Lecture 4 Absorption and transport of xenobiotics across cell membranes [2024].pptx

+ BCHEM 458 Absorption and transport of xenobiotics + MOVEMENT AND TRANSPORT ACROSS MEMBRANES Pharmacokinetics determines the fate of substances. administered to a living organism. Any chemical xenobiotic such as: pharmaceutical drugs, pesticides, food additives, co...

+ BCHEM 458 Absorption and transport of xenobiotics + MOVEMENT AND TRANSPORT ACROSS MEMBRANES Pharmacokinetics determines the fate of substances. administered to a living organism. Any chemical xenobiotic such as: pharmaceutical drugs, pesticides, food additives, cosmetics, etc + +The structure of the cell membrane + Membrane Function Membranes organize the chemical activities of cells. The outer plasma membrane forms a boundary between a living cell and its surroundings Exhibits selective permeability Controls traffic of molecules in and out  Internal membranes provide structural order for metabolism Form the cell's organelles Compartmentalize chemical reactions + Fluid Mosaic Model of the PM A membrane is a mosaic Proteins and other molecules are embedded in a framework of phospholipids A membrane is fluid Most protein and phospholipid molecules can move laterally Embedded in the bilayer are proteins Most of the membrane’s functions are accomplished by the embedded proteins. Integral proteins span the membrane Peripheral proteins are on one side or the other of the membrane Fig. 5-1a + Carbohydrate of glycoprotein Glycoprotein Glycolipid Integrin Phospholipid Microfilaments of cytoskeleton Cholesterol + Types of Membrane Proteins The following categories are also based on the function of these proteins 1.Cell-cell recognition proteins 2.Integrins 3.Intercellular junction proteins 4.Enzymes 5.Signal transduction proteins Aka - Receptor proteins 6.Transport proteins  Passive and active + Types of membrane proteins Cell-cell recognition proteins Identify type of cell and identify a cell as “self” versus foreign Most are glycoproteins Carbohydrate chains vary between species, individuals, and even between cell types in a given individual. Glycolipids also play a role in cell recognition  Integrins  are a type of integral protein  The cytoskeleton attaches to integrins on the cytoplasmic side of the membrane  Integrins strengthen the membrane Intercellular junction proteins Help like cells stick together to form tissues Enzymes Many membrane proteins are enzymes This is especially important on the membranes of organelles + Signal transduction (receptor) proteins  Signal transduction (receptor) proteins bind hormones and other substances on the outside of the cell.  Binding triggers a change inside the cell. Called signal transduction Example: The binding of insulin to insulin receptors activates glucose transport proteins. + Xenobiotic (drug) transport across cell membranes  Unless given IV, drugs must  Drug absorption is cross several semipermeable determined by: cell membranes before it  Drugs physicochemical reaches the systemic circulation properties  Regardless of route of  Dosage form administration, drug must be in solution to be absorbed  Route of administration  Therefore, solid drugs must be able to disintegrate and disaggregate + How xenobiotics are transported across cell membranes 1. PASSIVE TRANSPORT Drugs diffuse from higher concentration (e.g. GIT fluids)_ to lower concentration (blood) Diffusion is directly proportional to the concentration gradient Diffusion depends on: Lipid solubility Size Degree of ionisation Area of absorptive surface + Simple Diffusion  Nonpolar, hydrophobic molecules diffuse directly through the lipid bilayer  Simple diffusion does not require the use of transport proteins.  Examples: O2, CO2, steroids  Polar, hydrophilic substances cannot pass directly through the lipid bilayer  Examples: water, ions, carbohydrates  Degree of ionisation  Ionised drugs cannot diffuse across the cell membrnes  Non ionised drugs diffuse readily Simple Diffusion Polar molecules (ex. Glucose, water) small, nonpolar ions molecules (ex. H+, Na+, K+) (ex. O2, CO2) LIPID-SOLUBLE WATER-SOLUBLE LIPID-SOLUBLE + Factors Affecting Diffusion Rate Steepness of concentration gradient Steeper gradient, faster diffusion Molecular size Smaller molecules, faster diffusion Temperature Higher temperature, faster diffusion Lipid solubility Degree of ionisation  Many drugs are acidic or basic compounds, which are ionized to a certain degree in aqueous medium. Their degree of ionization depends on their dissociation constant (pKa) and the pH of the environment and extension of ionisation. Henderson-Hasselbach equation: For Acidic drug: Conc. Of nonionised Acid pKa = pH + log Conc.Of ionised Acid For Basic drug: Conc. Of ionised base pKa = pH + log Coc. Of non ionised base + Effect of degree of ionisation on xenobiotic absorption Most drugs are weak organic acids The