Characterization of prostanoid receptors present on adrenergic neurons innervating the porcine uterine longitudinal muscle.

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.

Metabolic barrier against bisphenol A in rat uterine endometrium.

Exposure to environmental chemicals with estrogenic activity during the early stage of pregnancy can seriously affect embryonic development and the maintenance of pregnancy. To estimate the metabolism and pharmacodynamics of a xenoestrogen, bisphenol A, in a reproductive organ, the metabolite of bisphenol A was analyzed after incubating a rat uterine sac in buffer solutions containing the chemical. When the inner or the outer side of the uterine sac was exposed to bisphenol A, the concentration of the parent chemical was decreased in buffer solution and then, only one metabolite, bisphenol A-glucuronide, was observed only in the outer, that is, the maternal, side. A small amount of the parent chemical could pass through the uterine sac without being modified. Uridine diphosphate (UDP)-glucuronosyltransferase (UGT) was shown by immunohistochemical staining analysis to be distributed in epithelial cells of the endometrium, oviduct, and uterine glands. Based on measurements of enzyme activity and on Western blot analysis, UGT activity toward bisphenol A and UGT protein were identified in the microsomal fractions prepared from rat uterus. UGT isoforms, such as UGT1A1, 1A2, 1A5, 1A6, and 1A7, were expressed, and MRP-1 (multidrug resistance-associated protein) and MRP-3, which are well-known to be transporters of various drug-glucuronides, were detected in the rat uterus by reverse transcription-PCR. These results elucidate the rat uterine barrier system by showing that most bisphenol A perfused into the uterus was glucuronidated in the epithelium, resulting in transport of glucuronides to the maternal side.

Expression, cellular localization, and functional role of monocarboxylate transporter 4 (MCT4) in the gastrointestinal tract of ruminants.

This is the first study to determine the precise cellular localization of monocarboxylate transporter 4 (MCT4), along with its co-existence with its chaperone, CD147 in the ruminant gastrointestinal tract. Quantitative Western blot analysis demonstrated that the abundance of MCT4 protein was in the order of forestomach > large intestine > abomasum >or= small intestine. Immunohistochemistry and immunofluorescence confocal laser microscopy showed that MCT4 in the forestomach was confined to the cell membranes of strata corneum and granulosum, while diffuse cytoplasmic staining for MCT4 was visualized in strata spinosum and basale. In the epithelium cells lining the abomasum, MCT4 immunoreactive positivities were predominantly localized on the basolateral membranes. In the small intestine, MCT4 was localized at the brush borders and the basolateral membranes of the epithelial cells lining the villi, however it was mostly found on the apical membranes of the crypt cells. In the large intestine, the immunoreactivity for MCT4 differed between the surface epithelium and the crypts; in the surface epithelium, MCT4 was mainly localized at the apical membranes, whereas in the crypts it was predominantly expressed on the basolateral membranes of the lining epithelial cells. MCT4 was remarkably co-existed with CD147 along the bovine gastrointestinal tract. Our results suggest that MCT4 can play an important role in the transport of SCFA. The study also explored the potential functional collaboration between MCT1 and MCT4 and provided new insights into the mechanisms that mediate the transport of SCFA and other monocarboxylates in the different segments of the ruminant gastrointestinal tract.

Detection of gamma-tubulin in spermatogonial cells of Bombyx mori (Lepidoptera) and Chortophaga viridifasciata (Orthoptera).

We detected a putative gamma-tubulin gene in silico and detected BACs containing the gene from a Bombyx mori BAC library. BAC-FISH mapping revealed that the gene is located on chromosome 5. To observe the distribution of gamma-tubulin, we employed antibodies against mammalian gamma-tubulin peptides. Western blot analysis disclosed a band very similar in size to gamma-tubulin protein in other species (approximately 48 kDa). In mitotic metaphase of B. mori spermatogonial cells, gamma-tubulin is exclusively localized in the spindle poles, where the centrosomes occur. We applied the same system to the grasshopper Chortophaga viridifasciata, as a representative of insect orders in which the gamma-tubulin distribution had not previously been studied. Gamma-tubulin was also found in the spindle poles during metaphase of spermatogonial cells in the grasshopper.

Isoform-specific expression and induction of udp-glucuronosyltransferase in immunoactivated peritoneal macrophages of the rat.

