Lifetime of secondary messengers such as Calcium or IP3

Lifetime of secondary messengers such as Calcium or IP3

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Can anyone suggest me literature on the lifetime of secondary messengers such as calcium or IP3? A book would be preferred. What I am specifically looking for is a validation that secondary messengers have a certain lifetime and undergo degradation. I need not know specifically how long their lifetime is but rather interested in the fact that they do undergo biodegradation.

[EDIT]: Replaced the word 'biomolecule' by 'secondary messengers' as per the suggestion of @WYSIWYG to narrow down the answer.

The lifetime of Calcium can be considered infinite in biological time scales, it is not a real biomolecule. The half-life time of other biomolecules like IP3, PIP2, receptors, hormones, etc. is species-dependent and within the species strongly depending on the cell and its metabolic state.

Short answer: There is no overview for lifetimes of biomolecules. It is easier to search for a specific molecule in a specific cell. Perhaps you are lucky and somebody measured it.

Update to the comments: All known second messengers, like Ca2+, IP3, PIP2, change their concentration in response to a stimulus, e.g. activation of muscarinic receptors causes activation of phospholipase C which cleaves PIP2 in DAG and IP3. IP3 causes release of Ca2+. Ca2+ never gets degraded or synthesized but the intracellular concentration changes. PIP2 gets degraded, i.e. its concentration becomes lower, while the concentration of its cleavage products, IP3 and DAG, increases. Those changes are temporary, after removal of the stimulus the 2nd messengers get degraded as well or e.g. Ca2+ is taken up into intracellular compartments.

A general overview about second messengers.

Inositol trisphosphate

Inositol trisphosphate or inositol 1,4,5-trisphosphate abbreviated InsP3 or Ins3P or IP3 is an inositol phosphate signaling molecule. It is made by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, by phospholipase C (PLC). Together with diacylglycerol (DAG), IP3 is a second messenger molecule used in signal transduction in biological cells. While DAG stays inside the membrane, IP3 is soluble and diffuses through the cell, where it binds to its receptor, which is a calcium channel located in the endoplasmic reticulum. When IP3 binds its receptor, calcium is released into the cytosol, thereby activating various calcium regulated intracellular signals.


There are three basic types of secondary messenger molecules:

  • Hydrophobic molecules: water-insoluble molecules such as diacylglycerol, and phosphatidylinositols, which are membrane-associated and diffuse from the plasma membrane into the intermembrane space where they can reach and regulate membrane-associated effector proteins.
  • Hydrophilic molecules: water-soluble molecules, such as cAMP, cGMP, IP3, and Ca 2+ , that are located within the cytosol.
  • Gases: nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) which can diffuse both through cytosol and across cellular membranes.

These intracellular messengers have some properties in common:

  • They can be synthesized/released and broken down again in specific reactions by enzymes or ion channels.
  • Some (such as Ca 2+ ) can be stored in special organelles and quickly released when needed.
  • Their production/release and destruction can be localized, enabling the cell to limit space and time of signal activity.

There are several different secondary messenger systems (cAMP system, phosphoinositol system, and arachidonic acid system), but they all are quite similar in overall mechanism, although the substances involved and overall effects can vary.

In most cases, a ligand binds to a membrane-spanning receptor protein molecule. The binding of a ligand to the receptor causes a conformation change in the receptor. This conformation change can affect the activity of the receptor and result in the production of active second messengers.

In the case of G protein-coupled receptors, the conformation change exposes a binding site for a G-protein. The G-protein (named for the GDP and GTP molecules that bind to it) is bound to the inner membrane of the cell and consists of three subunits: alpha, beta and gamma. The G-protein is known as the "transducer."

When the G-protein binds with the receptor, it becomes able to exchange a GDP (guanosine diphosphate) molecule on its alpha subunit for a GTP (guanosine triphosphate) molecule. Once this exchange takes place, the alpha subunit of the G-protein transducer breaks free from the beta and gamma subunits, all parts remaining membrane-bound. The alpha subunit, now free to move along the inner membrane, eventually contacts another membrane-bound protein - the "primary effector."

The primary effector then has an action, which creates a signal that can diffuse within the cell. This signal is called the "second (or secondary) messenger." The secondary messenger may then activate a "secondary effector" whose effects depend on the particular secondary messenger system.

Calcium ions are one type of second messengers and are responsible for many important physiological functions including muscle contraction, fertilization, and neurotransmitter release. The ions are normally bound or stored in intracellular components (such as the endoplasmic reticulum(ER)) and can be released during signal transduction. The enzyme phospholipase C produces diacylglycerol and inositol trisphosphate, which increases calcium ion permeability into the membrane. Active G-protein open up calcium channels to let calcium ions enter the plasma membrane. The other product of phospholipase C, diacylglycerol, activates protein kinase C, which assists in the activation of cAMP (another second messenger).

cAMP System Phosphoinositol system Arachidonic acid system cGMP System Tyrosine kinase system
First Messenger:
Epinephrine (α2, β1, β2)
Acetylcholine (M2)
Epinephrine (α1)
Acetylcholine (M1, M3)
Histamine (Histamine receptor) - -
First Messenger:
ACTH, ANP, CRH, CT, FSH, Glucagon, hCG, LH, MSH, PTH, TSH AGT, GnRH, GHRH, Oxytocin, TRH - ANP, Nitric oxide INS, IGF, PDGF
Signal Transducer GPCR/Gs (β1, β2), Gi (α2, M2) GPCR/Gq Unknown G-protein - RTK
Primary effector Adenylyl cyclase Phospholipase C Phospholipase A guanylate cyclase RasGEF (Grb2-Sos)
Second messenger cAMP (cyclic adenosine monophosphate) IP3 DAG Ca2+ Arachidonic acid cGMP Ras.GTP (Small G Protein)
Secondary effector protein kinase A PKC CaM 5-Lipoxygenase, 12-Lipoxygenase, cycloxygenase protein kinase G MAP3K (c-Raf)