proportion of the un-ionized or bases, existing in un-ionized and form present (and thus the drug’s ionized forms in an aqueous ability to cross a membrane) is environment. determined by the environmental pH and the drug’s pKa (acid The un-ionized form is usually lipid dissociation constant). soluble (lipophilic) and diffuses readily across cell membranes. The pKa is the pH at which concentrations of ionized and un- The ionized form has low lipid ionised forms are equal. solubility (but high water solubility —ie, hydrophilic) and high electrical resistance and thus cannot penetrate cell membranes easily. + When the pH is lower than the Therefore, when a weak acid is pKa, the un-ionized form of a given orally, most of the drug in the weak acid predominates, but the stomach is un-ionized, favoring ionized form of a weak base diffusion through the gastric predominates. mucosa. Thus, in plasma (pH 7.4), the ratio For a weak base with a pKa of 4.4, of un-ionized to ionized forms for the outcome is reversed; most of a weak acid (eg, with a pKa of 4.4) the drug in the stomach is ionized. is 1:1000; in gastric fluid (pH 1.4), Theoretically, weakly acidic drugs the ratio is reversed (1000:1). (eg, aspirin) are more readily absorbed from an acid medium (stomach) than are weakly basic drugs (eg, quinidine). + However, whether a drug is acidic or basic, most absorption occurs in the small intestine because the surface area is larger and membranes are more permeable (see Oral Administration). Sent from Yahoo Mail for iPhone + Lipid:water partition coefficient Is the ratio of concentrations of a compound in a mixture of two immiscible phases – Used to estimate the distribution of drugs within the body – Hydrophobic drugs with high octanol/water partition are preferentially more distributed in hydrophobic compartments like lipid bilayers – Hydrophilic drugs with low octanol/water partition coefficient are distributed in aqueous compartments like blood serum + Facilitated diffusion or carrier- mediated transport Polar compounds like sugar and amino acids and certain drugs of therapeutic interest cannot penetrate through membrane by passive diffusion but are moved by a carrier system present on the membrane surface Carrier molecules are usually proteins which combine with a drug substrate and form a complex After the complex crosses the membrane; carrier dissociates from the drug and carrier returns to the original side of membrane for reuse. e.g. GLUT 4 enhances the permeation of glucose across a muscle cell membrane + + Osmosis Osmosis – diffusion of water across a selectively permeable membrane Water moves from an area of _______ water concentration to an area of _____ water conc. Is energy required ? Watertravels in/out of the cell through aquaporins (integral membrane proteins that form ores in the membrane of biological cells) + Osmosis Terms Consider two solutions separated by a plasma membrane.  Hypertonic solution with a relatively high concentration of solute  Hypotonic solution with a relatively low concentration of solute  Isotonic solutions with the same solute concentration  Lower Higher Equal concentration concentration concentration of solute of solute of solute H2O Solute molecule Selectively permeable membrane Water molecule Solute molecule with cluster of water molecules Net flow of water + Osmosis and Animal Cells  Isotonic solution Hypotonic solution Hypertonic solution H2O H2O H2O H2O Animal cell (1) Normal (2) Lysed (3) Shriveled H2O H2 O Plasma H2O H2O membrane Plant cell (4) Flaccid (5) Turgid (6) Shriveled (plasmolyzed) See page 83  Osmosis Summary When a cell is placed in a Hypotonic solution: Cell gains water through osmosis Animal cell lyses; plant cell becomes turgid (firm) When a cell is placed a Hypertonic solution: Cell loses water through osmosis Animal cell shrivels; plant cell plasmolyzes + ACTIVE TRANSPORT Active transport proteins move substances across the PM against their concentration gradient. Requires energy (ATP) Carrier mediated transport against a concentration gradient Active transport proteins are highly selective Active transport is needed for proper functioning of nerves and muscles + Active Transport of “X”  Active transport proteins span the  The energized plasma membrane transport protein  They have openings changes shape and for “X” on only one releases “X” on the other side of the cell. side of the membrane  “X” enters the channel and binds to functional groups inside the transport  The phosphate group protein. is released from the  Cytoplasmic ATP binds to the transport transport protein and protein it resumes its original  A phosphate group is shape. transferred from ATP to the transport protein  protein is energized by the added –  Process repeats. P. + Active Transport of “X” Fig. 5-8-1 + Transport protein Solute 1 Solute binding Fig. 5-8-2 + Transport protein Solute 1 