Pharmacol Rev Research Articles

Dueling nucleosides: cross-regulation of extracellular adenosine by guanosine. Focus on “Extracellular guanosine regulates extracellular adenosine levels”

<i>Dueling nucleosides: cross-regulation of extracellular adenosine by guanosine</i>. Focus on “Extracellular guanosine regulates extracellular adenosine levels”

Hypertensive nephrosclerosis: not enough of a good thing?

glutathione s -transferases (GSTs) are multifunctional enzymes that are perhaps best known to participate in cell detoxification by catalyzing the conjugation of reduced glutathione to a wide variety of xenobiotics that have electrophilic centers. Production of these adducts promotes cytoprotection

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

Does oxygen tune cellular mechanotransduction?

Does oxygen tune cellular mechanotransduction?

Highly Accurate Quantification of the Oligomerization of the β2 Adrenergic Receptor using FRET

The oligomerization of G-protein coupled receptors, GPCRs, has in many cases shown to be important for transmembrane protein structure and function [1,2], however achieving quantitative parameters describing the process that governs oligomerization is difficult. Traditionally oligomerization is quantified by preparing distinct samples that carry different concentrations and by monitoring bulk spectroscopic signals e.g FRET efficiencies [3], still the limited amount of samples does not allow very accurate determination of the experimental parameters.Here we developed a nanoscale assay [4,5] that allows a highly accurate quantification of the interaction forces between transmembrane molecules. As a proof of concept we focused on the prototypical human β2-adrenergic receptor, β2AR, reconstituted in lipid vesicles. Our approach allowed us to extract the dimerization equilibrium constant of the β2AR to be −4 ± 0.2 KbT.[1] Bouvier, M. Oligomerization of G-protein-coupled transmitter receptors. Nat Rev Neurosci 2, 274-286 (2001).[2] Smith, N.J. & Milligan, G. Allostery at G protein-coupled receptor homo- and heteromers: uncharted pharmacological landscapes. Pharmacol Rev 62, 701-725 (2010).[3] Fung, J.J. et al. Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer. EMBO J 28, 3315-3328 (2009).[4] Stamou, D. et al. Amphipathic motifs in BAR domains are essential for membrane curvature sensing. Embo Journal 28, 3303-3314 (2009).[5] Stamou, D. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nature Chemical Biology 5, 835-841 (2009).

The Editor takes a closer look at some of this month's articles

The Editor takes a closer look at some of this month's articles

Counterpoint: Activation of the Intrarenal Renin-Angiotensin System is the Dominant Contributor to Systemic Hypertension

Our discussion regarding the dominant mechanism responsible for hypertension calls to mind the famous poem, “The Blind Men and the Elephant,” by John Godfrey Saxe based on an ancient fable from India. In essence, the blind men described the elephant based on their specific encounter, thus

Signaling through the Na/K-ATPase: implications for cardiac fibrosis

the cardiotonic effects of digitalis have been exploited in clinical practice for many years. However, while the existence of endogenous digitalis-like substances (also called cardiotonic steroids) has been accepted for some time, their exact chemical nature has been only recently demonstrated ([1][

LGIC

LGIC

Angiotensin II type-1 receptor regulates RhoA and Rho-kinase/ROCK activation via multiple mechanisms. Focus on “Angiotensin II induces RhoA activation through SHP2-dependent dephosphorylation of the RhoGAP p190A in vascular smooth muscle cells”

angiotensin ii, a crucial regulator of the cardiovascular system, is not only required for physiological functions, such as maintenance of blood pressure and plasma sodium concentration but also is involved in pathophysiological conditions such as hypertension and atherosclerosis (26). Angiotensin II exerts its effect by mainly binding to the angiotensin II type-1 (AT1) receptor. As its seven transmembrane structure suggests, the AT1 receptor couples to several heterotrimeric G proteins including Gq. In addition, the activation of the AT1 receptor leads to increased reactive oxygen species; activation of various protein tyrosine and serine/threonine kinases; and of small G proteins such as Ras, Rac, and RhoA, which may require heterotrimeric G protein-dependent and -independent mechanisms (6, 7, 11, 19).

International Union of Pharmacology. LXX. Subtypes of γ-Aminobutyric AcidAReceptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update

In this review we attempt to summarize experimental evidence on the existence of defined native GABA(A) receptor subtypes and to produce a list of receptors that actually seem to exist according to current knowledge. This will serve to update the most recent classification of GABA(A) receptors (Pharmacol Rev 50:291-313, 1998) approved by the Nomenclature Committee of the International Union of Pharmacology. GABA(A) receptors are chloride channels that mediate the major form of fast inhibitory neurotransmission in the central nervous system. They are members of the Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily and share structural and functional homology with other members of that family. GABA(A) receptors are assembled from a family of 19 homologous subunit gene products and form numerous, mostly hetero-oligomeric, pentamers. Such receptor subtypes with properties that depend on subunit composition vary in topography and ontogeny, in cellular and subcellular localization, in their role in brain circuits and behaviors, in their mechanisms of regulation, and in their pharmacology. We propose several criteria, which can be applied to all the members of the LGIC superfamily, for including a receptor subtype on a list of native hetero-oligomeric subtypes. With these criteria, we develop a working GABA(A) receptor list, which currently includes 26 members, but will undoubtedly be modified and grow as information expands. The list is divided into three categories of native receptor subtypes: "identified," "existence with high probability," and "tentative."

