H/K-ATPase molecules

Figure 6.1 Pseudocolour displays of fluorescence images of FITC-modified H/K-ATPase molecules. FITC-modified H/K-ATPase was solubilized by (a) CjjEs and (b) nOG, and observed by TIRFM.The initial fluorescence intensities with one, two and four units are indicated by single, double and triple arrowheads, respectively. A scale bar of 5 pm and a linear 0-255 pseudocolour scale of fluorescence intensity are shown. A colour version of this figure may be found in the authors original publication [1 ].Reprinted with permission from Abe ef a/.. Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase. Biochemistry 42 (2003) 15132-15138. Copyright 2003 American Chemical Society.

This Illustrates the general mechanism for benzimidazole inhibition of gastric acid secretion. The PPI is absorbed from the duodenum and passes via the blood Into the parietal cell. If the cell is secreting acid, the prodrug accumulates in the canaliculus and undergoes acid-catalyzed conversion to the sulfenamide, whereon It reacts with cysteines in the active H,K ATPase molecules. 

As a further difference, it takes several days for omeprazole to reach its steady-state bioavailability [5]. Corresponding data for lansoprazole and pantoprazole are not available, although unchanged values of C ax and area under the time concentration curve (AUC) indicate the absence of relevant increases after the first dose [7, 8]. At face value, this discrepancy would suggest that the latter drugs reach maximum acid inhibitory efficacy with the first dose and, by this, are apt to provide faster pain relief. Hence, it may be surprising that omeprazole as well as lansoprazole and pantoprazole take several days to reach steady-state inhibition of acid secretion [9-11]. For instance, 40 mg of pantoprazole reduced acid output by 51 and 85% after the first and seventh dose, respectively [11]. This type of behavior, however, is predictable from the way the parietal cell organizes its supply with active H, K" -ATPase molecules [12] unless acid secretion is maximally stimulated, a substantial portion of the pump molecules are stored in... 

Fig. 2. E]-E2 reaction cycle of H,K-ATPase, accounting for the transport of two H and two K ions per molecule of hydrolysed ATP. The ATPase reaction proceeds from 2H E, ATP through 2H E -P and 2H E2-P to 2K E. Details of the reaction cycle are described in the text.

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

The secretion of acid resumes only after new molecules of H+, K+-ATPase are inserted into the gastric mucosa. 

The pharmacokinetics of available proton pump inhibitors are shown in Table 63-2. Their bioavailability is decreased approximately 50% by food hence, the drugs should be administered on an empty stomach. In a fasting state, only 10% of proton pumps are actively secreting acid and susceptible to inhibition. Proton pump inhibitors should be administered approximately 1 hour before a meal (usually breakfast or dinner), so that the peak serum concentration coincides with the maximal activity of proton pump secretion. The drugs have a short serum half-life of about 1.5 hours however, the duration of acid inhibition lasts up to 24 hours due to the irreversible inactivation of the proton pump. At least 18 hours are required for synthesis of new H+/K+ ATPase pump molecules. Because not all proton pumps are inactivated with the first dose of medication, up to 3-4 days of daily medication are required before the full acid-inhibiting potential is reached. Similarly, after stopping the drug, it takes 3-4 days for full acid secretion to return. 

Two molecules of the active intermediate of omeprazole bind to one active site of gastric H /K -ATPase [63, 64], This binding is a disulphide linkage and can be prevented and reversed by the addition of mercaptan [65-67]. Detailed investigations of three reactions of H /K -ATPase enzyme cycle have shown that the K -stimulated ATPase-activity, / -nitrophenol-phosphatase(pNPPase)-activity and formation of phosphoenzyme are also inhibited [63, 68]... 

Figure 6.3 Histogram of the fluorescence intensity of solubilized H/K-ATPase. Distributions of fluorescence intensities of FITC- H/K-ATPase solubilized with SDS ((a) n = 189), Ci2Eg ((b) n = 283) and nOG ((c) n = 206) are shown. The fluorescence intensities of one to four dye molecules were in the linear range of the camera.The solid line indicates the sum of one to four Gaussian components fitted to the data. Single, double and quadruple arrowheads indicate the peak positions of each Gaussian distribution that is responsible for one, two or four fluorescence molecules, respectively. Reprinted with permission from Abe etal., Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase. Biochemistry 42 (2003) 1 5132-1 5138. Copyright 2003American Chemical Society.

