Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

Expression of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and PAC1 in the Periodontal Ligament After Tooth Luxation

Pituitary adenylate cyclase-activating peptide (PACAP) is widely distributed throughout the nervous system. PACAP not only acts as a neurotransmitter but also elicits a broad spectrum of biological action via the PACAP-specific receptor, PAC1. However, no studies have investigated PACAP and PAC1 in the periodontal ligament (PDL), so we aimed to perform this investigation in rats after tooth luxation. In the PDL of an intact first molar, there are few osteoclasts and osteoblasts. However, at days 3 and 5 after luxation, large PAC1-positive cells, thought to be osteoclasts because of their expression of the osteoclast marker, tartrate-resistant acid phosphatase, were detected in appreciable numbers. Osteoblast numbers increased dramatically on day 7 after luxation, and PAC1-positive mononuclear small cells were increased at day 14, many of which expressed the osteoblast marker, alkaline phosphatase. PACAP-positive nerve fibers were rarely detected in the PDL of intact first molars, but were increasingly evident at this site on days 5 and 7 after luxation. Double-immunofluorescence analysis demonstrated the relationship between PACAP-positive nerve fibers and PAC1-positive osteoclasts/-blasts in the PDL. At 5 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoclasts. At 7 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoblasts. These results suggest that PACAP may have effects on osteoclasts and osteoblasts in the PDL after tooth luxation and thus regulate bone remodeling after these types of injury.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Promotion of Flowering by DNA Base Analogues and Changes in Acid Phosphatase and Peroxidase Isozyme Composition in Dark-Grown Arabidopsis thaliana

A DNA base analogue, 5-bromodeoxyuridine (BUdR), promoted floweringof Arabidopsis thaliana in short and long photoperiods and evenin total darkness. The promotive effect of BUdR was nullifiedby thymidine which had a weak inhibitory effect by itself. AnotherDNA base analogue, 5-fluorodeoxyuridine (FUdR), inhibited theflowering at a low concentration (10^–8 M), but markedlyenhanced the promotive effect of BUdR if they were present togetherin the culture medium. In the flower-promoting medium containing both BUdR and FUdR,the number of acid phosphatase isozymes decreased temporarily,followed by an increase to the control level with a prolongedculture period. The number of peroxidase isozymes was greaterin plants grown in the medium with BUdR or BUdR $ FUdR thanin those without them. (Received October 22, 1987; Accepted March 25, 1988)     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.