IP3, DAG, and Ca 2+ are second messengers in the phosphoinositol pathway. The pathway begins with the binding of extracellular primary messengers such as epinephrine, acetylcholine, and hormones AGT, GnRH, GHRH, oxytocin, and TRH, to their respective receptors. Epinephrine binds to the α1 GTPase Protein Coupled Receptor (GPCR) and acetylcholine binds to M1 and M2 GPCR. [8]

Binding of a primary messenger to these receptors results in conformational change of the receptor. The α subunit, with the help of guanine nucleotide exchange factors (GEFS), releases GDP, and binds GTP, resulting in the dissociation of the subunit and subsequent activation. [9] The activated α subunit activates phospholipase C, which hydrolyzes membrane bound phosphatidylinositol 4,5-bisphosphate (PIP2), resulting in the formation of secondary messengers diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). [10] IP3 binds to calcium pumps on ER, transporting Ca 2+ , another second messenger, into the cytoplasm. [11] [12] Ca 2+ ultimately binds to many proteins, activating a cascade of enzymatic pathways.

What is Second Messenger System?

Second messengers are intracellular molecules that send signals from receptors to targets. The cell releases second messengers in response to exposure to extracellular signalling molecules, which are first messengers. Second messengers trigger physiological processes of the cell. Such processes are cell proliferation, differentiation, migration, survival, apoptosis, muscle contraction, fertilization, and neurotransmitter release, etc.

Figure 02: Second Messenger System

There are several different second messenger systems within the cell. Cyclic AMP, cyclic GMP, inositol trisphosphate, diacylglycerol, and calcium are several examples. Generally, second messengers are non-protein small molecules which are made from phospholipids. They are produced after the first messenger dependent receptor activation. Moreover, second messenger molecules are typically small molecules that can easily diffuse within the cell. They operate through the activation of protein kinases. In fact, each second messenger associates a particular type of protein kinase. Sometimes, second messengers couple with multi-cyclic kinases and amplify the strength of the original signal.

Function of G alpha proteins

Inhibits adenylyl cyclase, Activates potassium channel

Activates phospholipase C

Stimulates adenylyl cyclase present in eye

Activates adenylyl cyclase, Activates calcium channel

Mechanism of action of G-protein coupled receptors


In resting (inactive) state of G protein, GDP (guanosine diphosphate) is tightly bound to the α subunit. But when an agonist is bound to G protein-coupled receptor then the GDP bound to the α subunit gets replaced by GTP.

This α-GTP is then dissociated from the β and γ subunits and subsequently interact with the membrane bound effector(such as adenylyl cyclase). The GTPase activity of the α subunit increases on binding, leading to hydrolysis of the bound GTP to GDP which allows the α subunit to recombine with the βγ complex.

Chapters 6-10 Biology 1406

Active Site:
-A specific enzyme location that binds with the substrate.

The direct energy source for glycolysis

An end product that is modified to enter the citric acid cycle

A product that donates electrons to the electron transport chain

Radiant energy (energy from the sun)
Carbon Dioxide CO2 (gas taken from the air)
Water H2O (absorbed from the roots and the air)

Glucose (C6H12O6) Sugar used as food for the plant. Stores energy.

Molecules produced during the Calvin cycle that leaves the cycle.
-A few of the glyceraldehyde 3-phosphate (G3P)

Unique to Photosystem 2
-The hydrogen atoms from water molecules are split, producing H+ and replacement electrons for chlorophyll molecules.
-Energy from electrons excited in this photosystem is used to help pump H+ ions into the thylakoid for ATP production.

Calvin cycle:
Use CO2
Produce Sugars
Need Ribulose Bisphosphate

CO2 Dependent Reactions- Stroma

During Prophase, chromosomes condense, nuclear membranes break down, and the mitotic spindle forms.

During metaphase, chromosomes line up along the equatorial plane of the cell.

During anaphase, the two sister
chromatids are pulled apart and move to opposite poles of the cell.

Results & Discussion

DiscoverX Hithunter ® IP3 was prepared in 384-well format and standard curves were run on BMG LABTECH’s PHERAstar FS in fluorescence polarization mode. IP3 standard reagents were added according to the assay protocol and the fluorescence polarization signal was read one hour after the addition of the last reagent. The Hithunter ® IP3 standard curve is illustrated in the figure below (figure 3).

Fig. 3: Hithunter IP3 Assay: Standard curve data in 384-well format.

The Hithunter ® IP3 assay is a robust, sensitive and specific tool for measuring cellular D-myo-inositol 1,4,5-triphosphate. The signal is based on competitive binding between an IP3 fluorescent tracer and an unlabeled IP3 from cell lysate or IP3 standard. The signal is read as a change in fluorescence polarization and is inversely proportional to the amount of IP3 in cell lysates. The standard curve signal to background is >5, and the EC50 is 13 nM. The signal can be measured immediately or up to 16 hours later.

Neurotransmitters, Receptors, and Second Messengers Galore in 40 Years

The past four decades have witnessed extraordinary advances in the molecular understanding of neurotransmitters, their receptors, and second messengers. This essay highlights a selected group of particular notable discoveries, emphasizing seminal findings that have transformed thinking in the field.


To say that much about neurotransmission has changed in the past four decades is a gross understatement. In 1970 the biogenic amines were well established as neurotransmitters. Amino acids were making inroads with GABA accepted and glycine strongly suspected as inhibitory neurotransmitters. Hints abounded for glutamate, but the jury was still out. Few thought of peptides as transmitters. Substance P had been identified in the 1930s as an agent in brain extracts with smooth muscle activity, but its designation (P for powder) indicated our ignorance. In 1970 Susan Leeman isolated the substance P peptide and obtained its amino acid sequence, but there was still no evidence that it was a neurotransmitter. Of course, no one even dreamed of gases, “abnormal” isomers of amino acids, such as d -serine, or lipid-derived endocannabinoids as transmitters. Receptors were presumed to be proteins localized to synaptic membranes, but none had been biochemically identified. The only characterized second messenger was cAMP. So much has transpired in the intervening years that this brief review perforce highlights only a few themes and key investigators.

Neurotransmitter transporters

The founding of the Society for Neuroscience in 1970 coincided with an important event for neuroscience, especially for the world of neurotransmitters. The Nobel Prize in Physiology or Medicine was awarded to Julius Axelrod, Bernard Katz, and Ulf von Euler for “their discoveries concerning the humoral transmitters in the nerve terminals and the mechanisms for their storage, release and inactivation.” Katz was honored for elucidating the quantal release of acetylcholine from nerve terminals at the neuromuscular junction. Von Euler was cited for establishing definitively that norepinephrine is a neurotransmitter at nerve terminals of the sympathetic nervous system. The citation for Axelrod emphasized his discovery of “the mechanisms which are involved in the inactivation of noradrenaline, partly under the influence of an enzyme discovered by himself.” The Nobel Assembly did not specify Axelrod's discovery of the reuptake of norepinephrine by nerve terminals as a mode of synaptic inactivation nor did it comment on his discovery that tricyclic antidepressants act by inhibiting this reuptake process. This tells us that in 1970 these concepts, catechism today, were still controversial. In the early 1970s monitoring the uptake of radiolabeled neurotransmitters into pinched off nerve terminals, synaptosomes, of the brain led to fairly thorough characterization of the process and the ability to discriminate between transport of different biogenic amines, such as norepinephrine and serotonin (Iversen, 1999). Dogma held that nerve terminals selectively reaccumulated the transmitter molecules they had released. Thus, increasing appreciation that glia are the predominant mode of transmitter inactivation in the brain, especially for glutamate, was revolutionary.

In 1972 Lilly scientists used synaptosomal transport to identify fluoxetine (Prozac) as a selective inhibitor of serotonin uptake (Kramer, 1993 Torres and Amara, 2007). A series of serotonin-selective reuptake inhibitors were shortly followed by norepinephrine-selective antidepressants. Both classes of drugs are clinically effective, leading to resolution of the long fought battle between those favoring either serotonin or norepinephrine as critical in regulating mood. The answer—both are important.

The field of neurotransmitter uptake was somewhat dormant until 1991 when Susan Amara and coworkers cloned norepinephrine transporters, followed by cloning of transporters for all the transmitter amines, amino acids, and related neuroactive substances (Torres and Amara, 2007). Molecular genetic techniques identified a repeat of 20–23 bp within a polymorphic region of the serotonin transporter gene, occurring as two alleles, the “short” variant of 14 repeats and the “long” 16 repeat variant (Belmaker and Agam, 2008). The short form leads to decreased expression of the serotonin transporter and less serotonin uptake. This short allele interacts with stressful life events and correlates with the recurrence of depression. Thus, besides mediating actions of antidepressants, serotonin appears to have an etiologic role in some forms of affective illness. Buttressing this conclusion, polymorphisms in the gene for the brain-specific tryptophan hydroxylase-2, the rate-limiting enzyme in serotonin biosynthesis, are associated with depression in discrete populations.

Neurotransmitter localizations and actions

The ability to visualize neurons containing individual neurotransmitters has been a profoundly important advance in neuroscience, contributing mightily to the principal mantra of the neurosciences—to link neurochemistry, neuroanatomy, and neurophysiology. This effort commenced in the mid-1960s with the development by Nils-Åke Hillarp, Bengt Falck and collaborators of procedures whereby catecholamines and serotonin fluoresce in the microscope after reaction with formaldehyde vapors. Hillarp and his students Kjell Fuxe and Annica Dahlström then mapped norepinephrine, dopamine, and serotonin containing neurons in the brain (Hillarp et al., 1966). Immunohistochemical mapping of neuropeptides, pioneered by Tomas Hökfelt, was of comparable importance (Hokfelt et al., 1984). Although carried out just before 1970, this work laid the conceptual underpinnings for the integrated molecular/cellular/neurophysiologic world of recent decades.

The 1970s were the years of neuropeptides. Identification of opiate receptors raised the question, “Why have receptors for opiates? Man was not born with morphine in him.” Several groups purified peptides from the brain with selective opiate-like activity. In December 1975 John Hughes, Hans Kosterlitz, and collaborators reported the sequences of two five-amino acid containing peptides, methionine-enkephalin and leucine-enkephalin (Hughes et al., 1975). In the months leading up to this denouement, opiate researchers had conclaved to name the hypothetical morphine-like substances and selected “endorphins.” Hughes and Kosterlitz preferred the designation enkephalin from the Greek “in the head.” They wished to free the scientific community from prejudice that these substances would relate only to opiate-associated behavior. With the discovery of other opioid peptides incorporating the enkephalin sequence, the term endorphin came to be used generically for any peptide with opioid activity. Since “we are not addicted to ourselves,” many pharmaceutical concerns attempted to develop enkephalin derivatives as less addicting analgesics—they all failed. At opiate receptor complexes, stable drug-like enkephalin derivatives are just as addicting as most opiates. The rapid degradation of synaptically released enkephalins precludes their persistent receptor occupancy, which is necessary to initiate the addictive process.

Isolation of the enkephalins sparked a massive interest in peptides as potential transmitters. Immunohistochemical mapping revealed several dozen peptides highly localized to specific neuronal populations in the central and peripheral nervous systems. The localization of substance P, calcitonin gene-related peptide, and others in thin, unmyelinated pain fibers led to efforts to develop analgesics blocking receptors for these peptides.

Edward Herbert as well as Shosaku Numa elucidated how neuropeptides are generated, introducing some startling new concepts (Douglass et al., 1984). Many years earlier Donald Steiner had shown that insulin is generated from a large protein precursor, proinsulin, in two steps. Within the precursor, insulin is surrounded by pairs of basic amino acids. First, a trypsin-like enzyme cleaves to the right of each of these basic amino acids leaving insulin with a single lysine or arginine at its C terminus. A carboxypeptidase then removes the attached basic amino acid. A similar pattern was elucidated for the enkephalins and β-endorphin. But there were surprises. The β-endorphin precursor, pro-opiomelanocortin, contains within its sequence ACTH and melanocyte-stimulating hormone along with β-endorphin. The enkephalin precursors provided other surprises. One of the precursors contains six copies of methionine enkephalin and a single copy of leucine enkephalin, while the other contains three copies of leucine enkephalin.

Among the amino acids, the past four decades have witnessed enormous interest in glutamate as the principal excitatory neurotransmitter in the brain. Differentiation of glutamate receptor subtypes was key to this effort, especially the discrimination of NMDA and AMPA receptors. It is now well accepted that most “bread and butter” excitatory transmission involves AMPA receptors. NMDA receptors are both voltage and ligand gated. Thus, depolarization by AMPA receptor activation opens NMDA receptors permitting the influx of calcium, a critical event in the synaptic plasticity underlying long-term potentiation. Work of Roger Nicoll, Robert Malenka, and others showing that many AMPA receptors are “silent” led to numerous studies showing that cycles of internalization and externalization of AMPA receptors are also critical to synaptic plasticity (Kerchner and Nicoll, 2008).

Observations in the late 1980s that nitric oxide mediates endothelial dependent relaxation of blood vessels piqued the interest of ourselves and others in the possibility that NO might be a neurotransmitter in the brain (Bredt and Snyder, 1994). Stimulation of cyclic GMP by glutamate receptors was shown to be mediated by NO. Purification and cloning of the neuronal NO synthase (nNOS) permitted its characterization in depth and the identification of inducible NOS and endothelial NOS. Although nNOS occurs in only 1% of neurons, their processes ramify so extensively that every cell in the brain is probably exposed to NO. When released in excess following hyperstimulation of NMDA receptors, NO is neurotoxic and may mediate brain damage from vascular stroke. In the peripheral nervous system, NO is also an important neurotransmitter, well established as the transmitter that mediates penile erection. The alleviation of erectile dysfunction by inhibitors of phosphodiesterase-5 such as sildenafil (Viagra) reflects augmented levels of cyclic GMP, the second messenger for NO. Although the vascular actions of NO stem from activation of guanylyl cyclase, it is increasingly appreciated that most signaling by NO involves nitrosylation of numerous prominent cellular proteins such as the sodium pump, actin, and tubulin (Jaffrey et al., 2001 Hess et al., 2005).

NO is not the only gaseous neurotransmitter. Carbon monoxide is formed by cleavage of the heme ring by heme oxygenase (HO) (Mustafa et al., 2009). The neuronal form of the enzyme, HO2, occurs in discrete neuronal populations in the brain and periphery. In the myenteric plexus of the intestines, nNOS and HO2 are colocalized. Mice with deletion of nNOS and HO2, respectively, display 40–50% decreases in non-adrenergic non-cholinergic (NANC) transmission with NANC transmission virtually abolished in the double knock-outs.

Recently hydrogen sulfide (H2S) has been established as a major endothelial-derived relaxing factor (EDRF) (Yang et al., 2008). EDRF activity declines profoundly in arteries of mice with deletion of cystathionine-γ-lyase, the principal H2S forming enzyme in the periphery. In the brain, cystathionine-β-synthase generates H2S, also from cysteine. H2S appears to signal by forming a persulfide linkage to cysteines in target proteins, a process referred to as sulfhydration, analogous to NO nitrosylating proteins (Mustafa et al., 2009). Whether H2S is a bonafide neurotransmitter remains to be determined.


Most of us in the receptor field would regard 1970 as a banner year. I recall vividly the excitement attendant upon the identification of the nicotinic acetylcholine receptor in the electric organ of Torpedo, an electric eel, monitored by the binding of the pseudo-irreversible snake toxin α-bungarotoxin labeled with iodine-125 (Changeux and Taly, 2008). Jean Pierre-Changeux was a key figure in these events, while the laboratories of Ricardo Miledi and Michael Raftery were important contributors. This breakthrough was made possible by several important factors. The electric shock delivered by eels is mediated by massive numbers of the receptor, comprising up to 20% of the total protein of the electric organ. Nature evolved α-bungarotoxin to interact with extraordinarily high affinity and virtual irreversibility in order for snakes to attack their prey. Binding studies were facilitated by the ability to label the toxin with iodine-125 to extremely high specific radioactivity. Several neuropharmacologists noted that the very success of this effort portended the impossibility, for the foreseeable future, of biochemically labeling neurotransmitter/drug receptors in the brain, which were correctly estimated to represent no more than one millionth by weight of brain tissue.

In 1973 binding studies using [ 3 H]opiates enabled identification of opiate receptors in crude brain homogenates using reversibly binding ligands (Snyder and Pasternak, 2003). What contributed to the success of this unanticipated advance? Opiates labeled with tritium to high specific radioactivity permitted the use of very low radioligand concentrations, which would selectively interact with pharmacologically relevant receptors. Vigorous but extremely rapid washing permitted dissociation of nonspecifically bound radioligand while preserving interactions with the pharmacologically relevant receptor, which should have much higher affinity for the ligand than nonspecific binding sites. Within 3 years, appropriate radioligands, generally with low nanomolar dissociation constants for receptors, labeled receptors for the principal biogenic amine and amino acid neurotransmitters. This work permitted elucidation of the actions of many drugs. For the opiate receptor, heroin and codeine had negligible receptor affinity, indicating that they are only prodrugs, respectively, deacetylated and demethylated to form monacetylmorphine and morphine.

Important therapeutic effects of drugs were elucidated by receptor binding studies. Work of Arvid Carlsson on dopamine turnover had suggested that the antipsychotic effects of neuroleptic drugs might involve receptor blockade followed by feedback systems accelerating dopamine turnover (Carlsson and Lindqvist, 1963). The antipsychotic clinical potencies of a large series of neuroleptic drugs correlated closely with their affinities for dopamine receptors labeled by [ 3 H]haloperidol but not with receptors labeled by [ 3 H]dopamine (Creese et al., 1976 Seeman et al., 1976). We now know that the haloperidol and dopamine labeled binding sites, respectively, reflect D2 and D1 subtypes of dopamine receptors with blockade of D2 receptors being most therapeutically relevant. Muscarinic anticholinergic atropine-like side effects of first generation antipsychotic and antidepressant drugs hinder their use. Ligand binding to muscarinic cholinergic receptors afforded the drug industry a simple means for screening out such adverse actions leading to a new generation of safer and much more extensively used antidepressants.

Discrimination of binding sites by various ligands elucidated receptor subtypes. In the case of opiate receptors, Hans Kosterlitz differentiated binding sites for receptors designated μ, δ, and κ, which corresponded elegantly with evidence from pharmacologic studies in intact animals and various organ systems (Paterson et al., 1983). This advance led to efforts by the pharmaceutical industry to develop subtype-selective opiates that might be analgesics with less addictive potential, a goal that remains unfulfilled. Differentiation of serotonin receptor subtypes has facilitated the development of diverse agents, including 5-HT3 antagonists, in relieving the nausea of cancer chemotherapy and the triptan class of antimigraine agents.

A giant step forward in appreciation of receptors came from their molecular cloning, an effort in which the laboratory of Numa pioneered, with the cloning of the nicotinic acetylcholine receptor of the neuromuscular junction in 1982, an area in which Jean-Pierre Changeux also made major contributions (Numa, 1987). Cloning the receptor protein revealed that it harbors both the recognition site for the neurotransitter and the associated ion channel. This observation was presaged by work of Richard Huganir and Ephraim Racker who reconstituted the acetylcholine receptor protein, purified by conventional biochemistry, into vesicles loaded with radiolabeled sodium and demonstrated that the pure receptor protein contains the relevant sodium ion channel (Huganir and Racker, 1982). Today we take this concept for granted, but many investigators felt that ligand-binding proteins and ion channel proteins were separate entities that migrated through the membrane by lateral diffusion with binding of the neurotransmitter triggering their linkage. Robert Lefkowitz cloned the first biogenic amine receptor, the β-adrenoceptor (Dixon et al., 1986). Like Numa and others, Lefkowitz laboriously purified the receptor protein to homogeneity, obtaining partial amino acid sequence, then screened a cDNA library with a nucleotide probe. The β-adrenoceptor turned out to be a homolog of rhodopsin, cloned by Jeremy Nathans (Nathans and Hogness, 1984), who then cloned genes for the three visual pigments that mediate color vision (Nathans et al., 1986).

Intracellular messengers

In 1970, cAMP and cyclic GMP were the only established second messenger molecules. It was assumed that hormone/transmitter mediated deformation of receptors somehow impacts adenylyl cyclase. In 1969–1970 Martin Rodbell and Lutz Birnbaumer discovered that hormonal stimulation of receptor-coupled adenylyl cyclase required the addition of GTP leading to Rodbell's proposal of a “G protein,” which binds GTP and interfaces with the receptor (Rodbell, 1992). Alfred Gilman sought and isolated such proteins using the S49 leukemia cell line variant, which contains receptor and adenylyl cyclase but still does not respond to hormone treatment (Gilman, 1995). In 1994 Rodbell and Gilman shared the Nobel Prize in Physiology or Medicine for this effort. Today's scientists are often obsessed with publishing only in journals with high “impact factors.” Virtually all the key Rodbell and Gilman publications appeared in the Journal of Biological Chemistry.

In the mid-1980s Michael Berridge and colleagues identified inositol 1,4,5 trisphosphate (IP3) as a second messenger mediating the ability of hormones to release intracellular calcium (Berridge, 2009). It soon became evident that as many or more neurotransmitters and hormones act via IP3 as through cAMP. Since intracellular calcium is released in discrete quanta, it was assumed that intracellular calcium is stored in small vesicles whose surfaces must contain receptors for IP3. The “grind and bind” techniques that permitted identification of neurotransmitter receptors in the brain also facilitated labeling of IP3 receptors and their purification (Ferris and Snyder, 1992). Reconstitution of the purified IP3 receptor protein into lipid vesicles loaded with radioactive calcium revealed that the receptor contains both an IP3 recognition site and its associated calcium channel and permitted demonstration that the pure receptor protein contains the machinery to mediate quantal release of calcium. Receptor cloning by Katsuhiko Mikoshiba revealed one of the most exquisitely regulated proteins in biology (Mikoshiba, 2007). The IP3 receptor is a very large protein, >2700 aa, with the IP3 recognition site occupying only 200 aa at the N terminus and the calcium channel comprising a similar number of amino acids at the C terminus. The large intermediate area has binding sites whereby calcium release is modulated by factors such as NADH, ATP, the immunophilin FKBP12, ankyrin, Homer, Protein 4.1, myosin, calmodulin, caldendrin, chromogranins, cytochrome c, TRPC C calcium channels, heterotrimeric G proteins, and Irbit.