Ion Channels

Ion Channels

Nuclear Receptors

Nuclear Receptors

Delineating the contributions of AT1a and AT1b receptor-mediated uptake of ANG II in kidneys and adrenals

inappropriately elevated angiotensin ii (ANG II) levels induced by infusion of ANG II or overexpression of the renin-angiotensin system (RAS) commonly result in increased intrarenal and intra-adrenal ANG II content ([8][1]). Derangement of renal function due to inappropriate activation of intrarenal

Study on the Interaction between Florasulam and Bovine Serum Albumin

In this paper, the interaction between florasulam (FU, 2',6',8-trifluoro-5-methoxy [Kragh-Hansen U, Molecular aspects of ligand binding to serum albumin. Pharmacol Rev 33(1):17-53 1981; Carter DC and Ho JX, Structure of serum albumin. Adv Protein Chem 45:153-203 1994; He XM, and Carter DC, Atomic structure and chemistry of human serum albumin. Nature 358(6383):209-215 1992] triazolo [1,5-c]pyrimidine-2-sulfonanilide) and bovine serum albumin (BSA) was investigated by fluorescence, ultraviolet absorption (UV) and Far-UV circular dichroism (CD) spectrometries. A strong fluorescence quenching was observed and the quenching mechanism was considered as static quenching. The binding constant of FU with BSA at 299 and 309 K were obtained as 1.5 x 10(4) and 7.1 x 10(3) l mol(-1), respectively. There was one binding site between FU and BSA. The thermodynamic parameters enthalpy change (DeltaH) and entropy change (DeltaS) were calculated as -57.89 kJ mol(-1) and -113.6 J mol(-1) K(-1), respectively, which indicated that the acting force between FU and BSA was mainly hydrogen bond and Van der Waals force. According to the Förster non-radiation energy transfer theory, the average binding distance between donor (BSA) and acceptor (FU) was obtained (r = 1.59 nm). The investigations of the UV/Vis and CD spectra of the system showed that the conformation of BSA was changed in presence of FU.

Age-dependent changes in the functional expression of two nicotinic receptor subtypes in CA1 stratum radiatum interneurons in the rat hippocampus

Protein density measurements and mRNA analysis have provided valuable information on age-dependent changes in the distribution of different nicotinic receptor (nAChR) subtypes in various areas of the rat brain, including the hippocampus. However, very little is known regarding the functional expression of nAChRs in individual neuron types at various ages. Likewise, there is paucity of information regarding the functional and pharmacological profile of nAChRs in the mature rat hippocampus. To address these issues, we used the whole-cell patch-clamp technique to record nicotinic responses from CA1 stratum radiatum (SR) interneurons in hippocampal slices from rat pups (5–19 days old) and adult rats (2–5 months old). As previously observed in the hippocampus of rat pups, CA1 SR interneurons in the hippocampus of adult rats responded to choline (10 mM, 12 s) with whole-cell currents that decayed to the baseline within the agonist pulse, were sensitive to inhibition by methyllycaconitine (10 nM) or α-bungarotoxin (50 nM), and were, therefore, mediated by α7* 1 1 According to the nomenclature for nAChRs and their subunits [Lukas RJ, Changeux JP, Le Novere N, Albuquerque, EX, Balfour DJ, Berg, DK et al. International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 1999; 51: 397–401.], the asterisk next to nAChR subunits throughout text is meant to indicate that the exact subunit composition is not known. [1] nAChRs. Likewise, as previously observed in the hippocampus of young rats, in the adult rat hippocampus excitatory postsynaptic currents (EPSCs) were recorded from SR interneurons in response to a pulse of ACh (0.1 mM, 12 s) applied in the presence of the GABA A receptor antagonist bicuculline. ACh-triggered EPSCs were inhibited by mecamylamine (1 μM) or choline (1 mM) and were, therefore, likely to have resulted from activation of α3β4β2* nAChR. The magnitude of α7* nAChR-mediated responses increased with the age of the animals. In contrast, the magnitude of α3β4β2* nAChR-mediated responses was highest at the second postnatal week. The distinct age dependency of functional expression of α7* and α3β4β2* nAChRs strongly suggests that the excitability of CA1 SR interneurons is differentially regulated by the nicotinic cholinergic system in the hippocampus of rat pups and adult rats.

