Transport, active

Specificity The transporter has a similar specificity to hexokinase it transports several hexoses, glucose, mannose, 2-deoxyglucose. Similarly, the optical isomer, D-glucose, but not L-glucose, is transported (Chapter 3). 

Competition The rate of transport of one sugar is reduced by the transport of another sugar the kinetics are identical to that of competitive inhibition of hexokinase by the presence of other sugars. 

Specific activation or inhibition Transport of glucose can be increased or decreased by specihc compounds insulin increases the transport whereas phloridzin, a plant glycoside, inhibits glucose transport by muscle. Insulin increases glucokinase activity in liver, whereas a plant sugar, mannoheptulose, inhibits glucokinase activity. Hexokinase is inhibited by its product, glucose 6-phosphate. 

The HCOj is transported out of the red cell via a transporter that transports the chloride ion into the erythrocyte. The reverse process occurs in the lungs the hydrogencarbonate is transported into the erythrocyte in exchange for the chloride ion. Within the cell, the hydrogencarbonate ion is converted to CO2, via the same enzyme. 

The CO2 diffuses from the erythrocyte into the blood and across the membrane of the alveoli into the air in the alveoli, for loss to the enviroment. 

Active or carrier-mediated transport involves using membrane proteins to transport substances across the cell membrane (see Fig. 2-2). Membrane proteins that span the entire membrane may serve as some sort of carrier that shuttles substances from one side of the membrane to the other.41 Characteristics of active transport include the following  

Carrier specificity. The protein carrier exhibits some degree of specificity for certain substances, usually discriminating among different compounds according to their shape and electrical charge. This specificity is not absolute, and some compounds that resemble one another will be transported by the same group of carriers. 

Expenditure of energy. The term active transport implies that some energy must be used to fuel the carrier system. This energy is usually in the form of adenosine triphosphate (ATP) hydrolysis. 

Ability to transport substances against a concentration gradient. Carrier-mediated active transport may be able to carry substances uphill —that is, from areas of low concentration to areas of high concentration. 

Certain cells have the ability to transport substances across their membranes through processes such as endocytosis. Here the drug is engulfed by the cell via an invagination of the cell membrane. Although limited in scope, this method does allow certain large, nonlipid-soluble drugs to enter the cell. 

The main factor that governs the transport of a compound by an active carrier system is the interaction of this compound with a carrier protein. In this case the description of the molecular structure should be similar to that used in ligand-based design to describe the interaction of the compound with any other protein using pharmacophoric representation or 3D-QSAR. 

Sometimes it is useful to apply a combination of the descriptors based on the global properties of the molecule and those based on a pharmacophoric representation. Conceptually, the global properties would better describe the imtial passive membrane permeation required to reach the site of action. Then, the specific protein interactions could be explained by the pharmacophoric descriptors. This has been demonstrated successfully in the P-glycoprotein case [26], where two processes are important for the transport passive transport to the cell and active 

The distribution of a compound in the human body can also be partially related to the absorption properties. There are specific transport systems that are expressed in certain tissues that can influence the distribution of the compound. For example, rosuvastatin, a new member of the statin family is transported by the OATP-C carrier system, which is selectively expressed in the liver, making this compound selectively distributed into this organ [27]. In general it is not possible to derive computational models for these selective transport systems since there is not yet enough experimental information and data to support the model building and validation. Nevertheless, there are three properties that are commonly used to describe the distribution of a compound in the human body the solubility, the unspecific binding of the compound to plasma proteins and the volume of distribution. 

Solubility is a key property in the distribution of the compound from the gastrointestinal tract to the blood. There have been several modeling efforts to predict the solubility, based on different type of descriptors. The intrinsic solubility (thermodynamic solubility of the neutral species) for a set of 1028 compounds has been modeled using the VolSurf descriptors based on GRID-MIFs (Fig. 10.9(a)) and PLS multivariate analysis [20]. The interpretation of the model can be based on the PLS coefficients the ratio of the surface that has an attractive interaction with the water probe contributes positively to the solubility, while the hydrophobic interactions and log P have a negative contribution. 

Although several models to predict the solubility have been published, none take into account the crystal packing of the compounds. Neglecting the crystal packing could be relevant for some compounds, causing the solubility prediction to fail. 

Some laboratories have found an alternative to the short-term cultures by using cell lines other than Caco-2 cells. The most popular of these is Madin-Darby canine kidney (MDCK) cells, an epithelial cell line from the dog kidney. MDCK cells have been suggested to perform as well as Caco-2 cells in studies of passive drug permeability [56]. These cells have also been used to optimise the conditions for studies of low-solubility drugs [53]. However, as noted previously, the active transport processes of this cell line can be quite different to those of Caco-2 cells [28-30], Another cell line that only requires short-term culture is 2/4/A1, which is a conditionally immortalised rat intestinal epithelial cell line [86]. The 2/4/A1 cell line is discussed in Section 4.3.2.2 below. 

