Month: September 2014

G Protein-Coupled Receptors and Second Messengers

September 26, 2014 Pharmacology No comments , , ,

UCSFWhen the ligand binds to its receptor, subsequent signaling mechanism must transfer the binding or occupation of receptor into transmembrane singling, which we call occupancy-response. Most transmembrane signaling is accomplished by a small number of different molecular mechanisms to transduce many different signals. These structures in the process of transmembrane signaling involve receptors on the cell surface and within in the cell, as well as enzymes and other components that generate, amplify, coordinate, and terminate postreceptor signaling by chemical second messengers in the cytoplasm.

Generally, five basic mechanisms of transmembrane signaling are well understood. Each uses a different strategy to circumvent the barrier posed by the lipid bilayer of the plasma membrane. Today here we will discuss about the G protein receptor, which is a transmembrane receptor protein that stimulates a GTP-binding signal transducer protein (G protein) that in turn modulates production of an intracellular second messenger.

Many extracellular ligands act by increasing the intracellular concentrations of second messengers such as cAMP (adenylyl cyclase as the effector element), calcium ion, or the phosphoinositides. In most cases, they use a transmembrane signaling system with three separate components. First, the extracellular ligand is selectively detected by a cell-surface receptor, which causes the change in conformation. Second, the change  in conformation of the receptor in turn triggers the activation of a G protein located on the cytoplasmic face of the plasma membrane. Finally, the activated G protein then changes the activity of an effector element, usually an enzyme or ion channel, and this activity change of the effector element changes the concentration of the intracellular second messenger. It is the intracellular second messenger that makes the cellular response.

Common intracellular second messengers include cAMP, calcium ion, and phosphoinositides. There are many examples of such receptors, such as β adrenoceptors, glucagon receptors, thyrotropin receptors, and certain subtypes of dopamine and serotonin receptors.

G Protein ReceptorsG protein-coupled receptors (GPCRs) use a molecular mechanism that involves binding and hydrolysis of GTP, like shown in the figure on the left. This mechanism allows the transduced signal to be amplified. Becasue once the G-GTP formed, the duration of the activity of effector element like adenylyl cyclase depends on the longevity of G-GTP complex rather than on the receptor’s binding for its ligand. For example, norepinephrine may encounter its membrane receptor for only a few milliseconds, but the product of G-GTP from its binding may persist for tens of seconds before becoming inactive, which certainly amplifies the transduced signal of binding between norepinephrine and its receptor. Also, the molecular mechanism helps to explain the spare receptor theory in pharmacodynamics (for detail please refer to

Receptor Regulation

G protein-mediated responses to drugs and hormonal agonists often attenuate with time. After reaching an initial high level, the response diminishes over seconds or minutes, even in the continued presence of the agonist. This “desensitization” is often rapidly reversible; a second exposure to agonist, if provided a few mintues after termination of the first exposure, results in a response similar to the initial response.

The mechanism of “desensitization” include two ways: phosphorylation of GPCRs’ cytoplasmic loops (residues, as shown in the figure below), and the endocytosis of GPCRs from the plasma membrane. The agonist-induced change in conformation of the receptor causes it to bind, activate, and serve as a substrate for a family of specific receptor kinases, called G protein-coupled receptor kinases (GRKs). The activated GRK then phosphorylates serine residues in the receptor’s carboxyl terminal tail. The presence of phosphoserines increases the receptor’s affinity for binding a third protein, β-arrestin, and the binding of β-arrestin to cytoplasmic loops of the GPCR diminishes the receptor’s ability to interact with G protein, thereby reducing the agonist response.

Upon removal of agonist, GRK activation is terminated, the β-arrestin binding affinity  is reduced, and the desensitization process can be reversed by cellular phosphatases (the receptor must be engulfed into cytoplasm first, where it can touch the cellular phosphatases), which turn the phosphorylated serine residues back to what they were before.

On the other hand, the β-arrestin binding also accelerates endocytosis of receptors from the plasma membrane. However, this endocytosis is helpful since endocytosis of GPCRs promotes their dephosphorylation (need the termination of GRK activity and to be dissociated with β-arrestin first) by a receptor phosphatease that is present at high concentration on endosome membranes. Then, the dephosphorylated GPCRs can return to the plasma membrane.

