Inhibition of Interactions Between NDPK-B and NDPK-C in Presence of Graphene Oxide

During a heart failure, higher amount of nucleoside diphosphate kinase (NDPK) enzyme in the sarcolemma membrane inhibits the synthesis of second messenger cyclic adenosine monophosphate (cAMP), which is required for the regulation of the calcium ion balance for normal functioning of the heart. In a dependent pathway, NDPK normally phosphorylates the stimulatory guanosine diphosphate, GDP(s), to a guanosine triphosphate, GTP(s), on the heterotrimeric (α, β and γ subunits) guanine nucleotide binding protein (G protein), resulting in the stimulation of the cAMP formation. In case of a heart failure, an increased quantity of NDPK also reacts with the inhibitory GDP(i), which is converted to a GTP(i), resulting in the inhibition of the cAMP formation. Typically, the βγ dimer of the G protein binds with hexameric NDPK-B/C complex and receives the phosphate at the residue His266 from residue His118 of NDPK-B. It is known that NDPK-C is required for NDPK-B to phosphorylate the G protein. In this work, the interactions between NDPK-B and NDPK-C are quantified in the presence and absence of graphene oxide (GO) as well as those between NDPK-B and GO through stability analysis involving hydrogen bonds, center of mass (COM), root mean square deviation (RMSD), and salt bridges, and energetics analysis involving van der Waals (VDW) and electrostatic energies. Furthermore, the role of water molecules at the interface of NDPK-B and NDPK-C as well as between NDPK-B and GO is investigated to understand the nature of interactions. It is found that the adsorption of NDPK-B on GO triggers a potential conformational change in the structure of NDPK-B, resulting in a diminished interaction with NDPK-C. This is confirmed through a reduced center of mass (COM) distance between NDPK-B and GO (from 40 Å to 30 Å) and an increased COM distance between NDPK-B and NDPK-C (from 50 Å to 60 Å). Furthermore, this is also supported by fewer salt bridges between NDPK-B and NDPK-C, and an increased number of hydrogen bonds formed by the interfacial water molecules. As NDPK-C is crucial to be complexed with NDPK-B for successful interaction of NDPK-B with the G protein, this finding shows that GO can suppress the interactions between NDPK-B/C and G proteins, thereby providing an additional insight into the role of GO in the heart failure mechanism.

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation

Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

Analysis of β2AR-Gs and β2AR-Gi complex formation by NMR spectroscopy

The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the β2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the β2AR-Gs complex revealed the interaction interface of β2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549–555 (2011)], yet, the dynamic process of the β2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the β2AR in response to the full agonist BI-167107 and Gs and Gi1. These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the β2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the β2AR-Gs and the β2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs. These differences between the β2AR-Gs and β2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.

Ranitidine: recent regulatory issues

Ranitidine is histamine 2 receptor blocker which became commercial in 1981 as an antacid by Glaxosmithkline Pharamceuticals by the brand name of Zantac in various formulations. The drug accelerated in the market as amongst most commonly used drug for peptic ulcer disease, acid reflux and sooner than later it became available as an over-the-counter drug in 1996 for adults and children. Ranitidine’s mechanism of action involves competitive block of histamine 2 receptor leading to decrease cAMP formation which reduces acid secretion from parietal cells of stomach thereby healing the peptic ulcer.

α 2-Adrenoceptors: Challenges and Opportunities—Enlightenment from the Kidney

It was indeed a Don Quixote-like pursuit of the mechanism of essential hypertension when we serendipitously discovered α2-adrenoceptors (α2-ARs) in skin-lightening experiments in the frog. Now α2-ARs lurk on the horizon involving hypertension causality, renal denervation for hypertension, injury from falling in the elderly and prazosin’s mechanism of action in anxiety states such as posttraumatic stress disorder (PTSD). Our goal here is to focus on this horizon and bring into clear view the role of α2-AR-mediated mechanisms in these seemingly unrelated conditions. Our narrative begins with an explanation of how experiments in isolated perfused kidneys led to the discovery of a sodium-retaining process, a fundamental mechanism of hypertension, mediated by α2-ARs. In this model system and in the setting of furosemide-induced sodium excretion, α2-AR activation inhibited adenylate cyclase, suppressed cAMP formation, and caused sodium retention. Further investigations led to the realization that renal α2-AR expression in hypertensive animals is elevated, thus supporting a key role for kidney α2-ARs in the pathophysiology of essential hypertension. Subsequent studies clarified the molecular pathways by which α2-ARs activate prohypertensive biochemical systems. While investigating the role of α1-adrenoceptors (α1-ARs) versus α2-ARs in renal sympathetic neurotransmission, we noted an astonishing result: in the kidney α1-ARs suppress the postjunctional expression of α2-ARs. Here, we describe how this finding relates to a broader understanding of the role of α2-ARs in diverse disease states. Because of the capacity for qualitative and quantitative monitoring of α2-AR-induced regulatory mechanisms in the kidney, we looked to the kidney and found enlightenment.

Exploring Biased Agonism at FPR1 as a Means to Encode Danger Sensing

Ligand-based selectivity in signal transduction (biased signaling) is an emerging field of G protein-coupled receptor (GPCR) research and might allow the development of drugs with targeted activation profiles. Human formyl peptide receptor 1 (FPR1) is a GPCR that detects potentially hazardous states characterized by the appearance of N-formylated peptides that originate from either bacteria or mitochondria during tissue destruction; however, the receptor also responds to several non-formylated agonists from various sources. We hypothesized that an additional layer of FPR signaling is encoded by biased agonism, thus allowing the discrimination of the source of threat. We resorted to the comparative analysis of FPR1 agonist-evoked responses across three prototypical GPCR signaling pathways, i.e., the inhibition of cAMP formation, receptor internalization, and ERK activation, and analyzed cellular responses elicited by several bacteria- and mitochondria-derived ligands. We also included the anti-inflammatory annexinA1 peptide Ac2-26 and two synthetic ligands, the W-peptide and the small molecule FPRA14. Compared to the endogenous agonists, the bacterial agonists displayed significantly higher potencies and efficacies. Selective pathway activation was not observed, as both groups were similarly biased towards the inhibition of cAMP formation. The general agonist bias in FPR1 signaling suggests a source-independent pathway selectivity for transmission of pro-inflammatory danger signaling.