The gastric mucosa is covered with a viscoelastic and lubricant mucous layer. The chief determinants of the physiological role of the mucous layer are mucins, which are high molecular weight glycoproteins. Gastric mucins are produced in and secreted from specialized differentiated mucous cells located in the epithelium lining the gastric pits, and play an important role in the protection of gastric mucosa from acid-peptic injury. The production and release of mucins are regulated by neurotransmitters, hormones and biologically active peptides. Mucins are synthesized in the Golgi apparatus, and stored in intracellular granules which are transported to the luminal surface of the cell (Forstner & Forstner, 1994). These mucin granules finally discharge their contents through holes in the plasma membrane. This process is generally known as exocytosis. The first event in exocytosis is the fusion of a granule with the plasma membrane at the fusion pore, and is mediated by the exocytosis-related proteins, which are activated by intracellular Ca¥, protein kinase A (PKA) and protein kinase C (PKC) (Forstner & Forstner, 1994). Intracellular Ca¥, in particular, is widely accepted as playing a key role in exocytosis in epithelial cells, endocrine cells and nerve endings. In the gastric mucosa, elevation of intracellular Ca¥ concentration, [Ca¥]é, also increases mucin release (Seidler & Sewing, 1989), and cholinergic stimulation is well known to increase [Ca¥]é. The present experiments were designed to investigate the effects of acetylcholine (ACh) on the frequency of exocytotic events.

Effects of cAMP accumulation on ATP-dependent priming and Ca(2+)-dependent fusion in Ca(2+)-regulated exocytosis were examined in antral mucous cells of guinea pigs by using video-enhanced contrast microscopy. The Ca(2+)-regulated exocytosis activated by 1 microM ACh consisted of two phases, an initial transient phase followed by a sustained phase, which were potentiated by cAMP accumulation. Depletion of ATP by 100 microM dinitrophenol (uncoupler of oxidative phosphorylation) or anoxia induced the sustained phase without the initial transient phase in Ca(2+)-regulated exocytosis. However, accumulation of cAMP before depletion of ATP induced and potentiated an initial transient phase followed by a sustained phase in Ca(2+)-regulated exocytosis. This suggests that the initial transient phase of Ca(2+)-regulated exocytosis is induced by fusion of all primed granules maintained by ATP and that accumulation of cAMP accelerates ATP-dependent priming of the exocytotic cycle. Moreover, ACh and Ca(2+) dose-response studies showed that accumulation of cAMP shifted the dose-response curves to the low concentration side, suggesting that it increases Ca(2+) sensitivity in the fusion of the exocytotic cycle. In conclusion, cAMP accumulation increases the number of primed granules and Ca(2+) sensitivity of the fusion, which potentiates Ca(2+)-regulated exocytosis in antral mucous cells.

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 microM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 microM; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 microM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE(2) (1 microM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). ACh and ionomycin increased PGE(2) release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca(2+)](i) activates Ca(2+)-regulated exocytotic events and PGE(2) release mediated by COX-1. The released PGE(2) induces the accumulation of cAMP, which enhances the Ca(2+)-regulated exocytosis. The autocrine mechanism mediated by PGE(2) maintains the high-level mucin release from antral mucous cells during ACh stimulation.