The H, K -ATPase is, except for the kiiiey, only localized to the parietal cell. In vivo the kidney H, H -ATPase is not inhibited by PPIs, probably due to the fact that there is no acidic milieu in the vicinity of the enzyme and thus no transformation to the active molecule, the sulphenamide. 

There are, however, various types of active transport systems, involving protein carriers and known as uniports, symports, and antiports as indicated in Figure 3.7. Thus, symports and antiports involve the transport of two different molecules in either the same or a different direction. Uniports are carrier proteins, which actively or passively (see section "Facilitated Diffusion") transport one molecule through the membrane. Active transport requires a source of energy, usually ATP, which is hydrolyzed by the carrier protein, or the cotransport of ions such as Na+ or H+ down their electrochemical gradients. The transport proteins usually seem to traverse the lipid bilayer and appear to function like membrane-bound enzymes. Thus, the protein carrier has a specific binding site for the solute or solutes to be transferred. For example, with the Na+/K+ ATPase antiport, the solute (Na+) binds to the carrier on one side of... 

Active transport of a molecule across a membrane against its concentration gradient requires an input of metabolic energy. In the case of ATP-driven active transport, the energy required for the transport of the molecule (Na+, K+, Ca2+ or H+) across the membrane is derived from the coupled hydrolysis of ATP (e.g Na+/K+-ATPase). In ion-driven active transport, the movement of the molecule to be transported across the membrane is coupled to the movement of an ion (either Na+ or H+) down its concentration gradient. If both the molecule to be transported and the ion move in the same direction across the membrane, the process is called symport (e.g. Na+/glucose transporter) if the molecule and the ion move in opposite directions it is called antiport (e.g. erythrocyte band 3 anion transporter). 

In this case, the energy required for the transport of the molecule across the membrane is derived from the coupled hydrolysis of ATP, for example the movement of Na+ and K+ ions by the Na+/K+-ATPase. All cells maintain a high internal concentration of K+ and a low internal concentration of Na+. The resulting Na+/K+ gradient across the plasma membrane is important for the active transport of certain molecules, and the maintenance of the membrane electrical potential (see Topic N3). The movement across the membrane of Na+, K+, Ca2+ and H+, as well as a number of other molecules, is directly coupled to the hydrolysis of ATP. 

Transport of many compounds including drugs across cell membranes is mediated by membrane proteins called carrier proteins or channel proteins. Some of these proteins transport only one substrate molecule at a time across the membrane (uniport systems), while others act as cotransport systems (Figure 9.4). Depending on the direction of the second substrate, the proteins are also called symporters or antiporters, for example, Na /glucose cotransporter, H " /peptide cotransporter, or Na /K antiporter (—Na /K -ATPase). 

Fig. 3. Schematic presentation of the binding of AMPPNP to a dimeric model of (K. + H l-ATPase and the effect of Mg on this process. Symbol represents a dimeric enzyme molecule, O stands Cor AMPPNP, for Mg ".

The plasma membrane contains an enzyme that catalyzes the export of Ca + from the cytoplasm at the expense of ATP hydrolysis. The Ca +-ATPase has features that place it in the category of plasma membrane enzymes that also includes the Na+/K+-ATPase and the H+-ATPase. The Ca +-ATPase functions to keep the cytosolic Ca +concentration low (< 1 fiM). It is not a major contributor to the generation of the membrane potential or to the energetics of the transport of bioorganic molecules. 

Asokan and Cho [83] reviewed the distribution of pH environments in the cell. Much of what is known in the physiological literature was determined using pH-sensitive fluorescent molecules and specific functional inhibitors. The physiological pH in the cytosol is maintained by plasma membrane-bound H+-ATPases, ion exchangers, as well as the Na+/K+-APTase pumps. Inside the organelles, pH microenvironments are maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Table 2.1 lists the approximate pH values of the various cellular compartments. 

---