Of note is that several GPCRs if persistently activated instead would be traffic to lysosomes after endocytosis and degraded. This process effectively attenuates (rather than restores) cellular responsiveness. So, depending on the particular receptor, duration of activation and the particular conformational change caused by a specific agent (e.g., etorphine and enkephalins cause rapid internalization of the receptor whereas morphine dose not cause MOR internalization), endocytosis can contribute to either rapid recovery or prolonged attenuation of cellualr responsiveness.Regulation of GPCRs

Well-Established Second Messengers

The second messengers are not the patent of G Protein-Coupled Receptors. Other receptor strategies such as receptor tyrosine kinases (e.g., receptor of ANP) also involve second messengers.

Cyclic Adensine Monophosphate (cAMP)

Acting as an intracellular second messenger, cAMP mediates such hormonal responses as the mobilization of stored energy (the breakdown of carbohydrates in liver or triglycerides in fat cells stimulated by β-adrenomimetic catecholamines), conservation of water by the kidney (mediated by vasopressin), Ca2+ homeostasis (regulated by parathyroid hormone), and increased rate and contractile force of heart muscle (β-adrenomimetic catecholamines). It also regulates the production of adrenal and sex steroids (in response to corticotropin or follicle-stimulating hormone), relaxation of smooth muscle, and many other endocrine and neural processes.

cAMP exerts most of its effects by stimulating cAMP-dependent protein kinases. These kinases are composed of a cAMP-binding regulatory (R) dimer and two catalytic (C) chains. When cAMP binds to the R dimer, active C chains are released to diffuse through through the cytoplasm and nucleus, where they transfer phosphate from ATP to appropriate substrate proteins, often enzymes. When the hormonal stimulus stops, the intracellular actions of cAMP are terminated by an elaborate series of enzymes. cAMP-stimulated phosphorylation of enzyme substrates is rapidly reversed by a diverse group of specific and nonspecific phosphateases. cAMP itself is degraded to 5′-AMP by several cyclic nucleotide phosphodiesterases (PDE).

Phosphoinositides and Calcium

Another well-studied second messenger system involves hormonal stimulation of phosphoinositide hydrolysis. Some of the hormones, neurotransmitters, and growth factors that trigger this pathway bind to receptors linked to G proteins, whereas others bind to receptor tyrosine kinases. In all cases, the crucial step is stimulation of a membrane enzyme, phospholipase C (PLC), which splits a minor phospholipid component of the plasma membrane, phosphatidylinositol-4,5-bisphosphate (PIP2), into two second messengers, diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3 or InsP3). Diacylglycerol is confined to the membrane, where it activates a phospholipid- and calcium-sensitive protein kinase called protein kinase C. IP3 is water-soluble and diffuses through the cytoplasm to trigger release of Ca2+ by binding to ligand-gated calcium channels in the limiting membranes of internal storage vesicles. Elevated cytoplasmic Ca2+ concentration resulting from IP3-promoted opening of these channels promotes the binding of Ca2+ to the calcium-binding protein calmodulin, which regulates activities of other enzymes, including calcium-dependent protein kinases. With its multiple second messengers and protein kinases, the phosphoinositide signaling pathway is much more complex than the cAMP pathway.

As in the cAMP system, multiple mechanisms damp or terminate signalling by this pathway. IP3 is inactivated by dephosphorylation. Diacylglycerol is either phosphorylated to yield phosphatidic acid, which is then converted back into phospholipids, or it is deacylated to yield arachidonic acid. Ca2+ is actively removed from the cytoplasm by Ca2+ pumps.

Cyclic Guanosine Monophosphate (cGMP)

Unlike cAMP, the ubiquitous and versatile carrier of diverse messages, cGMP has established signalling roles in only a few cell types. In intestinal mucosa and vascular smooth muscle, the cGMP-based signal transduction mechanism closely parallels the cAMP-mediated signalling mechanism. Ligands detected by cell-surface receptors stimulate membrane-bound guanylyl cyclase to produce cGMP, and cGMP acts by stimulating a cGMP-dependent protein kinase. The actions of cGMP in these cells are terminated by enzymatic degradation of the cyclic nucleotide and by dephosphorylation of kinase substrates.

Increased cGMP concentration causes relaxation of vascular smooth muscle by a kinase-mediated mechanism that results in dephospho rylation of myosin light chains. Examples include atrial natriuretic peptide (ANP), nitric oxide (NO) induced by vasodilator agents such as acetylcholine and histamine.