Solute binding 2 Phosphorylation Fig. 5-8-3 + Transport protein Protein changes shape Solute 1 Solute binding 2 Phosphorylation 3 Transport Fig. 5-8-4 + Transport protein Protein Phosphate changes shape detaches Solute 1 Solute binding 2 Phosphorylation 3 Transport 4 Protein reversion + Active Transport tell the story… ATP ATP P ADP +FORMS OF ACTIVE TRANSPORT Depending upon the driving force. the active transport can be subdivided into primary or secondary active transport Primary active transport : The bio­transportation of drugs is directly coupled with ATP hydrolysis for deriving the energy and is usually carried by ABC group of biotransportrs Secondary active transport : One ion or the solute (X) supplies the driving force for the transport of other ion/solute (Y). Depending on the direction of flux of X and Y, the transporter can be called either a symporter or an antiporter + Symporter transports X and Y in the same direction. Eg: Na+/K+ /2Cl- Antiporter transports X and Y in the opposite directions Eg: Na+ and H+ exchanger + Bulk Flow Exocytosis Cytoplasmic vesicle merges with the PM and releases its contents Example: Golgi body vesicles merge with the PM and release their contents How nerve cells release neurotransmittors  Endocytosis Vesicle forming Endocytosis can occur in three ways Phagocytosis ("cell eating") Pinocytosis ("cell drinking") Receptor-mediated endocytosis + Endocytosis - terms Endocytosis  PM sinks inward, pinches off and forms a vesicle  Vesicle often merges with Golgi for processing and sorting of its contents Phagocytosis – cell eating Membrane sinks in and captures solid particles for transport into the cell Examples: Solid particles often include: bacteria, cell debris, or food Pinocytosis – cell drinking Cell brings in a liquid Phagocytosis and pinocytosis are not selective Membrane sinks inward and captures whatever particles/fluid present. Vesicle forms and merges with the Golgi body… + Receptor Mediated Endocytosis  Receptor Mediated Endocytosis is a highly specific form of endocytosis.  Receptor proteins on the outside of the cell bind specific substances and bring them into the cell by endocytosis  Receptor proteins on PM bind specific substances (vitamins, hormones..) 2. Membrane sinks in and forms a pit  Called a coated pit 3. Pit pinches closed to form a vesicle around bound substances Cytoskeleton aids in pulling in the membrane and vesicle formation + Pinocytosis : Process where a cell drink or engulfs a fluid or a drug in solution. E.g: -Insulin crosses the BBB by this process Phagocytosis: Process were particulate matter transferred by local invagination of cell membrane Eg: -Poisoning by botulinum toxin Fig. 5-9c + Receptor-mediated endocytosis Plasma membrane Coat protein Receptor Coated vesicle Coated pit Coated pit Specific molecule Material bound to receptor proteins + Fig. 5-9 Phagocytosis + EXTRACELLULAR CYTOPLASM Food FLUID being Pseudopodium ingested “Food” or other particle Food vacuole Pinocytosis Plasma membrane Vesicle Receptor-mediated endocytosis Plasma membrane Coat protein Receptor Coated vesicle Coated pit Coated pit Specific molecule Material bound to receptor proteins  Membrane Transporter Proteins: Classification Membrane Transport Proteins Selective Channels Specific Carriers Primary Active Transport Facilitated Diffusion Secondary Active Transpor ATP-powered pumps Uniporters Symporters Antiporters ATPases: Glut1-5 Pept1 NHE -type, F-type and ABC-type ATPases (ABC transporters) Secondary active transport Facilitated diffusion Use of energy from another source-another Primary active transport Like any diffusion, transport from secondary diffusion gradient set up across the Energy derived from hydrolysis of an area of higher concentration to membrane using another ion. Because this ATP to ADP liberating energy lower concentration. Passive secondary diffusion gradient initially established from high energy phosphate transport is powered by the using an ion pump, as in primary active bond potential energy of a concentration transport, the energy is ultimately derived from gradient and does not require the the same source-ATP hydrolysis. expenditure of metabolic energy + + Channels Transport water or specific types of ions down their concentration or electric potential gradients Energetically favorable reaction Form protein-lined passageway across the membrane through which multiple water molecules or ions move simultaneously at a very rapid rate—up to 108 per second Plasma membrane of all animal cells contains potassium-specific channel proteins that are generally open and are critical to generating the normal, resting electric potential across the plasma membrane Many other types of channel proteins are usually closed, and open only in response to specific signals + Carrier proteins Carrier proteins cycle between conformations in which a solute binding