Vitamin B6 suppresses growth and expression of albumin gene in a human hepatoma cell line HepG2.

The effect of vitamin B6 on the growth of a human hepatoma cell line HepG2 in culture was studied. The growth of HepG2 cells and protein synthesis were almost completely inhibited in medium supplemented with 5 mM pyridoxine. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed no inhibitory action. The growth inhibition of HepG2 cells by pyridoxine was accompanied by a marked inhibition of secretion of plasma proteins, particularly albumin. Northern blot analysis of albumin mRNA showed that pyridoxine caused a rapid decrease in the expression of albumin gene. The electron-microscopic examination of pyridoxine-treated HepG2 cells revealed a smoothing of nuclear membrane, a decrease in the number of nucleoli, and an appearance of aggregated heterochromatin structures. These morphological features are compatible with the depressed transcriptional activity in the pyridoxine-treated cells. The mechanism by which vitamin B6 exerts its inhibitory effect was discussed in terms of our recent finding that vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding proteins. Binding of pyridoxal phosphate with tissue-specific transcription factors may reduce the capacity of these factors to interact with the regulatory region of albumin gene, resulting in the inhibition of the gene expression.

Morphological changes in rat aortic endothelial cell line stimulated by endothelin.

We have reported that endothelin (ET)-1 activates interleukin (IL)-6 production in rat aortic endothelial cell line (WAE-1 cell). In this experiment, we investigated the morphological changes induced by ET-1 in cultured WAE-1 cells. The cells were treated with ET-1 for 8 h or 24 h, and then compared with untreated cells by light and electron microscopies. The WAE-1 cells treated with ET-1 for 8 h showed remarkable alterations as following: by light microscopic observation, the cells were enlarged and filled with many vesicles in the cytoplasm, and by electron microscopic observation, the cells showed the increases of nuclear membrane infoldings, increased density of chromatin granules just inside the nuclear membrane, and multivesicular bodies in the cytoplasm. These morphological changes were hardly observed in WAE-1 cells-treated with ET-1 for 24 h. It was suggested that ET-1 stimulated DNA synthesis and secretion of cytoplasmic products in WAE-1 cells.

Migration of LHRH neurons derived from the olfactory placode in rats.

Using the olfactory placode of 12.5- and 14.5-day-old (E12.5, E14.5) rat embryos, we examined the migration of LHRH neurons by in vivo intraventricular transplantation and in vitro organotypic culture systems. In the transplantation, the olfactory placode of E12.5 embryos was co-transplanted with the cerebral cortex and also with medial basal hypothalamus (MBH). LHRH neurons that had migrated into the co-transplanted brain tissues were fusiform, but those that had moved into the neuro-mesenchymal tissue were polyhedral. The migration occurred most conspicuously in the MBH. In our in vitro studies, we used E14.5 embryos; their vomeronasal organ was cultured with MBH, the olfactory cortex, and the septum of the telencephalon in two systems (piled-culture with an intervening transferrable membrane and co-culture). Among these brain tissues, the MBH was the most effective in inducing the development and migration of LHRH neurons. We further found synaptic junctions of immunonegative nerve fibers on immunoreactive LHRH neurons located in the septum of E16.5 and 17.5 embryos. These findings suggest that the MBH may lead the intraseptal migration of LHRH neurons by yielding certain substances after introducing the neurons into the medial aspect of forebrain vesicles. The early development of the neuronal connection may further promote the migration of LHRH neurons.

Reciprocal synaptic relations between CRF-immunoreactive- and TRH-immunoreactive neurons in the paraventricular nucleus of the rat hypothalamus.

By double immunoelectron microscopy, we studied synaptic relations between corticotropin-releasing factor (CRF)-immunoreactive (ir) and thyrotropin-releasing hormone (TRH)-ir neurons in the paraventricular nucleus (PVN) of the rat hypothalamus. CRF-ir and TRH-ir neurons made reciprocal synaptic connections in the medial and periventricular parvocellular regions. These results may suggest that both the parvocellular neurons interplay on their hypophysiotropic functions within the PVN.