Limitations Related to Transport Studies and Their Solutions 4.3.2.1 Active Transport 

Another limitation is that there is no quantitative relationship between active drug transport in the cell culture models and in vivo e.g. [92, 93]. The reason may be that the expression level of the transporter in Caco-2 cells is not comparable to that in vivo or that there is a difference in effective surface area (see Section 4.3.2.2 below). One solution to this problem is to determine the apparent transport constants, Km and Vmax, for each transporter and subsequently, to determine a scaling factor. However, this is not readily done. In addition these studies are further complicated by the lack of specific substrates. For example, there are almost no specific substrates for the drug efflux transporters [18]. Therefore, other epithelial 

2 cells may therefore remain a viable future alternative also in the future in some of these situations, provided that sufficiently specific substrates or inhibitors can be identified (Fig. 4.4). 

In conclusion, there are several drawbacks to the use of Caco-2 cells in studies of active drug transport. Despite these drawbacks, we note that a recent comprehensive study comparing various P-glycoprotein drug efflux assays in drug discovery came to the conclusion that the Caco-2 transport assay is the method of choice, since it displays a biased responsiveness towards compounds with low or moderate permeability - in other words, towards compounds whose intestinal permeability is most likely to be significantly affected by drug efflux mechanisms [101]. 

With LqB = LBq = LbbAH, this equation is transformed into the facilitated form 

At static head (JB. / 0), we have the maximum mass load A imax. From equation 

By using LqB = LBq = LBBAH, this equation can be transformed into 

Diffusion occurs spontaneously from a region of higher chemical potential mi to a region of lower chemical potential P-2- and the direction of flow is the same as the direction of decreasing chemical potential. The total Gibbs energy change for such a system is expressed by 

Almost all cells have an active transport system to maintain nonequilibrium concentration levels of substrates. For example, in the mitochondrion, hydrogen ions are pumped into the intermembrane space of the organelle as part of producing ATP. Active transport concentrates ions, minerals, and nutrients inside the cell that are in low concentration 

The standard free energy change for the movement of an uncharged molecule from one side of a membrane at concentration Ci to the other at concentration Cs is given by the usual equation  

for a simple diffusion process is unity. That is, at diffusion equilibrium, the concentration of the solute is the same on both sides of thfe membrane, Thus, the AC for the movement of the solute from side 1 to side 2 under nonequilibrium conditions is given by  

Z = the net charge on the molecule (+1 for Na ) and A P = the membrane potential in volts (internal relative to external) 

The concentration of chloride ion in blood serum is out 0. lOM. The concentration of chloride ion in urine is about 0.16 M. (a) Calculate the energy expended by the kidneys in transp orting chloride from plasma to urine, (b) How many moles of Cl ions could be transported per mole of ATP hydrolyzed  

oxidation-reduction reactions (or ATP hydrolysis) within the cell membrane generates a ApH of 1 (interior higher by one unit) and a A P of — I20mv (interior negative). Alqng with El ions, /3-galactosides are cotransported in response to the total proton-motive force. (a) How much 

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Special demands are made to the laboratories that perform radiographic testing. They must observe sanitary norms and rules of radiation safety in their activities. Transportation of the equipment for implement works on site has to ensure observance of the requirements of the radiation safety. 

Calcium is absorbed from the intestine by facilitated diffusion and active transport. In the former, Ca " moves from the mucosal to the serosal compartments along a concentration gradient. The active transport system requires a cation pump. In both processes, a calcium-binding protein (CaBP) is thought to be required for the transport. Synthesis of CaBP is activated by 1,25-DHCC. In the active transport, release of Ca " from the mucosal cell into... 

CycHc adenosine monophosphate (cAMP), produced from ATP, is involved in a large number of ceUular reactions including glycogenolysis, Hpolysis, active transport of amino acids, and synthesis of protein (40). Inorganic phosphate ions are involved in controlling the pH of blood (41). The principal anion of interceUular fluid is HP (Pig. 3) (41). 

Active Transport. Maintenance of the appropriate concentrations of K" and Na" in the intra- and extracellular fluids involves active transport, ie, a process requiring energy (53). Sodium ion in the extracellular fluid (0.136—0.145 AfNa" ) diffuses passively and continuously into the intracellular fluid (<0.01 M Na" ) and must be removed. This sodium ion is pumped from the intracellular to the extracellular fluid, while K" is pumped from the extracellular (ca 0.004 M K" ) to the intracellular fluid (ca 0.14 M K" ) (53—55). The energy for these processes is provided by hydrolysis of adenosine triphosphate (ATP) and requires the enzyme Na" -K" ATPase, a membrane-bound enzyme which is widely distributed in the body. In some cells, eg, brain and kidney, 60—70 wt % of the ATP is used to maintain the required Na" -K" distribution. 