Phase I drug-metabolizing enzymes such as cytochrome P450 in immunocytes are known to play a role in metabolic activation of toxic and immunosuppressive compounds such as polycyclic aromatic hydrocarbon (PAH). UDP-glucuronosyltransferase (UGT), a drug-metabolizing phase II enzyme, accelerates elimination of these compounds; however, there is little information on the expression and function of UGT in immunocytes. In this study, we investigated the expressions of UGT isoforms in rat peritoneal macrophages and the role of UGT in macrophage functions. Expressions of UGT1A1, 1A6, and 1A7 were observed in macrophages by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. When macrophage cells cultured in plates were exposed to 1-naphthol and 3-hydroxybenzo-[a]pyrene (3-OH-B[a]P), these glucuronides increased in the medium, indicating that macrophages glucuronidated the chemicals. The production of the glucuronides of 1-naphthol and 3-OH-B[a]P was induced by lipopolysaccharide (LPS) treatment of the cultured macrophage cells. Northern blot analysis revealed that UGT1A7 mRNA was induced by LPS treatment. This result is the first evidence that a drug-metabolizing enzyme is induced by immunoactivation. The results indicated that macrophages can detoxify various toxic and immunosuppressive compounds with UGT, and that ability is enhanced by immunoactivation. We propose that macrophages contribute to protection against not only macromolecules as immunocytes but also small molecules such as the immunosuppressive agents PAHs in peripheral blood and interstitial tissues.

Presence of phenol UDP-glucuronosyltransferase in bovine alveolar macrophages and bronchial epithelial cells.

Pulmonary organ and cells are the primary target of atmospheric pollutants. Phenol UDP-glucuronosyltransferase (UGT) was found in bovine alveolar macrophage cells by immunohistochemical staining and also was observed in bronchial epithelial cells of the lung. A high level of activity of UGT, which is one of the phase II drug-metabolizing enzymes, toward 1-naphthol was observed in the microsomes of both cell types. By Western blotting analysis, a 54-kDa band was detected in alveolar macrophage cells and in bovine lung using polyclonal antibodies against a purified rat UGT, which catalyze the glucuronidation of various phenolic xenobiotics such as 1-naphthol and have the same molecular mass (54 kDa). Reverse transcriptase-polymerase chain reaction (RT-PCR) amplified the common cDNA region in UGT1A subfamily isoforms, indicating that UGT1A subfamily isoform was expressed in alveolar macrophages and in bronchial epithelial cells of the lung. These results suggest that phenol UGT act as a primary barrier against various phenolic chemicals in the lung.

The Determination of Spindle Polarity in Early Mitotic Stages of the Dividing Grasshopper Neuroblasts: (unequal cytokinesis/neuroblast/spindle axis/electron dense layer/microdissection).

Although the spindle body of the grasshopper neuroblasts at the early mitotic stages does not have any mechanical linkage to the surrounding cell cortex, a spindle axis is inevitably oriented in parallel with the original division axis. The present study analyzes how the definite orientation of spindle axis along the cap cell (CC)-ganglion cell (GC) axis of the neuroblast is maintained during these stages by use of the microdissection technique and electron microscopy. After removing a microneedle from the cell, metaphase spindles approximately 90°. rotated were able to return autonomously to the original axis. After the middle anaphase, however, the rotated spindle could not return at all. The electron microscopic observations revealed a characteristic behavior of an electron dense layer (EDL) in the CC-side cortex during neuroblast mitosis. The EDL first appeared at very late prophase and became most conspicuous at metaphase. It became discontinuous by the beginning of middle anaphase and then completely disappeared at middle anaphase. So long as an EDL existed in the CC-side polar cortex, 90° rotated spindle bodies were able to return autonomously to the original axis. After the disappearance of the EDL, the autonomous return of the rotated spindle no longer occurred. From these circumstantial evidence, it is conceivable that the orientation of the spindle body along the CC-GC axis is maintained by the interaction between the EDL and the spindle pole.

Microdissection Studies on the Polarity of Unequal Division in Grasshopper Neuroblasts: II. Cell Division in Binucleate Neuroblasts: (unequal division/grasshopper neuroblasts/binucleate cells).

The grasshopper neuroblast divides unequally to produce two types of cells: a large daughter neuroblast that contains a doughnut-shaped nucleus and repeats unequal division with definite polarity, and a small daughter ganglion cell that has a spherical nucleus with low mitotic activity. Binucleate neuroblasts were induced by preventing cytokinesis in the course of microdissection experiments, and subsequent divisions were traced to analyze the factors that determine the polarity of unequal division. In binucleate neuroblasts, both daughter chromosome groups developed into neuroblast-type nuclei. Mitosis of the two nuclei proceeded synchronously. Although the axes of the two mitotic apparatuses formed at late prophase were random in direction, they became parallel with the original division axis at metaphase. The two mitotic apparatuses shifted simultaneously toward the ganglion cell side during anaphase, just as in normal neuroblasts, and the binucleate cell divided unequally. These findings showed that the poearity of unequal division is strictly maintained in grasshpper neuroblasts, even when they contain two nuclei.