Somatostatin-like immunoreactive axon terminals on oxytocin-like immunoreactive neurons in the paraventricular nucleus of the rat hypothalamus.

By double label immunohistochemistry, we studied the synaptic relation between somatostatin (SS) and oxytocin (OT) or vasopressin (VP) neurons in the paraventricular nucleus (PVN) of the rat hypothalamus. In the light microscopic observation, SS-immunoreactive (ir) fibers were seen very frequently in contact with OT-ir neurons, but not with VP-ir neurons. Immunoelectron microscopic analysis revealed that somata and their dendrites of OT-ir magnocellular neurons were in synaptic contacts with SS-ir axon terminals. No SS-ir synaptic terminals were found on VP-ir magnocellular neurons. The results suggest that OT-containing magnocellular neurons in the PVN are under regulatory influences of SS-containing neurons.

Further evidence of the presence of rat embryonic hypothalamic factors that induce the differentiation of gonadotrophic hormone-releasing hormone-containing secretory neurons.

We examined the presence of factor(s) in the embryonic medial basal hypothalamus (MBH) that may influence nasal placode (NAP)-derived luteinizing hormone-releasing hormone (LHRH) neurons in determining their secretory phenotype. In this study, we performed organotypic culture and transplantation of the NAP from 12.5-day-old embryos of rats and vomeronasal organ (VNO) from 14.5-day-old embryos. Surgical operations, however, were performed on 16.5-day-old embryos. The NAP and VNO were cultured singly or with the MBH obtained from the embryos of the same age and, further, in a medium with a nerve growth factor or fibroblast growth factors. Although LHRH neurons were derived from the NAP and VNO in all the cultures, judging from numbers and cellular morphologies, the MBH was most effective. The VNO was transplanted into the third ventricle of adult female rats singly or with the cerebral cortex, the mesencephalon-myelencephalon complex, or the MBH from 14.5-day-old embryos. All the grafts gave rise to LHRH neurons, but the number of the neurons was far greater in the grafts cotransplanted with the MBH, in which the neurons projected long processes to blood capillaries and formed neurovascular complexes, the feature of which may suggest the occurrence of the secretory activity in the fibers. The animals were examined 5 days after the surgical operations. In rhinoectomized embryos, LHRH neurons were distributed throughout the brain in the same pattern as found in intact rats, showing normal cellular morphology. In the encephalectomized rats, immunoreactive LHRH cells were present only in the terminal ganglia. These findings indicate that the embryonal MBH has a factor (s) that is essential to the development of secretory LHRH neurons.

Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal polypeptide have synaptic contacts with axons immunoreactive for neuropeptide Y: an immunoelectron microscopic study in the rat.

An electron microscopic study showed by using a dual immunolabeling technique that in the suprachiasmatic nucleus of the rat, axon terminals immunoreactive for neuropeptide Y (NPY) made synaptic contacts upon neurons immunoreactive for vasoactive intestinal polypeptide (VIP). Diaminobenzidine (DAB)-labeled NPY axon terminals made synaptic contacts on silver-gold-labeled VIP perikarya and dendritic processes. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles labeled with DAB chromogen. At the synaptic portion, a symmetrical thickening of the pre- and post-synaptic membranes was evident.

Ultrastructural evidence for neuronal regulation of growth hormone secretion.