Phosphorylation is likely the most important posttranslational modification of proteins. The pioneering work of Paul Greengard in this arena commenced in 1969–1970 (Greengard, 2001). Up to that time phosphorylation was an arcane process uniquely associated with glycogen metabolism. Greengard demonstrated that large numbers of proteins are physiologically phosphorylated, especially in the brain. He characterized a number of these in depth, such as the synapsins that regulate synaptic vesicle disposition, and DARPP32, which is highly concentrated in dopamine enriched areas of the brain and is a principal target of cAMP-dependent kinase phosphorylation. Greengard identified a series of phosphatases and phosphatase inhibitors that regulate the various phosphorylation cascades.

Summing up

Where have we been and where are we going? The molecular characterization of synaptic transmission in terms of neurotransmitters, their receptors and intracellular messenger molecules advanced enormously in the past 40 years. The goal of all biomedical research is to understand human biology and thereby find causes and cures for disease. How do we fare when judged by these criteria? The principal drugs used in psychiatry—the antipsychotics, antidepressants and antianxiety agents—were all identified in the 1950s and the early 1960s. There have since been incremental advances but no major breakthroughs. Monitoring transmitter uptake in synaptosomes permitted the development of norepinephrine- and serotonin-selective antidepressants, which are substantive advances, but not transformational. The high-throughput monitoring of drug candidates at neurotransmitter receptors has greatly facilitated the drug development process. Systematic structure–activity analysis has permitted the design of drugs with extremely high affinity for receptors and with selectivity. For instance, the first-generation tricyclic antidepressants displayed troublesome muscarinic anticholinergic side effects. Screening chemicals at muscarinic receptors led to new generations of antidepressants devoid of these adverse actions.

What about insights into causation? Polymorphisms in enzymes of serotonin synthesis and serotonin transporters do predict susceptibility to depression indicating that serotonin is more than just a mediator of antidepressant drug effects. The explosion in technology permitting gene monitoring in humans will likely lead to further insights into the underpinnings of the major mental illnesses. Whether these genetic aberrations will involve neurotransmitters, receptors, and intracellular messengers is unknown. Thus far extensive genetic analysis of patients with schizophrenia and affective disorder has identified large numbers of rare sequence alterations that each contribute in a small way to the disease phenotype. Conceivably these illnesses will turn out to be extremely heterogeneous with multiple causal gene defects. If so, our hopes of finding “causes and cures” will be disappointed. But I am an optimist. I am confident that our molecular insights into synaptic communication will escalate in coming decades and that, 40 years from today, we will both have vastly greater insights into normal function as well as profound new understanding of disease etiology and associated therapies.

2. Calcium regulation of ROSਏormation

2.1. ROS formation

ROS are derived from molecular oxygen by electron transfer reactions resulting in the formation of superoxide anion radical (O2 •− ), and subsequently hydrogen peroxide (H2O2), either spontaneously, or by the action of superoxide dismutases (SOD). In the presence of iron, superoxide and H2O2 can lead to the formation of highly reactive hydroxyl radicals, which can damage cellular proteins, RNA, DNA and lipids. Interaction of ROS with nitric oxide or fatty acids can lead to the formation of peroxynitrite or peroxyl radicals, respectively, that are also highly reactive. In the presence of chloride, peroxidases can catalyze the generation of hypochlorous acid (HOCl) and singlet oxygen ( 1 O2) from H2O2.

Superoxide is not freely diffusible, but can cross membranes via ion channels. Extracellular superoxide has been shown to enter the cell via the anion blocker sensitive chloride channel-3 [69], while mitochondrial outer membrane´s voltage-dependent anion channels can direct superoxide flux from mitochondria to the cytosol [64]. On the other hand, hydrogen peroxide, which is not a radical, is diffusible over membranes and therefore has been frequently considered to act as a second messenger. Efficient transmembrane diffusion of hydrogen peroxide can be directed by aquaporins, which probably fine tune hydrogen peroxide levels in the cytoplasm, intracellular organelles, and the extracellular space [16]. High ROS levels in the cell can be achieved endogenously (e.g. in several cardiac pathologies), or exogenously (e.g. by administration of some types of chemotherapeutics). There is increasing evidence that in addition to the detrimental effects of high ROS levels exceeding the cellular antioxidant capacity, the cell is able to generate ROS in lower amounts that act as important signaling molecules controlling cell proliferation and cell death, cellular migration, vascular tone, and other cellular functions [75,99].

2.2. Calcium and mitochondrial ROS generation

Although the role of mitochondrial ROS is not completely understood, it is proposed that mitochondrial dysfunction causing excessive ROS production may be a prominent feature of several diseases [2]. Newer evidence suggests that mitochondrial ROS can also act as signaling molecules to activate pro-growth responses [139].

Generation of ROS by mitochondria has been considered for a long time to be only a byproduct of oxidative metabolism in the course of ATP production [151,164]. However, clear evidence exists that mitochondrial ROS might also have a function in signaling within mitochondria or between mitochondria and other organelles [130,34]. Under normal conditions, up to 1% of the electrons flowing to molecular oxygen through the electron transport chain may be diverted to form superoxide. Superoxide can be generated at different sites within the mitochondria [22]. Among them, the ubiquinone-binding sites in complex I (site IQ) and complex III (site IIIQo) of the respiratory chain, glycerol 3-phosphate dehydrogenase, the flavin in complex I (site IF), the electron transferring flavoprotein:Q oxidoreductase (ETFQOR) of fatty acid beta-oxidation, and pyruvate and 2-oxoglutarate dehydrogenases have the highest capacity to generate superoxide. Interestingly, only site IIIQo (on complex III) and glycerol 3-phosphate dehydrogenase can release superoxide into the intermembrane space suggesting that these sites are of a high importance of mitochondrial ROS release into the cytosol ([102], Fig. 2 ).

