms, 8 and 16 Hz) elicited an increase in perfusion pressure reflecting contraction of the bed and CCK inhibited neurogenic contractions elicited by 8 and 16 Hz TNS. The inhibition of neurogenic contractions was blocked by the CCK2 receptor (CCK2R) antagonist, L-365,260 (10 and 100 nM), but not by the CCK1R antagonist, SR-27897. The inhibition of neurogenic contractions was reversed by the non-specific NOS inhibitor, L-NAME as well as by the specific nNOS inhibitor, S-methyl-L-thiocitrulline. In whole-mount segments of mesenteric arteries, CCK2R was detected in the adventitia, in nerve terminals, where it co-localized with synaptophysin and nNOS. CCK-8 immunoreactive fibers were also detected. These results suggest that CCK mediates vasodilatation of the mesenteric vascular bed through the release of NO via its presynaptic CCK2R. Our findings provide, for the first time, a neural mechanism by which CCK may increase mesenteric blood flow.

GABAergic striatal neurons exhibit caspase-independent, mitochondrially mediated programmed cell death.

GABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3‐morpholinylsydnoneimine (SIN‐1), 3‐nitropropionic acid (3‐NP), NMDA, 3,5‐dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow‐onset kinetics with time‐dependent annexin‐V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis‐inducing factor and Omi, suggestive of early recruitment of both caspase‐dependent and caspase‐independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN‐1 produced mainly calpain activation, and 3‐NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult‐dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.