Infectious Diseases – Sites of Infections and the Virulence

September 14, 2014 Infectious Diseases, Physiology and Pathophysiology No comments , , , ,

CaduceusThe topics of sites of infections and virulence are two fundamental content in the discipline of infectious diseases. As a pharmacist and clinician we have to read and understand them, which can direct our clinical practice at the right direction.

Site of Infection

The type of pathogen (the number of infectious agents absorbed by the host and the virulence of the pathogen), the portal of entry, and the competence of the host’s immunologic defense system ultimately determine the site of an infectious disease. Some pathogens cause local infectious diseases, some tend to disseminate (though the circulatory system) from the primary site of infection to involve other locations and organ systems, which we call systemic infections. In particular situation, the abscess or pus, is a purulent exudate or a localized pocket rich in leukocytes (mostly neutrophils), devitalized tissue and debris of dead cells, and in many cases, microbes.

In this case, the dissemination of the pathogen has been contained by the host, but white cell function within the toxic environment of the abscess is hampered, and the elimination of microorganisms is slowed if not actually stopped. Similarly, infections of biomedical implants such as catheters, artificial heart valves, and prosthetic bone implants are seldom cured by the host’s immune response and antimicrobial therapy (due to the biofilm formed on these devices), which necessitates the removal of the device.


Virulence factors are substances or products generated by infectious agents that enhance their ability to cause disease. Generally, virulence factors can be summarized into four primary categories including toxins, adhesion factors, evasive factors, and invasive factors.


Toxins are substances that alter or destroy the normal function of the host or host’s cells. Toxin production is a trait chiefly monopolized by bacterial pathogens, although certain fungal and protozoan pathogens also elaborate substances toxic to humans. Bacterial toxins have diverse spectrum of activity and exert their effects on a wide variety of host target cells. For classification purposes, however, the bacterial toxins can be divided into two main types: exotoxins, and endotoxins.

Exotoxins are proteins released from the bacterial cell during growth. Bacterial exotoxins enzymatically inactivate or modify key cellular constituents, leading to cell death or dysfunction. Diphtheria toxin, for example, inhibits cellular protein synthesis; botulism toxin decreases the release of neurotransmitter from cholinergic neurons, causing flaccid paralysis; tetanus toxin decreases the release of neurotransmitter from inhibitory neurons, producing spastic paralysis; and cholera toxin induces fluid secretion into the lumen of the intestine, causing diarrhea. Other examples of exotoxin-induced diseases include pertussis (whooping cough), anthrax, traveler’s diarrhea, toxic shock syndrome, and a host of food-borne illnesses (i.e., food poisoning).

In contrast to exotoxins, endotoxins do not contain protein, are not actively released from the bacterium during growth, and have no enzymatic activity. Rather, endotoxins are complex molecules composed of lipid and polysaccharides found in the cell wall of gram-negative bacteria. Studies of different endotoxins have indicated that the lipid portion of the endotoxin confers the toxic properties to the molecule. Endotoxins are potent activators of a number of regulatory systems in humans. A small amount of endotoxin in the circulatory system (endotoxemia) can induce clotting bleeding, inflammation, hypotension, and fever.

Adhesion Factors

No interaction between microorganisms and humans can progress to infection or disease if the pathogen is unable to attach and colonize the host. The process of microbial attachment may be site specific (e.g., mucous membranes, skin surfaces), cell specific (e.g., T lymphocytes, respiratory, epithelium, intestinal epithelium), or nonspecific (e.g., moist areas, charged surfaces). In any of these cases, adhesion requires a positive interaction between the surfaces of host cells and the infectious agent.

The site to which microorganisms adhere is called a receptor, and the reciprocal molecule or substance that binds to the receptor is called a ligand or adhesin. Receptors may be proteins, carbohydrates, lipids, or complex molecules composed of all three. Similarly, ligands may be simple or complex molecules and, in some cases, highly specific structures. After initial attachment, a number of bacterial agents become embedded in a gelatinous matrix of polysaccharides called a slime or mucous layer. The slime layer serves two purposes: It anchors the agent firmly to host tissue surfaces, and it protects the agent from the immunologic defenses of the host. Many viral agents produce filamentous appendages or spikes that recognize carbohydrate receptors on the surfaces of specific cells in human body.

Evasive Factors

A number of factors produced by pathogens enhance virulence by evading various components of the host’s immune system. The are briefly summarized below.

1.Extracellular polysaccharides, including capsules, slime, and mucous layer, discourage engulfment and killing of pathogens by the host’s phagocytic white blood cells.