Heat shock transcription factors (HSFs) play roles not only in heat shock response but also in development of the reproductive organs, brain, and lens. Here, we analyzed sensory organs and found abnormalities of the olfactory epithelium in adult HSF1-null mice, which is developmentally related to the lens. The olfactory epithelium was normal until postnatal 3 weeks but was not maintained later than 4 weeks in HSF1-null mice. The olfactory epithelium was atrophied with increased cell death of olfactory sensory neurons. Analysis of the epithelium revealed that induction of HSP expression and reduction of LIF expression are lacking in adult HSF1-null mice. We found that DNA binding activity of HSF1 is induced in the olfactory epithelium later than 4 weeks and that HSF1 binds directly to Lif gene and inhibits its expression. HSF4 has opposing effects on LIF expression and olfactory neurogenesis. These data indicate that HSF1 is required for the precise expression of Hsp and cytokine genes that is obligatory for maintenance of olfactory neurogenesis in adult mice and suggest that stress-related processes are involved in its maintenance.Heat shock response is characterized by induction of a set of heat shock proteins (HSPs) 2 and is an evolutionary conserved response inherent in all organisms from bacteria to human. In eukaryotes, this response is regulated mainly by heat shock transcription factor (HSF), which can bind to a heat shock element (HSE) composed of at least three inverted repeats of consensus sequence nGAAn (1). In vertebrates, three mammalian Hsf genes (Hsf1, Hsf2, and Hsf4) and three avian HSF genes (HSF1, HSF2, and HSF3) have been identified (2, 3), and their expression profiles are temporally and spatially different during development (3, 4). In unstressed cells HSF1 is present mostly as a monomer and is converted to a trimer that binds to HSE in response to heat shock, whereas HSF4 is constitutively a trimer. A single mutation of Hsf1, Hsf2, or Hsf4 genes or combined mutations of Hsf genes in mice showed that HSFs are involved in development of the reproductive organs (5-10), brain (6, 10, 11), and lens (12-14) and in immune response (15,16). In addition to regulating HSP expression, HSFs are involved in development partly by regulating expression of growth factor genes such as Fgf and Il-6 genes directly (13,15). Although HSF1 binds to many genes in vivo in unstressed cells (17), we have little information about when and how HSF activity is regulated during development and maintenance of tissues.We previously identified that HSF4 is required for development of lens in the eye partly by regulating Hsp and Fgf genes (13). HSF1 is also involved in lens development and competes with HSF4 for expression of the same Fgf genes. These observations highlighted sensory organs that receive stimuli from the external environment and are susceptible to stress. Here, we examined development of other sensory organs in HSF1-and HSF4-null mice. Surprisingly, we found abnormalities in the nasal cavity in adult HSF1-nu...

No abstract

Mutations in EFHC1 gene have been previously reported in patients with epilepsies, including those with juvenile myoclonic epilepsy. Myoclonin1, also known as mRib72-1, is encoded by the mouse Efhc1 gene. Myoclonin1 is dominantly expressed in embryonic choroid plexus, post-natal ependymal cilia, tracheal cilia and sperm flagella. In this study, we generated viable Efhc1-deficient mice. Most of the mice were normal in outward appearance, and both sexes were found to be fertile. However, the ventricles of the brains were significantly enlarged in the null mutants, but not in the heterozygotes. Although the ciliary structure was found intact, the ciliary beating frequency was significantly reduced in null mutants. In adult stages, both the heterozygous and null mutants developed frequent spontaneous myoclonus. Furthermore, the threshold of seizures induced by pentylenetetrazol was significantly reduced in both heterozygous and null mutants. These observations seem to further suggest that decrease or loss of function of myoclonin1 may be the molecular basis for epilepsies caused by EFHC1 mutations.

This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an β-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca concentration ([Ca]) caused by switching to an EGTA-containing Ca-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia.

Effects of prostaglandin E2 (PGE2) on exocytosis of mucin were studied in mucous cells isolated from guinea‐pig antrum using video‐microscopy. Stimulation with PGE2 elicited a sustained increase in the frequency of exocytotic events in a dose‐dependent manner, which was under regulation by both Ca2+ and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO‐AEI‐329, 10 μM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30% of that evoked by 1 μM PGE2). Stimulation with an EP1 agonist (17‐P‐T‐PGE2, 1 μM), which activates Ca2+ signals, increased the frequency of exocytotic events to a lesser extent (5% of that evoked by 1 μM PGE2), while addition of an EP1 antagonist (ONO‐8713, 10 μM) decreased the frequency of exocytotic events (approximately 40% of that evoked by 1 μM PGE2). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca2+ signal activated via the EP1 receptor potentiates the cAMP‐regulated exocytotic events activated via the EP4 receptor during PGE2 stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE2‐stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors.