Isosmotic modulation of Ca²⁺‐regulated exocytosis in guinea‐pig antral mucous cells: role of cell volume

Abstract

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.

<b>PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca</b>^<b>2+</b><b>-regulated </b><b>exocytosis </b>

“…The volume of perfusion chamber was approximately 20 μL, and the rate of perfusion was 200 μL/min. Exocytotic events, which were detected by rapid changes in the light intensity of the granules (8,9,14,15,19,(22)(23)(24), were counted in two to four cells every 30 s and were normalized to the number of cells (events/cell/30s). The frequency of exocytotic events measured using six to eleven coverslips from two or three animals was expressed as the mean ± SE based on the number of experiments.…”

“…In antral mucous cells, the Ca 2+ -regulated exocytosis which is activated by ACh (acetylcholine) is the main mechanism for mucin release (8,14,15,19) and is modulated by many substances, such as cAMP, NO, cGMP, intracellular Cl − , and arachidonic acid (AA) (8,14,19,(21)(22)(23)(24). NO is synthesized by nitric oxide synthase 1 (NOS1) and enhances the Ca 2+ -regulated exocytosis mediated via cGMP accumulation in antral mucous cells (28).…”

“…Guinea pigs were sacrificed by intraperitoneal injection of pentobarbital sodium (100 mg/kg), and then, the stomach was removed from the animal. The procedures for the cell preparation have been previously described (8,9,14,15,19,(22)(23)(24). Briefly, the gastric antrum was excised from the stomach, and the mucosal layer was stripped from the muscle layer in cold phosphate buffered saline (4°C) using glass slides.…”

“…The isolated antral tubular shaped glands were mounted on a coverslip pre-coated with neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA, USA) for firm attachment of the cells. The coverslip with the cells was set in a perfusion chamber mounted on the stage of a differential interference contrast microscope (BX50Wi; Olympus, Tokyo, Japan), which was connected to a videoenhanced contrast system (ARGUS-20; Hamamatsu Photonics, Hamamatsu, Japan) (8,9,14,15,19,(22)(23)(24). Images were recorded continuously using a video recorder.…”

Constitutive Activity of Inwardly Rectifying K+ Channel at Physiological [Ca]i Is Mediated by Ca2+/CaMK II Pathway in Opossum Kidney Proximal Tubule Cells

“…They were resuspended and stored in a control bath solution containing 2% BSA at 4°C and placed on a coverslip precoated with neutralized Cell-Tak to allow the cells to adhere firmly to the coverslip. The coverslip with cells was set in a perfusion chamber, which was then mounted on the stage of an inverted microscope (IX70, Olympus, Tokyo, Japan) connected to an image analysis system (ARGUS/HiSCA, Hamamatsu Photonics, Hamamatsu, Japan) [22,23]. All the experiments were performed at room temperature.…”