site is accessible on one side of the membrane or the other. With carrier proteins, there is never an open channel all the way through the membrane. + The transport rate mediated by carriers is faster than in the absence of a catalyst, but slower than with channels. A carrier transports one or few solute molecules per conformational cycle, whereas a single channel opening event may allow flux of many thousands of ions. + Uniporters Transport is specific and saturable Facilitated “low resistance” diffusion: – Down the concentration gradient – Accelerates reaction that is already thermodynamically favored Reversible Rate much higher than passive diffusion: - Molecule never in contact with hydrophobic core of the membrane An example is the GLUT1 glucose carrier, in plasma membranes of various cells, including erythrocytes. GLUT1 is a large integral protein, predicted via hydropathy plots to include 12 transmembrane a-helices. + Uniporters: Example GLUT1 Facilitated vs. passive diffusion Mechanism of transport Primary active transporters + Active transport enzymes couple net solute movement across a membrane to ATP hydrolysis. An active transport pump may be a uniporter or antiporter. ATP dependent ATP-dependent ion pumps are grouped into classes based on transport mechanism, as well as genetic & structural homology. Examples include:  P-class pumps  F-class (e.g., F1Fo-ATPase) & related V-class pumps.  ABC (ATP binding cassette) transporters, which catalyze transmembrane movements of various organic compounds including amphipathic lipids and drugs  V-class pumps + ABC transporters ATP-binding cassette (ABC) transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane- bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. + P-class ion pumps They are a gene family exhibiting sequence homology. They include:  Na+,K+-ATPase, in plasma membranes of most animal cells is an antiport pump. It catalyzes ATP-dependent transport of Na+ out of a cell in exchange for K+ entering.  (H+, K+)-ATPase, involved in acid secretion in the stomach is an antiport pump. It catalyzes transport of H+ out of the gastric parietal cell (toward the stomach lumen) in exchange for K+ entering the cell.  Ca++-ATPases, in endoplasmic reticulum (ER) and plasma membranes, catalyze ATP-dependent transport of Ca++ away from the cytosol, into the ER lumen or out of the cell. Some evidence indicates that these pumps are antiporters, transporting protons in the opposite direction. Ca++-ATPase pumps function to keep cytosolic Ca++ low, allowing Ca++ to serve as a signal. + + Secondary active transporters Couple the movement of one type of ion or molecule against its concentration gradient to the movement of a different ion or molecule down its concentration gradient Ability to transport two different solutes simultaneously  also called co-transporters Mediate coupled reactions in which an energetically unfavorable reaction coupled to energetically favorable reaction Catalyze “uphill” movement of certain molecules  often referred to as “active transporters”, but unlike pumps, do not hydrolyze ATP (or any other molecule) Symport + Cotransport carriers bind two dissimilar solutes & transport them together across a membrane. Transport of the two solutes is obligatorily coupled. A gradient of one substrate, usually an ion, may drive uphill (against the gradient) transport of a co-substrate. It is sometimes referred to as secondary active transport. E.g:  glucose-Na+ symport, in plasma membranes of some epithelial cells  bacterial lactose permease, a H+ symport carrier. +Lactose permease catalyzes uptake of the disaccharide lactose into E. coli bacteria, along with H+, driven by a proton electrochemical gradient. Lactose permease has been crystallized with thiodigalactoside (TDG), an analog of lactose. + Symporters: Example Pept1 + Antiport Carriers exchange one solute for another across a membrane. A substrate binds & is transported. Then another substrate binds & is transported in the other direction. The carrier protein cannot undergo the conformational transition in the absence of bound substrate. Example of an antiport carrier: Adenine nucleotide translocase (ADP/ATP exchanger) catalyzes 1:1 exchange of ADP for ATP across the inner mitochondrial membrane. + Antiporters: Example NHE + ABC transporters transporter molecules such as -ions, sugars, amino acids, vitamins, peptides, polysaccharides, hormones, lipids. ABC transporters are involved in diverse cellular processes such as - maintenance of osmotic homeostasis, - nutrient uptake, - antigen processing, - cell division, - bacterial immunity, - pathogenesis + References :  Pharmacological basis of Therapeutics – Goodman & Gilman 12th Edition.  Basic & Clinical Pharmacology 11th Edition.  Essential Medical Pharmacology– K. D. Tripathi 7th Edition.  Principles of pharmacology – HL Sharma & KK Sharma.