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Aquaporins in rat pancreatic interlobular ducts. Am J Physiol Gastrointest Liver Physiol 282: G324-G331, 2002; 10.1152/ajpgi.00198. 2001.--The aquaporin (AQP) family of water channels is distributed ubiquitously in many epithelia and plays a fundamental role in transmembrane water transport. The aim of this study is to identify the water transport pathway in pancreatic duct cells where most of the HC[O.sup.-.sub.3]-rich fluid originates. Using digital videomicroscopy, we measured the osmotic water permeability ([P.sub.f]) of pancreatic duct epithelium by exposing isolated rat interlobular ducts to the hypotonic solution (145 mosM). To identify mRNA and protein of AQPs expressed in duct cells, we conducted RT-PCR analysis and immunohistochemistry of the isolated duct and pancreas. The calculated [P.sub.f] (160-230 [micro]m/s) of the isolated ducts was significantly reduced to 16-35 [micro]m/s by 80-90% with either basolateral or luminal applications of Hg[Cl.sub.2]. Fluid secretion evoked by secretin was almost completely abolished by a basolateral or luminal application of Hg[Cl.sub.2]. A large amount of AQP1 and small amounts of AQP5 transcripts were detected in the isolated duct cells by RT-PCR. AQP1, but not AQP5, immunoreactivity was present in both luminal and basolateral membranes of the interlobular duct cells. Mercury-sensitive water channels are present in both luminal and basolateral membranes of rat pancreatic ducts. AQP1 of the known AQPs appears to be the main water pathway in interlobular ducts. isolated pancreatic duct; mercury-sensitive water channel; water permeability; fluid secretion

Recent crystallographic studies revealed that G-protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8, at the proximal region of the C-terminal tail. However, the significance of helix 8 in GPCR functions and signaling is not fully understood. Helix 8 mutants of leukotriene B4 receptor type 1 (BLT1) exhibit prolonged activation after ligand stimulation, suggesting some regulatory roles of helix 8 in GPCR signaling. Here, we report the inhibitory role of BLT1 helix 8 on ligand-dependent internalization using BLT1 and platelet-activating factor receptor as model GPCRs. Mutating die dileucine motif in helix 8 of BLT1 to alanines (BLT1 LLAA) enhanced LTB4-dependent internalization of BLT1, whereas wild-type (WT) BLT1 exhibited minimal internalization. Mutational studies revealed diat phosphorylation of 5 serine/threonine residues between amino acids 308 and 319 of BLT1 was responsible for enhanced ligand-dependent internalization of BLT1 LLAA. BLT1 LLAA showed enhanced basal and ligand-dependent phosphorylation compared to WT BLT1. Taken together, helix 8 of BLT1 inhibits receptor internalization by suppressing the excessive phosphorylation of the C-terminal tail. [ABSTRACT FROM AUTHOR]

The morphological effect of endothelin-1 (ET-1) and the distribution of endothelin-binding sites on the kidney and lung was investigated ultrastructually by intravenous injection of [I]-ET-1 into rats. About 10% decrease of the diameter of glomeruli was observed at 10 min after the injections of ET-1 or [I]-ET-1 (1.3-2.4 nmole/kg). When localization of [I]-ET-1 in the kidney was examined by light and electron microscopic radioautography, silver grains were preferably localized on the fenestrated endothelial cells of glomeruli and peritubular capillary endothelial cells. Some grains were also localized on the interdigitating processes of urinary tubules. Quantitative analysis of silver grains in the glomeruli showed that 83% of grains were located on the fenestrated endothelial cells, 12% on the podocytes of visceral cells, and 5% on mesangial cells at 10 min. After 60 min, 50% of silver grains were incorporated into the cytoplasm of fenestrated endothelial cells. In contrast to glomeruli, silver grains were rare on the arteries and large arterioles. However, a few silver grains were often observed on the smooth muscle cells of small arterioles (8-20 μm in diameter). In the lung, 70% of silver grains were located on the alveolar capillary endothelial cells. These results indicate the abundance of ET receptors on the glomerular fenestrated endothelium, peritubular fenestrated endothelium and alveolar capillary endothelium. [ABSTRACT FROM AUTHOR]

