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The cellular distribution of parchorin, a new chloride intracellular channel family member, was investigated in rabbit tissues by immunohistochemistry using an antibody recognizing the sequence containing a parchorin-specific repeat. Parchorin preferentially resides in the epithelium of the ducts of the lacrymal, parotid, sub-mandibular, and mammary glands and the pancreas, prostate, and testis. In the trachea and lung, parchorin was found in the airway epithelium and type II alveolar cells. In the kidney, parchorin was distributed mainly from the thick ascending limb to the distal convoluted tubule. In the eye, both pigment and nonpigment epithelia of the ciliary body were positive, whereas only the pigment epithelium was positive in the retina. Parchorin was also present in the cochlea and semicircular canal. The amount of parchorin in the gastric mucosa, but not in the submandibular glands, increased after weaning. In the mammary gland, parchorin expression was greater in a lactating rabbit (1 wk after delivery) compared with a pregnant (3 wk) rabbit. The cellular distribution and changes in expression indicate that parchorin plays an important role, possibly in chloride transport, in the cells that create an ion gradient for water movement. chloride channel; duct; rabbit; immunohistochemistry

Author(s): Motohiro Nishida [1]; Yoshiko Maruyama [1]; Rie Tanaka [1]; Kenji Kontani [2]; Taku Nagao [1]; Hitoshi Kurose (corresponding author) [1] Reactive oxygen species (ROS) have been identified as central [...]

The role of endothelial calcium in the regulation of vascular tone was investigated using intact coronary arteries in dogs. Calcium channel and nitric oxide synthase studies were carried out to determine the mechanism of calcium-mediated regulation of vascular tone in coronary arteries depolarized by high potassium concentration. Results indicate that endothelial calcium mediates vascular relaxation by activating nitric oxide synthase.

The antisecretory mechanisms that mediate the action of the antiulcer drug, ME-3407, were characterized in vivo in isolated gastric glands and purified H+-K+-adenosine triphosphatase (ATPase). The presence of ME-3407 in isolated gastric glands induced alterations in the incorporation of tubulovesicle-rich H+-K+-ATPase into the apical plasma membrane. The presence of ME-3407 in gastric glands also resulted in the displacement of the ezrin membrane cytoskeletal linker protein. Furthermore, the drug affected membrane recruitment events mediating parietal cell stimulation.

Calyculin A (CLA) stimulates acid secretion in isolated rabbit gastric glands without affecting their adenosine 3',5'-cyclic monophosphate contents. CLA-stimulated secretion is due to an increase in the phosphorylation of protein kinase A substrates, such as ezrin, through the inhibition of protein phosphatase (PrPase) type 1. The inhibition of protein kinase A suppresses the acid secretion. CLA and okadaic acid (OKA) are potent inhibitors of PrPase, but OKA does not stimulate acid secretion.

Abstract Background Transcriptome data from quantitative PCR (Q-PCR) and DNA microarrays are typically obtained from a fixed amount of RNA collected per sample. Therefore, variations in tissue cellularity and RNA yield across samples in an experimental series compromise accurate determination of the absolute level of each mRNA species per cell in any sample. Since mRNAs are copied from genomic DNA, the simplest way to express mRNA level would be as copy number per template DNA, or more practically, as copy number per cell. Results Here we report a method (designated the "Percellome" method) for normalizing the expression of mRNA values in biological samples. It provides a "per cell" readout in mRNA copy number and is applicable to both quantitative PCR (Q-PCR) and DNA microarray studies. The genomic DNA content of each sample homogenate was measured from a small aliquot to derive the number of cells in the sample. A cocktail of five external spike RNAs admixed in a dose-graded manner (dose-graded spike cocktail; GSC) was prepared and added to each homogenate in proportion to its DNA content. In this way, the spike mRNAs represented absolute copy numbers per cell in the sample. The signals from the five spike mRNAs were used as a dose-response standard curve for each sample, enabling us to convert all the signals measured to copy numbers per cell in an expression profile-independent manner. A series of samples was measured by Q-PCR and Affymetrix GeneChip microarrays using this Percellome method, and the results showed up to 90 % concordance. Conclusion Percellome data can be compared directly among samples and among different studies, and between different platforms, without further normalization. Therefore, "percellome" normalization can serve as a standard method for exchanging and comparing data across different platforms and among different laboratories.