Care should be exercised when attempting to interpret in vivo pharmacological data in terms of specific chemical—biological interactions for a series of asymmetric compounds, particularly when this interaction is the only parameter considered in the analysis (10). It is important to recognize that the observed difference in activity between optical antipodes is not simply a result of the association of the compound with an enzyme or receptor target. Enantiomers differ in absorption rates across membranes, especially where active transport mechanisms are involved (11). They bind with different affinities to plasma proteins (12) and undergo alternative metaboHc and detoxification processes (13). This ultimately leads to one enantiomer being more available to produce a therapeutic effect. 

Biochemically, most quaternary ammonium compounds function as receptor-specific mediators. Because of their hydrophilic nature, small molecule quaternaries caimot penetrate the alkyl region of bdayer membranes and must activate receptors located at the cell surface. Quaternary ammonium compounds also function biochemically as messengers, which are generated at the inner surface of a plasma membrane or in a cytoplasm in response to a signal. They may also be transferred through the membrane by an active transport system. 

Materials may be absorbed by a variety of mechanisms. Depending on the nature of the material and the site of absorption, there may be passive diffusion, filtration processes, faciHtated diffusion, active transport and the formation of microvesicles for the cell membrane (pinocytosis) (61). EoUowing absorption, materials are transported in the circulation either free or bound to constituents such as plasma proteins or blood cells. The degree of binding of the absorbed material may influence the availabiHty of the material to tissue, or limit its elimination from the body (excretion). After passing from plasma to tissues, materials may have a variety of effects and fates, including no effect on the tissue, production of injury, biochemical conversion (metaboli2ed or biotransformed), or excretion (eg, from liver and kidney). 

The influx of Ca(Il) across the presynaptic membrane is essential for nerve signal transmission involving excitation by acetylcholine (26). Calcium is important in transducing regulatory signals across many membranes and is an important secondary messenger hormone. The increase in intracellular Ca(Il) levels can result from either active transport of Ca(Il) across the membrane via an import channel or by release of Ca(Il) from reticulum stores within the cell. More than 30 different proteins have been linked to regulation by the calcium complex with calmoduhn (27,28). 

Because bretylium is poody absorbed from the GI tract (- 10%), it is adrninistered iv or im. Very litde dmg is protein bound in plasma. Bretylium is taken up by an active transport mechanism into and concentrated in postganglionic nerve terminals of adrenergicahy innervated organs. Peak plasma concentrations after im injections occur in about 30 min. Therapeutic plasma concentrations are 0.5—1.0 p.g/mL. Bretylium is not metabolized and >90% of the dose is excreted by the kidneys as unchanged dmg. The plasma half-life is 4—17 h (1,2). 

Fig. 2. Schematic representation of relevant electrolyte transport through the renal tubule, depicting the osmolar gradient ia medullary iaterstitial fluid ia ywOj yW where represents active transport, —passive transport, hoth active and passive transport, and passive transport of H2O ia the presence of ADH, ia A, the cortex, and B, the medulla. An osmole equals a mole of solute divided by the number of ions formed per molecule of the solute. Thus one mole of sodium chloride is equivalent to two osmoles, ie, lAfNaCl = 2 Osm NaCl. ADH = antidiuretic hormone.

From a thermodynamic and kinetic perspective, there are only three types of membrane transport processes passive diffusion, faeilitated diffusion, and active transport. To be thoroughly appreciated, membrane transport phenomena must be considered in terms of thermodynamics. Some of the important kinetic considerations also will be discussed. 

All Active Transport Systems Are Energy-Coupling Devices... 

The gradients of H, Na, and other cations and anions established by ATPases and other energy sources can be used for secondary active transport of various substrates. The best-understood systems use Na or gradients to transport amino acids and sugars in certain cells. Many of these systems operate as symports, with the ion and the transported amino acid or sugar moving in the same direction (that is, into the cell). In antiport processes, the ion and the other transported species move in opposite directions. (For example, the anion transporter of erythrocytes is an antiport.) Proton symport proteins are used by E. coU and other bacteria to accumulate lactose, arabinose, ribose, and a variety of amino acids. E. coli also possesses Na -symport systems for melibiose as well as for glutamate and other amino acids. 

Fructose is present outside a cell at 1 /iM concentration. An active transport system in the plasma membrane transports fructose into this cell, using the free energy of ATP hydrolysis to drive fructose uptake. Assume that one fructose is transported per ATP hydrolyzed, that ATP is hydrolyzed on the intracellular surface of the membrane, and that the concentrations of ATP, ADP, and Pi are 3 mM, 1 mM, and 0.5 mM, respectively. T = 298 K. What is the highest intracellular concentration of fructose that this transport system can generate Hint Kefer to Chapter 3 to recall the effects of concentration on free energy of ATP hydrolysis.)... 

Active Transport of Ions Using Synthetic Ionophores... 

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