Localization of intravenously injected [I]-endothelin-1 was examined in rat liver by light and electron microscopic radioautography. At 10 min after injection, silver grains were localized along the sinusoidal wall, i.e., mostly on the thin processes of fat-storing cells and sinusoidal endothelial cells, and also on the Kupffer cells and the microvilli of hepatocytes. About 35% of the total silver grains were located on the processes of fat-storing cells at 10 min. The grain density (number of silver grains/cell area) of fat-storing cells was three-fold that of Kupffer cells, and 18-fold that of hepatocytes. At 60 min, 60% of the total grains were observed on the fat-storing cells, though the value of grain density was not changed. Silver grains were internalized into the cytoplasm of fat-storing cells and often associated with multivesicular bodies. In contrast, the grain density of endothelial cells and Kupffer cells decreased with time. These results indicate that hepatic fat-storing cells have a considerable number of endothelin-binding sites, and incorporate bound endothelin into cytoplasm. [ABSTRACT FROM AUTHOR]

Mammary myoepithelial cells were isolated and cultured to characterize their properties. The intracellular calcium concentration (Cai 2+) was measured using the ratio of fura-2 fluorescence at 340 nm to that at 360 nm ( F340/ F360), and the contraction was simultaneously monitored by the change of fluorescence intensity at 360 nm ( F360). Cultured myoepithelial cells retained their contractile machinery as in the intact tissue. NBD-phallacidin fluorescence, which marks F-actin, and electron microscopy showed abundant bundles of microfilaments in the cytoplasm. Oxytocin (> 0.1 nM) induced an increase in Cai 2+ and contraction. The amplitude and time course of the Cai 2+ increase were not markedly affected in the Ca2+-free solution. Nifedipine (10 μM) did not affect the response to oxytocin. ATP (>1 μM) induced an increase in Cai 2+ and contraction. The response to ATP was not affected by Ca2+ removal, but was suppressed by suramin (100 μM), an antagonist of P2-purinergic receptors. The order of potency of nucleotides to increase Cai 2+ was ATP = ADP > UTP > UDP. These findings indicate the presence of P2-purinergic receptors in mammary myoepithelial cells. The results suggest that stimulant-induced Cai 2+ increases and contractions are due to release of Ca2+ from intracellular stores in these cells. In addition to the hormonal action of oxytocin, extracellular nucleotides may function as paracrine agents to contract myoepithelial cells in the mammary gland. [ABSTRACT FROM AUTHOR]

Rats were intravenously injected with synthetic I-endothelin, and then were fixed by intracardiac perfusion at 10min, 30min and 1 hr after the injection. Localization of I-endothelin was examined in the small intestine by light and electron microscopic radioautography. Through the time course examined, more than seventy percent of total silver grains were localized in the fibroblasts, especially beneath the basal lamina of epithelium of the intestinal villi. Silver grains were observed mostly on the thin processes, and also on the vacuoles. At lOmin, 80% of silver grains on the fibroblasts were located on the plasmalemma. By the chase, grains on the plasmalemma decreased, and silver grains in the cytoplasm increased gradually. [ABSTRACT FROM PUBLISHER]

Exocytosis of cultured bovine adrenal chromaffin cells was examined in a balanced salt solution containing Ficoll using rapid freezing followed by freeze-substitution. In solution without Ficoll, exposure to Ba produced many exocytotic lumina between the cells; in most cases, these lumina were large and empty. When chromaffin cells were exposed to Ba in Ficoll, it was possible to observe a small pore between the plasmalemma and each granule. The granule retained the dense contents when the pore was approximately 20 nm in diameter, and became empty when the pore was widened in 14% Ficoll. The addition of Ficoll made it easy to catch the events occurring during exocytosis of individual bovine chromaffin granules. [ABSTRACT FROM PUBLISHER]

Recent crystallographic studies revealed that G-protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8, at the proximal region of the C-terminal tail. However, the significance of helix 8 in GPCR functions and signaling is not fully understood. Helix 8 mutants of leukotriene B4 receptor type 1 (BLT1) exhibit prolonged activation after ligand stimulation, suggesting some regulatory roles of helix 8 in GPCR signaling. Here, we report the inhibitory role of BLT1 helix 8 on ligand-dependent internalization using BLT1 and platelet-activating factor receptor as model GPCRs. Mutating the dileucine motif in helix 8 of BLT1 to alanines (BLT1 LLAA) enhanced LTB4-dependent internalization of BLT1, whereas wild-type (WT) BLT1 exhibited minimal internalization. Mutational studies revealed that phosphorylation of 5 serine/threonine residues between amino acids 308 and 319 of BLT1 was responsible for enhanced ligand-dependent internalization of BLT1 LLAA. BLT1 LLAA showed enhanced basal and ligand-dependent phosphorylation compared to WT BLT1. Taken together, helix 8 of BLT1 inhibits receptor internalization by suppressing the excessive phosphorylation of the C-terminal tail.