The morphological substrate for the central mechanisms that control growth hormone (GH) release in the rat hypothalamus was investigated immunohistochemically by light and electron microscopy. In electron-microscopic studies, a dual immunolabeling technique was employed to demonstrate pairs of peptides, i.e. rat hypothalamic growth hormone-releasing factor (rhGRF) and somatostatin (SRIH), rhGRF and substance P (SP), and rhGRF and methionine-enkephalin-Arg6-Gly7-Leu8 (Enk-8), in different neuronal structures. Immunoreactivity of rhGRF was detected as silver-gold particles and those of the other substances as diaminobenzidine products by preembedding immunostaining procedures. In the external layer of the median eminence, axonal terminals immunolabeled for rhGRF and for SRIH showed the same pattern of distribution and close proximity. The neuronal inputs to GRF cell bodies in the arcuate nucleus were examined, and SRIH, SP and Enk-8 fibers with varicosities were found to form dense networks around the perikarya of GRF neurons, suggesting the presence of synaptic associations. Axonal terminals immunolabeled for SRIH, SP or Enk-8, and unlabeled terminals appeared to form coincidental synaptic junctions on GRF perikarya. These findings suggest that the central regulation of GH release occurs at the levels of the median eminence and the cell bodies.

Development of hypothalamic neurons in intraventricular grafts: expression of specific transmitter phenotypes.

The anlages of the medial-basal hypothalamus (MBH), septopreoptic area (POA), Rathke's pouch, and the parietal cortex (CC) of rats (at 12.5, 14.5 and 16.5 days of gestation) were transplanted singly or in combination into the third ventricle of adult female rats, and the development of neurons in the grafts was investigated immunohistochemically with the use of antisera to tyrosine hydroxylase (TH), somatostatin (SRIH), ACTH, methionine enkephalin-Arg6-Gly7-Leu8 (Enk-8), rat corticotropin-releasing factor (rCRF), rat hypothalamic growth hormone-releasing factor (rhGRF), and luteinizing hormone-releasing hormone (LHRH). TH and all the peptides examined except LHRH were detected in distinct neurons in MBH grafts and in cografts of MBH plus Rathke's pouch from 12.5-day-old embryos. SRIH, rCRF, Enk-8, and TH were found in POA grafts from embryos of the same age. Although immunoreactive LHRH was first detected in neurons in POA grafts from 16.5-day-old embryos, it appeared in cografts of POA and MBH from 12.5-day-old embryos. The immunoreactive fibers developed in the grafts expressed the same characteristic behaviors as in intact brain; the fibers containing hormonal substances formed complexes with the vasculature like in the organum vasculosum laminae terminalis (OVLT) or in the median eminence, while the fibers containing neurotropic signals formed fiber networks surrounding other nerve cell bodies as if they synaptically associate. In CC grafts, the neurons contained TH, SRIH, rCRF, or Enk-8, and their axonal processes formed fiber networks. These findings suggest that all the hypothalamic neurons examined are committed by 12.5 days of gestation to develop maintaining transmitter phenotype and target recognition capacity.

Synaptic association between enkephalin-containing axon terminals and proopiomelanocortin-containing neurons in the arcuate nucleus of rat hypothalamus.

By employing an electron microscopic dual immunolabeling technique, a synaptic association between neurons containing immunoreactive adrenocorticotropin (ACTH) and axonal terminals containing immunoreactive methionine-enkephalin octapeptide (Enk-8) was found in the arcuate nucleus of the rat hypothalamus. The axonal terminals contained many small clear vesicles and some large cored vesicles. At the synaptic portions, membrane specialization was asymmetric.

Differential immunolabeling for electron microscopy of diverse peptidergic neurons.

We describe a simple and reliable method for differential immunolabeling of pre- and post-synaptic signal peptides at the ultrastructural level. Hypothalamic tissues of rats, including the suprachiasmatic nucleus, were cut on a Vibratome. Visualization of the immunolabeling of somatostatin (SRIH) and vasoactive intestinal polypeptide (VIP) was performed with avidin-biotin-peroxidase-diaminobenzidine (DAB). The end product of the DAB to VIP was further silver-intensified in a physical processing using silver nitrate, and the silver grains were finally substituted for gold. DAB-labeled SRIH fibers synapse on gold-labeled VIP perikarya and dendrites in the suprachiasmatic nucleus.

Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus.

By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered diffusely throughout the neuroplasma at various densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptic membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.