2.Some pathogens can avoid phagocytosis by excreting leukocidin C toxins, which cause specific and lethal damage to the cell membrane of host neutrophils and macrophages, etc.

3.Some pahogens are adapted to survive and reproduce within phagocytic white blood cells after ingestion, avoiding or neutralizing the usually lethal products contained within the lysosomes of the cell. An extreme example is the Helicobacter pylori, which produces a urease enzyme on its outer cell wall. The urease converts gastric urea into ammonia, thus neutralizing the acidic environment of the stomach and allowing the organism to survive in this hostile environment.

4.Some pathogens evading immunologic surveillance have evolved ways to avoid recognition by host antibodies. Strains of S. aureus produce a surface protein (protein A) that immobilizes immunoglobulin G (IgG), holding the antigen-binding region harmlessly away from the organisms. Also, this pathogen secretes a unique enzyme called coagulase which converts soluble human coagulation factors into solid clot, which envelops and protects the organism from phagocytic host cells and antibodies.

5.Some agents secrete enzymes that cleave and inactivate secretory IgA, neutralizing the primary defense of the respiratory and genital tracts at the site of infection.

6.Some agents can alter surface antigens during the disease course so that the immunological detection has been avoided.

7.Some viruses, such as HIV, impair the function of immunoregulatory cells. Although this property increases the virulence of these agents, it is not considered a virulence factor in the true sense of the definition.

Invasive Factors

Invasive factors are products produced by infectious agents that facilitate the penetration of anatomic barriers and host tissue. Most invasive factors are enzymes capable of destroying cellular membranes (e.g., phospholipases), connective tissue (e.g., elastases, collagenases), intercellular matrices (e.g., hyaluronidase), and structural protein complexes (e.g., proteases).

At the end, I want to emphasize that it is the combined effects of these factors above, the amount of pathogen the host absorbing, and the antimicrobial and inflammatory substances released by host cells mediate the pathophysiology of the infectious diseases.

The Disease Course of An Infectious Disease

September 9, 2014 Infectious Diseases, Physiology and Pathophysiology No comments , , , , , ,

The course of any infectious disease can be divided into several distinguishable stages after the point when the potential pathogen enters the host. These stages are the incubation period, the prodromal stage, the acute stage, the convalescent stage, and the resolution stage. These stages are based on the progression and intensity of the host’s symptoms over time. The duration of each phase and the pattern of the overall illness can be specific for different pathogens, thereby aiding in the diagnosis of an infectious disease.

Stages of A Primary Infectious DiseaseHowever, several notable exception to the classic presentation of an infectious process have existed. Chronic infectious diseases have a markedly protracted and sometime irregular course. The host may experience symptoms of the infectious process continuously or sporadically for months or years without a convalescent phase. In contrast, subclinical or subacute illness progresses from infection to resolution without clinically apparent symptoms. A disease is called insidious if the prodromal phase is protracted. A fulminant illness is characterized by abrupt onset of symptoms with little or no prodrome. Fatal infections are variants of the typical disease course.

The incubation period is the phase during which the pathogen begins active replication without producing recognizable symptoms in the host. The incubation period may be short, as in the case of salmonellosis (6 to 24 hours), or prolonged, such as that of hepatitis B (50 to 180 days) or HIV (months to years). The duration of the incubation period can be influenced by additional factors, including the general health of the host, the portal of entry, and the degree of the infectious dose of the pathogen.

The hallmark of the prodromal stage is the initial appearance of symptoms in the host, although the clinical presentation during this time may be only a vague sense of malaise. The host may experience mild fever, myalgia, headache, and fatigue. These are constitutional changes shared by a great number of disease processes. The duration of the prodromal stage can vary considerably from host to host.

The acute stage is the period during which the host experiences the maximum impact of the infectious process corresponding to rapid proliferation and dissemination of the pathogen. During this phase, toxic by-products of microbial metabolism, cell lysis, and the immune response mounted by the host combine to produce tissue damage and inflammation. These symptoms of the host are pronounced and more specific than in the prodromal stage, usually typifying the pathogen and sites of involvement (see another thread about the site of infection).

The convalescent period is characterized by the containment of infection, progressive elimination of the pathogen, repair of damage tissue, and resolution of associated symptoms. Similar to the incubation period, the time required for complete convalescence may be days, weeks, or months, depending on the type of pathogen and the voracity of the host’s immune response.

The resolution is the total elimination of a pathogen from the body without residual signs or symptoms of disease.