Kiyoshi Kusano

Cdna Cloning, Characterization, and Brain Region Specific Expression of a Neuromedin-b Preferring Bombesin Receptor

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.

Maintenance of LHRH and Oxytocin Neurons in Slice Explants Cultured in Serum-Free Media: Effects of Tetrodotoxin on Gene Expression

A variety of neuroendocrine cells survive and express specific neuropeptide genes for long periods of time in slice explant cultures in the presence of serum. However, before use of these slice explant cultures as experimental models for physiological and pharmacological studies on the regulation of neuropeptide gene expression, it is first necessary to evaluate their characteristics in defined (e.g. serum free) media and to control for the spontaneous electrical and synaptic activity of neurons in these cultures. In this study, brain slices from postnatal day 4 rats were cultured in serum-containing media (SCM) for 12 days to allow thinning, and then maintained in a serum-free, defined media (SFM) for 6 days. Culture slices transferred to SFM appeared healthy and numerous neuroendocrine neurons containing messenger RNA (mRNA) encoding for LHRH and magnocellular neurons containing mRNA encoding for oxytocin (OT) were detected using in situ hybridization histochemistry (ISHH). Each of these neuronal subtypes robustly produced their appropriate gene products as determined by immunocytochemical analysis. Abundant magnocellular OT neurons were found in cultures grown in either SCM or SFM. In contrast, magnocellular vasopressin (VP) neurons were rarely detected under these conditions. Inhibition of spontaneous electrical and synaptic activity in these slice explant cultures was effectively achieved by incubation for the last 2.5 days of culture in the presence of tetrodotoxin (TTX; 10(-6) M). Densitometric single cell analyses after ISHH was performed on both LHRH and OT cells. Comparisons of the density values (corresponding to mRNA levels), from these slice explants, found that: (1) cellular LHRH mRNA levels decreased in the absence of serum, whereas cellular OT mRNA levels did not significantly change under these conditions; (2) the presence of TTX in the media resulted in an overall decrease in cellular LHRH mRNA values in both SCM and SFM, and (3) the OT neurons in these slice cultures appear to be composed of a heterogeneous population, with one cell subtype responding to TTX with an increase in cellular OT mRNA levels. These data show that factors in serum and spontaneous electrical activity can differentially influence mRNA levels of LHRH cells and magnocellular OT neurons in culture.

Interactions of cyclic adenosine monophosphate, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor treatment on the survival and growth of postnatal mesencephalic dopamine neurons in vitro

The survival of rat postnatal mesencephalic dopamine (DA) neurons in dissociated cell cultures was studied by examining the combinatorial effects of dibutyryl cyclic adenosine monophosphate (db-cAMP), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), as well as selective inhibitors of protein kinase A (PKA), and mitogen-activated protein kinase (MAPK). Postnatal DA neurons were maintained for 14 days in vitro, and were identified by immunohistochemistry using tyrosine hydroxylase antibody. The survival and growth of DA neurons was significantly increased by the inclusion of either >100 microM db-cAMP or 10 microM Forskolin plus 100 microM IBMX in the culture medium. Neither 10-50 ng/ml GDNF nor 50 ng/ml BDNF alone significantly increased DA neuron survival in vitro. However, the combined use of GDNF and BDNF did increase DA neuron survival, and the addition of either db-cAMP or IBMX/Forskolin to media containing these neurotrophins markedly increased DA neuron survival and growth. The cAMP inhibitor Rp-cAMP, the cAMP-dependent protein kinase A inhibitor H89, and the MAP kinase (MAPK) pathway inhibitor PD98059 significantly reduced the survival of DA neurons when applied alone in the absence of added growth factors. Application of GDNF plus BDNF, or db-cAMP significantly protected the DA neurons from the deleterious effects on survival of either 20 microM H89 or 20 microM PD 98059. The results suggest that BDNF, GDNF, and cAMP produce convergent signals to activate PKA and MAPK pathways which are involved in the survival of postnatal mesencephalic DA neurons in vitro.

Distinguishing bombesin receptor subtypes using the oocyte assay

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.