Calcium and ROS crosstalk between endoplasmic reticulum and mitochondria. The endoplasmic reticulum (ER) is a major site of calcium storage. Calcium from ER cisternae is flowing mainly through calcium release channels as inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR). These channels are accumulated in mitochondrial associated membranes (MAMs), which associate with the mitochondrial outer membrane. Calcium ions from the cytoplasm enter the mitochondria through voltage dependent anion channels (VDAC) or calcium uniporter. High levels of calcium stimulate respiratory chain activity leading to higher amounts of reactive oxygen species (ROS). ROS can further target ER-based calcium channels leading to increased release of calcium and further increased ROS levels. Increased ROS and calcium load can open the mitochondrial permeability transition pore (mPTP) resulting in the release of pro-apoptotic factors. Abbreviations: SERCA – sarco/endoplasmic reticulum Ca 2+ ATPase RyR – ryanodine receptors IP3R – IP3 receptor VDAC – voltage-dependent anion channel ANT – adenine-nucleotide transporter mPTP – mitochondrial permeability transition pore mNCX – mitochondrial sodium/calcium exchanger.

The metabolic state of the cell has an important impact on ROS production capacity of mitochondria. The chemical nature of the substrates fueling the respiratory chain, the amplitude of the membrane potential in mitochondria (ΔΨm), the pH of the matrix, and the oxygen tension in the surroundings are important factors controlling ROS production in mitochondria [5].

Since Ca 2+ primarily promotes ATP synthesis by stimulating enzymes of the Krebs cycle and oxidative phosphorylation in the mitochondria, it has been suggested that the increased metabolic rate would consume more oxygen resulting in increased respiratory chain electron leakage and ROS levels [26]. Indeed, mitochondrial ROS generation correlated with metabolic rate [136]. Under normal conditions, Ca 2+ diminished ROS from both complexes I and III while it enhanced ROS generation when these complexes were inhibited by pharmacological agents. One explanation has been that Ca 2+ induces a three-dimensional conformation change of the respiratory chain complexes which leads to mitochondrial ROS generation [26].

There is further evidence that the metabolic state of the mitochondria determines the effects of calcium on mitochondrial ROS levels. When the membrane potential is high (no ATP synthesis), Ca 2+ uptake results in decreased ROS generation. When the membrane potential is set to a depolarized range (ATP synthesis), ROS generation is stimulated, or not influenced by Ca 2+ , depending on the amount of the Ca 2+ load [1]. When mitochondria are overloaded with Ca 2+ , ROS production might increase independently of the metabolic state of mitochondria [94].

Mitochondrial permeability transition pore (mPTP) is a voltage and Ca 2+ -dependent, cyclosporin A sensitive, high conductance channel, whose prolonged opening leads to a brisk increase in the permeability of the inner mitochondrial membrane to solutes with molecular mass up to 1500ꃚ [13]. As a consequence, a bioenergetic catastrophe occurs: equilibration of the proton gradient causes mitochondrial depolarization, followed by respiratory inhibition and generation of ROS, massive release of matrix Ca 2+ , and swelling of mitochondria which leads to breaches in the outer mitochondrial membrane that induce the release of intermembrane proteins. Thus, mPTP opening prompts the demise of the cell, and its (dys)regulation turned out to be a crucial step in the pathogenesis of a variety of diverse diseases, encompassing ischemia-reperfusion damage, lysosomal storage diseases, liver damage, many acute and chronic disorders of the central nervous system and cancer (for review see [119]).

2.3. Calcium and NADPH oxidases

The family of NADPH oxidases (NOXes) has been considered unique in that their sole function is to generate superoxide or hydrogen peroxide, respectively, and that they are responsive to receptor stimulation [115,9]. Up to date, this family comprises 7 members, which differ in their catalytic subunits as well as in the requirement of regulatory proteins. The initially identified NADPH oxidase contains the NOX2 core unit, which builds together with the p22phox subunit the cytochrome b558. It is also known as the “respiratory burst” enzyme of neutrophils and is a part of the innate immune response. Upon binding of particles, bacteria, fungi or soluble inflammatory mediators to specific receptors on the neutrophil cell surface, NOX2 is activated and mediates release of large amounts of ROS [108]. This activation is regulated by cytosolic subunits p47phox, p67phox, p40phox and the Rac GTPase, which need to be phosphorylated by calcium activated protein kinase C (PKC) in order to translocate to the plasma membrane and join the NOX2/p22phox complex [30].

The majority of neutrophil-activating receptors induce extracellular calcium entry as an early signaling response to activate effector functions, including phagocytosis, degranulation, and chemotaxis [108]. These membrane receptors induce generation of inositol 1,4,5-trisphosphate (IP3) which activates IP3Rs and Ca 2+ release from the intracellular stores which is important for phagocytosis [137]. Depleted stores are reloaded by the sarco/endoplasmic reticulum Ca 2+ ATPase SERCA, whereby calcium influx into the cell is enhanced through store-operated calcium channels [23]. This Ca 2+ influx is also required for neutrophil ROS generation by stimulating Ca 2+ -dependent recruitment of S100A8/A9 proteins which act as Ca 2+ sensors and can interact with flavocytochrome b558 and p67phox to promote ROS generation [25]. Moreover, Hv1 voltage-gated proton channels have been shown to extrude the protons and compensate the charge generated by NADPH oxidases, thereby enhancing the driving force for extracellular Ca 2+ entry and sustaining NADPH oxidase activity [46].

