Syntaxin6 separates from GM1a-rich membrane microdomain during granule maturation.

Since it was reported that components of immature secretory granules (ISGs) are different from those of mature secretory granules (MSGs) in rat parotid acinar cells, we have been considering that components of secretory granules (SGs) change dynamically during granule maturation. As the first step to understand the mechanism of granule maturation, we separated low-density detergent-resistant membrane fractions (DRMs) from purified SGs of rat parotid gland. When SGs were lysed by the detergent Brij-58, syntaxin6 and VAMP4 were found in DRMs that were different from the GM1a-rich DRMs containing VAMP2.

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells.

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar.

A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules.

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes.

Phosphodiesterases 1 and 2 regulate cellular cGMP level in rabbit submandibular gland cells.

In rabbit salivary glands, stimulation of muscarinic cholinergic receptors causes production of cGMP through intracellular Ca2+ and nitric oxide. In this study, we investigated a role of cyclic nucleotide phosphodiesterase (PDE) in regulating the cellular cGMP level by using cells dispersed from the submandibular gland. Methacholine, a cholinergic agonist, rapidly elevated the cGMP level.

Charge-based separation of detergent-resistant membranes of mouse splenic B cells.

Current biochemical characterization for cholesterol- and glycolipid-rich membrane microdomains largely depends on analysis of detergent-resistant membranes (DRMs). In the present study, we succeeded in separation of DRMs of similar density-based on their electrical charge using free-flow electrophoresis (FFE). After crosslinking of B cell receptor (BCR), mouse splenic B cells were lysed with 1% Brij-58 and the resulting lysate was subjected to sucrose density gradient ultracentrifugation.

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta.

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression.

Identification of FGF2-response element in the rat bone sialoprotein gene promoter.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Basic fibroblast growth factor (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. In skeletal tissues, FGF2 produced by osteoblasts accumulates in the bone matrix and acts as an autocrine/paracrine regulator of bone cells.

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts.

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry.

Prostaglandin E2 stimulates bone sialoprotein (BSP) expression through cAMP and fibroblast growth factor 2 response elements in the proximal promoter of the rat BSP gene.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Prostaglandin E2 (PGE2) has anabolic effects on proliferation and differentiation of osteoblasts via diverse signal transduction systems. Because PGE2 increases the proportion of functional osteoblasts in fetal rat calvarial cell cultures, we investigated the regulation of BSP, as an osteoblastic marker, by PGE2.

Evidence that nitric oxide does not directly contribute to methacholine-induced amylase secretion in rabbit parotid acinar cells.

Nitric oxide (NO) is a short-lived free radical and is a widespread intra- and intercellular messenger molecule involved in various physiological functions. We have demonstrated previously that the muscarinic agonist methacholine induces endogenous generation of NO in rabbit parotid acinar cells. Since methacholine also simultaneously evokes amylase secretion, we investigated the effect of NO on the methacholine-induced exocytotic amylase secretion in rabbit parotid acinar cells.

Angiotensin II induces prostaglandin E(2) release in human gingival fibroblasts.

We investigated the effect of angiotensin II on prostaglandin E(2) release in human gingival fibroblasts. Stimulation of human gingival fibroblasts with angiotensin II elicited prostaglandin E(2) release in a concentration- and time-dependent manner. Angiotensin III also induced prostaglandin E(2) release, but the effect was weaker than that of angiotensin II.

Involvement of phospholipase D in the cAMP-regulated exocytosis of rat parotid acinar cells.

The activation of beta-adrenergic receptors in rat parotid acinar cells causes intracellular cAMP elevation and appreciably stimulates the exocytotic release of amylase into saliva. The activation of Ca(2+)-mobilizing receptors also induces some exocytosis. We investigated the role of phospholipase D (PLD) in regulated exocytosis in rat parotid acinar cells.

TNF-alpha suppresses bone sialoprotein (BSP) expression in ROS17/2.8 cells.

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammatory responses in many diseases that inhibits bone formation and stimulates bone resorption. To determine molecular mechanisms involved in the suppression of bone formation we have analyzed the effects of TNF-alpha on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone.

Tumor necrosis factor alpha (TNF-alpha)-induced prostaglandin E2 release is mediated by the activation of cyclooxygenase-2 (COX-2) transcription via NFkappaB in human gingival fibroblasts.

Nuclear factor kappaB (NFkappaB) is a transcription factor and plays a key role in the expression of several genes involved in the inflammatory process. Cyclooxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid synthetic pathway. COX-2 is a highly inducible enzyme by proinflammatory cytokines, of which gene expression is regulated by NFkappaB.

Methacholine-induced cGMP production is regulated by nitric oxide generation in rabbit submandibular gland cells.

Guanosine 3',5'-monophosphate (cGMP) is an intracellular messenger in various kinds of cell. We investigated the regulation of cGMP production by nitric oxide (NO) in rabbit submandibular gland cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose- and time-dependent manner, but the alpha-agonist phenylephrine, substance P and the beta-agonist isoproterenol failed to evoke cGMP production.

4-Bromophenacyl bromide induces Ca2+ influx in human gingival fibroblasts.

4-Bromophenacyl bromide (BPB) is generally used as a phospholipase A(2) (PLA2) inhibitor. In the present study, we demonstrate that BPB induces Ca2+ influx in human gingival fibroblasts. In fura-2-loaded human gingival fibroblasts, BPB evoked a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner.