H+, K+-ATPase is a proton pump responsible for gastric acid secretion. It actively transports proton and K+ coupled with the hydrolysis of ATP, resulting in the formation of a 106 fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K+-ATPase) and the Ca2+ pump (Ca2+-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K+-ATPase. One of the antibodies, HK2032 inhibited the H+, K+-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K+-ATPase molecule, and that The H+, K+-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K+-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K+-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K+-ATPase. This compound inhibited the H+, K+-ATPase activity by stabilizing the K+-form of the enzyme. Third we studied the conformational changes of the H+, K+-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K+-ATPase did not utilize acetylphosphate instead of ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K+-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.
Most peptide drugs are hydrophilic molecules with a molecular weight between 300 and 20000 and such molecules are usually given by parenteral administration. In many cases, enteral administration of these peptides via the gastrointestinal tract is preferred. However, oral administration of peptides and proteins is often limited by their instability in the gastrointestinal environment and/or poor absorption from the gut. To promote the absorption of these drugs, we first discovered unsaturated fatty acids with absorption enhancing activities and less harmful properties to the gastrointestinal membranes in hydrolysates of natural oil. The mechanisms whereby the permeability of drugs was enhanced by the fatty acids are associated with the disorder in the membrane's interior and the interaction of these fatty acids with the polar head group of phospholipid. Furthermore, we suggested that a SH-related substance was involved in the permeability enhancing effect of these fatty acids. Secondly, we developed a lympho-targeting delivery system for bleomycin by the combined effects of an ion-pair complex with dextran sulfate (DS) and an absorption enhancer. We found a very high lymphatic concentration when administered bleomycin-DS together with the absorption enhancer. Its mechanism may be due to a molecular sieving in the blood-lymph barrier in the intestinal tissues. Finally, to improve the intestinal absorption of peptides, we synthesized novel lipophilic derivatives of peptides including TRH (thyrotropin releasing hormone), tetragastrin, enkephalin, calcitonin and insulin by a chemical modification with fatty acids, while maintaining their pharmacological activities. The stability and permeability of these peptides were improved by acylation with some fatty acids having appropriate carbon numbers. Thus, we have established the strategies for improving the delivery of peptide drugs by various approaches. In future, the combination use of these approaches will be expected to develop the delivery systems of these drugs for therapeutic treatment.
It has long been thought that intestinal absorption of most of the drugs proceeds by passive diffusion mechanism, in which lipid solubility of the drug molecule is a determinant factor. However, water-soluble natural compounds such as amino acids and sugars can move across cell membranes by the specialized carrier-mediated transport mechanisms. Although some drugs which are structurally analogous to natural compounds have been suggested to be absorbed by such transporters, no clear evidence for the involvement of carrier-mediated transport mechanisms has been obtained. In the present study, through the approachs by means of the molecular cloning and functional expression of drug transporters as well as membrane physiological analysis for the drug transport across the intestinal epithelial cell membranes, participation of the carrier-mediated transport mechanisms for the drug absorption was clarified. They include peptide transporter, monocarboxylic acid transporter, anion antiporter, and P-glycoprotein. Most of them have a function for the uptake of drugs into epithelial cells, leading to the increased absorption of drugs, whereas P-glycoprotein excludes drugs into the lumen, thereby decreasing the apparent absorbability of drugs. A rat intestinal monocarboxylic acid-proton cotransporter, MCT1, and an anion antiporter, AE2, were suggested to contribute to the pH-dependent intestinal absorption of monocarboxylic acids such as benzoic acid, lactic acid, nicotinic acid, and valproic acid. An involvement of such pH-dependent transporters in the intestinal absorption of weak organic acids is important, because they may have an alternative mechanism against passive diffusion according to the pH-partition hypothesis. PepT1 cloned from rat intestinal epithelial cells as a peptide transporter was clarified to localize at the intestinal epithelial brush-border membrane and to function for the absorption of β-lactam antibiotics by the proton-gradient energized mechanism. In contrast, P-glycoprotein functions for the secretion of drugs into the intestinal lumen, thereby decreasing intestinal absorption of an immunosuppressive, cyclosporin A and a 5-HT3 receptor antagonist, azasetron. These lines of studies on the clarification of carrier-mediated drug absorption mechanisms will provide new knowledge for the strategies to the enhancement of intestinal absorption of drugs.
This review describes the physiological and pharmacological effects of simple alkyl- and arylpyrazines. The description is made in the following order 1) the presence of 2, 5-dimethylpyrazine in the urine samples of human being and rodents 2) the effects of tetramethyl- and tetraethylpyrazines on the vascular smooth muscles 3) the activity of alkyl- and arylpyrazines on the aggregation of arachidonic acid- and collagen-induced platelets 4) the effects of 2, 5-dimethylpyrazine on the reproductive and the accessory reproductive organs of female and male rats 5) the effects of 2, 5-dimethylpyrazine on the plasma and on the contents of polyamines and fructose in the accessory reproductive organs of rats.
The antiallergic effects of green tea, oolong tea, and black tea extracts by hot water were examined. These extracts inhibited the passive cutaneous anaphylaxis (PCA) reaction of rat after oral administration. Three tea catechins, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) isolated from green tea showed stronger inhibitory effects than that of a green tea extract on the PCA reaction. The inhibitory effects of EGC and EGCg on the PCA reaction were greater than that of ECg. Caffeine also showed a inhibitory effect on the PCA reaction. These results indicate that tea could provide a significant protection against the type-I allergic reaction. These findings also suggest that tea catechins and caffeine play an important role in having an inhibitory effect on the type-I allergic reaction.