Gastric vesicles purified from acid-secreting rabbit stomach display [K.sup.+] permeability manifested by the valinomycin-independent proton pumping of [H.sup.+]-[K.sup.+]-ATPase as monitored by acridine orange quenching. This apparent [K.sup.+] permeability is attenuated by the treatment of the membrane with 5 mM [Mg.sup.2+], and this phenomenon has been attributed to membrane-bound phosphoprotein phosphatase. However, with the exception of the nonspecific inhibitor pyrophosphate, protein phosphatase inhibitors failed to inhibit the loss of [K.sup.+] permeability. Preincubation of the membrane with neomycin, a phospholipase C inhibitor, surrogated the effect of [Mg.sup.2+], whereas another inhibitor, U-73122, did not. Phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) restored the attenuated [K.sup.+] permeability by treatment with either [Mg.sup.2+] or neomycin. Furthermore, either phosphatidylinositol bound to phosphatidylinositol transfer protein or phosphatidylinositol 4,5,6-trisphosphate ([PIP.sub.3]) surrogated the effect of [PIP.sub.2]. [Mg.sup.2+] and neomycin reduced [K.sup.+] permeability in the membrane as determined by [Rb.sup.+] influx and [K.sup.+]-dependent [H.sup.+] diffusion. Treatment with [Mg.sup.2+] reduced the contents of [PIP.sub.2] and [PIP.sub.3] in the membrane. These results suggest that [PIP.sub.2] and/or [PIP.sub.3] maintain [K.sup.+] permeability, which is essential for proton pumping in the apical membrane of the secreting parietal cell. hydrogen; potassium; adenosinetriphosphatase; neomycin

A proposed mechanism for the potentiation of cAMP-mediated acid secretion by carbachol. Am J Physiol Cell Physiol 280: C155-C165, 2001.--Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [[sup.14]C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular [Ca.sup.2+] and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 [micro]M preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+].sub.i]) increase due to release from the [Ca.sup.2+] store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular [Ca.sup.2+] release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the [Ca.sup.2+] store and the receptor is maintained by actin microfilaments. inositol 1,4,5-trisphosphate receptor; cytoskeleton; cytochalasin D; calcium; parietal cell; rabbit

Akagi, Keiko, Taku Nagao, and Tetsuro Urushidani. Responsiveness of [Beta]-escin-permeabilized rabbit gastric gland model: effects of functional peptide fragments. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G736-G744, 1999.--We established a [Beta]-escin-permeabilized gland model with the use of rabbit isolated gastric glands. The glands retained an ability to secrete acid, monitored by [[sup.14]C] aminopyrine accumulation, in response to cAMP, forskolin, and histamine. These responses were all inhibited by cAMP-dependent protein kinase inhibitory peptide. Myosin light-chain kinase inhibitory peptide also suppressed aminopyrine accumulation, whereas the inhibitory peptide of protein kinase C or that of calmodulin kinase II was without effect. Guanosine-5'-O-(3-thiotriphosphate) (GTP[Gamma]S) abolished cAMP-stimulated acid secretion concomitantly, interfering with the redistribution of [H.sup.+]-[K.sup.+]-ATPase from tubulovesicles to the apical membrane. To identify the targets of GTP[Gamma]S, effects of peptide fragments of certain GTP-binding proteins were examined. Although none of the peptides related to Rab proteins showed any effect, the inhibitory peptide of Arf protein inhibited cAMP-stimulated secretion. These results demonstrate that our new model, the [Beta]-escin-permeabilized gland, allows the introduction of relatively large molecules, e.g., peptides, into the cell, and will be quite useful for analyzing signal transduction of parietal cell function. parietal cell; acid secretion; small GTP-binding protein; protein kinases