Competitive Antagonism between the Nicotinic Allosteric Potentiating Ligand Galantamine and Kynurenic Acid at α7* Nicotinic Receptors

Galantamine, a drug used to treat Alzheimer's disease, is a nicotinic allosteric potentiating ligand, and kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is an endogenous noncompetitive inhibitor of alpha7* nicotinic receptors (nAChRs) [the asterisk next to the nAChR subunit is intended to indicate that the exact subunit composition of the receptor is not known (Pharmacol Rev 51:397-401, 1999)]. Here, possible interactions between KYNA and galantamine at alpha7* nAChRs were examined in vitro and in vivo. In the presence of tetrodotoxin (TTX), approximately 85% of cultured hippocampal neurons responded to choline (0.3-30 mM) with alpha7* nAChR-subserved whole-cell (type IA) currents. In the absence of TTX and in the presence of glutamate receptor antagonists, choline triggered inhibitory postsynaptic currents (IPSCs) by activating alpha7* nAChRs on GABAergic neurons synapsing onto the neurons under study. Galantamine (1-10 microM) potentiated, whereas KYNA (10 nM-1 mM) inhibited, choline-triggered responses. Galantamine (1 microM), applied before KYNA, shifted to the right the concentration-response relationship for KYNA to inhibit type IA currents, increasing the IC(50) of KYNA from 13.9 +/- 8.3 to 271 +/- 131 microM. Galantamine, applied before or after KYNA, antagonized inhibition of choline-triggered IPSCs by KYNA. Local infusion of KYNA (100 nM) in the rat striatum reduced extracellular dopamine levels in vivo. This effect resulted from alpha7* nAChR inhibition and was blocked by coapplied galantamine (1-5 microM). It is concluded that galantamine competitively antagonizes the actions of KYNA on alpha7* nAChRs. Reducing alpha7* nAChR inhibition by endogenous KYNA may be an important determinant of the effectiveness of galantamine in neurological and psychiatric disorders associated with decreased alpha7* nAChR activity in the brain.

Role of Na/Ca exchange and the plasma membrane Ca 2+ ‐ATPase in cell function

The Fifth International Conference on Na/Ca Exchange took place between 23 and 27 August 2006 in Brussels, Belgium, and was organized by A. Herchuelz. Image taken from the meeting poster showing hypothetical models for the Na/Ca exchanger (left) and the plasma membrane Ca2+‐ATPase (right), courtesy of M. Hilge. ![][1] Cytosolic Ca2+ regulates several cellular processes and its concentration is, in turn, finely regulated by various channels, pumps and exchangers. The Na/Ca exchanger (NCX) and the plasma membrane Ca2+‐ATPase (PMCA) pump are two concurrent mechanisms for Ca2+ extrusion from the cell (Carafoli, 1994; Blaustein & Lederer, 1999; Philipson & Nicoll, 2000; Strehler & Treiman, 2004; Prasad et al , 2004). PMCA and NCX were cloned 17 and 19 years ago, respectively; four mammalian isoforms have been identified for the former (PMCA1–4) and three have been identified for the latter (NCX1–3; Carafoli, 1994; Philipson & Nicoll, 2000). Na/Ca exchange (NCX) is a unique mechanism that allows Ca2+ extrusion from the cell against its gradient without energy consumption. Indeed, it is the entry of Na+ along its electrochemical gradient that provides the energy for Ca2+ extrusion. In addition, because NCX is electrogenic and voltage sensitive, it can reverse during cellular activation and contribute to Ca2+ entry into the cell (Blaustein & Lederer, 1999). Recently, the crucial role of NCX in cellular Ca2+ homeostasis has been definitively shown by the generation of mice with functional destruction of one Ncx gene. Homozygous Ncx1 ‐deficient mice are not viable, and die between embryonic days 9 and 10, owing to the lack of development of the cardiovascular system (Wakimoto et al , 2000); therefore, NCX is essential for life. PMCA belongs to the P‐type family of transport ATPases, which form a phosphorylated intermediate … [1]: /embed/graphic-1.gif

Notice of Concern

It has come to our attention that the review by Suh and Checler [Suh Y-H and Checler F (2002) Amyloid precursor protein, presenilins, and α-synuclein: molecular pathogenesis and pharmacological applications to Alzheimer's disease. Pharmacol Rev 54: [469][1]–525] contains several paragraphs that

Muscarinic receptors and salivary secretion

The release of acetylcholine from parasympathetic nerves and its interaction with muscarinic acetylcholine receptors (mAChRs) regulates many fundamental functions in the periphery (smooth muscle contraction, glandular secretion, modulation of cardiac output) and central nervous system (motor control