Similar to the NOX2 containing enzyme in neutrophils, NOX1 activity in keratinocytes has been described to be dependent on calcium in response to UVA light [153]. NOX1 activity requires the recruitment of cytosolic activators similar to NOX2, suggesting that calcium might also act in resembling way. Moreover, it has been recently shown that NOX1 can directly be phosphorylated by the calcium activated PKC㬡 suggesting that calcium may via this way enhance NOX1 activity [138].

Apart from these more indirect ways of calcium-dependent NOX activation, the NOX5 as well as the DUOX1 and DUOX2 containing enzymes have been shown to be calcium-binding proteins, which require calcium for ROS generation. NOX5 contains an N-terminal regulatory domain (called NOX5-EF) with four EF-hands. When Ca 2+ binds to this domain, hydrophobic residues can interact with the C-terminal catalytic domain and activate the enzyme [7]. Besides of EF-hands, NOX5 can bind calcium-activated calmodulin to the C-terminal domain, leading to a conformational change and increased N-terminal enzymatic activity. Furthermore calcium-activated calcium/calmodulin-dependent kinase II (CAMKII) can positively regulate NOX5 activity via the phosphorylation of Ser475 [111]. Calcium-dependent NOX5 activity has been found to contribute to vascular proliferation and vessel formation [10], to proliferation in different cancer cell lines [3] and also might play a role in kidney disease [76] and in coronary artery disease [61].

Two other family members, dual oxidase 1 (DUOX1) and 2 (DUOX2) have been originally identified in the mammalian thyroid gland. DUOX1 is also highly expressed in airway epithelial cells and DUOX2 in the salivary glands and gastrointestinal tract. Dual oxidases contain an EF-hand calcium-binding cytosolic region similar to that in NOX5 and an N-terminal, extracellular domain with considerable sequence identity with mammalian peroxidases. DUOX enzymes are activated by calcium and release hydrogen peroxide rather than superoxide. In the thyroid, hydrogen peroxide produced by DUOX2 is utilized by thyroperoxidase as an electron acceptor to generate protein-bound iodothyronines (T3 and T4) [109,27,88]. Recently, it was shown that epidermal wounding induces a calcium flash which activates hydrogen peroxide production via DUOX1 and subsequently the recruitment of immune cells to migrate to the wound [122]. Similarly, calcium flashes have been shown to trigger DUOX-dependent hydrogen peroxide in zebrafish after mechanical injury, resulting in leukocyte recruitment [107]. Genetic studies in Drosophila have demonstrated that DUOX can generate microbicidal ROS in the gut epithelia [91].

Recent studies suggested a cross-talk between NADPH oxidases and mitochondrial ROS generation. For example, NOX2 was shown to stimulate mitochondrial ROS production by activating reverse electron transfer in angiotensin-II induced hypertension, while mitochondrial superoxide induced by activation of mitochondrial ATP-sensitive K + channels has been demonstrated to stimulate NOX2, contributing to the development of endothelial oxidative stress and hypertension [106,43]. Although the exact mechanisms of this cross-talk are not clear yet, these findings might explain some discrepancies found in the literature regarding the sources of ROS. Since both ROS generating systems are sensitive to calcium, they show the importance of the calcium-ROS cross-talk under (patho)physiological conditions.

Second messenger

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Second messenger, molecule inside cells that acts to transmit signals from a receptor to a target. The term second messenger was coined upon the discovery of these substances in order to distinguish them from hormones and other molecules that function outside the cell as “first messengers” in the transmission of biological information. Many second messenger molecules are small and therefore diffuse rapidly through the cytoplasm, enabling information to move quickly throughout the cell. As elements of signaling pathways, second messengers can serve to integrate information when multiple independent upstream inputs influence the rates of synthesis and degradation of the second messenger. In addition, second messengers can have multiple downstream targets, thereby expanding the scope of signal transmission.

A large number of second messenger molecules have been characterized, including cyclic nucleotides (e.g., cyclic adenosine monophosphate, or cAMP, and cyclic guanosine monophosphate, or cGMP), ions (e.g., Ca 2+ ), phospholipid-derived molecules (e.g., inositol triphosphate), and even a gas, nitric oxide (NO). The calcium ion Ca 2+ has a critical role in the rapid responses of neurons and muscle cells. At rest, cells maintain a low concentration of Ca 2+ in the cytoplasm, expending energy to pump these ions out of the cell. When activated, neurons and muscle cells rapidly increase their cytoplasmic Ca 2+ concentration by opening channels in the cell membrane, which allow Ca 2+ ions outside the cell to enter rapidly.

The cyclic nucleotide cAMP is synthesized by adenylyl cyclase enzymes, which are downstream of heterotrimeric G-proteins (guanine nucleotide binding proteins) and receptors. For example, when epinephrine binds to beta-adrenergic receptors in cell membranes, G-protein activation stimulates cAMP synthesis by adenylyl cyclase. The newly synthesized cAMP is then able to act as a second messenger, rapidly propagating the epinephrine signal to the appropriate molecules in the cell. This stimulatory signaling pathway leads to the production of effects such as increasing rate and force of contraction of the heart that are characteristic of epinephrine. Caffeine also enhances the action of cAMP by inhibiting the enzyme phosphodiesterase, which degrades cAMP the enhancement of cAMP activity contributes to the general stimulatory action of caffeine. As a gas, nitric oxide (NO) is distinct among second messengers in being able to diffuse across cell membranes, which allows signal information to cross into neighbouring cells.

Watch the video: Calcium acting as secondary messenger in hormonal activity (May 2022).