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Norepinephrine transporter (NET; encoded by SLC6A2) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic noradrenaline level 1 . Genetic mutations and dysregulation of NET are associated with a spectrum of neurological conditions in humans, making NET an important therapeutic target 1 . However, the structure and mechanism of NET remain unclear. Here we provide cryogenic electron microscopy structures of the human NET (hNET) in three functional states-the apo state, and in states bound to the substrate meta-iodobenzylguanidine (MIBG) or the orthosteric inhibitor radafaxine. These structures were captured in an inward-facing conformation, with a tightly sealed extracellular gate and an open intracellular gate. The substrate MIBG binds at the centre of hNET. Radafaxine also occupies the substrate-binding site and might block the structural transition of hNET for inhibition. These structures provide insights into the mechanism of substrate recognition and orthosteric inhibition of hNET., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

K ATP channels are metabolic sensors for intracellular ATP/ADP ratios, play essential roles in many physiological processes, and are implicated in a spectrum of pathological conditions. SUR2A-containing K ATP channels differ from other subtypes in their sensitivity to Mg-ADP activation. However, the underlying structural mechanism remains poorly understood. Here we present a series of cryo-EM structures of SUR2A in the presence of different combinations of Mg-nucleotides and the allosteric inhibitor repaglinide. These structures uncover regulatory helix (R helix) on the NBD1-TMD2 linker, which wedges between NBD1 and NBD2. R helix stabilizes SUR2A in the NBD-separated conformation to inhibit channel activation. The competitive binding of Mg-ADP with Mg-ATP to NBD2 mobilizes the R helix to relieve such inhibition, allowing channel activation. The structures of SUR2B in similar conditions suggest that the C-terminal 42 residues of SUR2B enhance the structural dynamics of NBD2 and facilitate the dissociation of the R helix and the binding of Mg-ADP to NBD2, promoting NBD dimerization and subsequent channel activation., (© 2023. The Author(s).)

Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions that participate in pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the physiological importance of this complex, its structure remains elusive. Here, we reported the cryo-EM structure of the functional human NOX2-p22 complex in nanodisc in the resting state. NOX2 shows a canonical 6-TM architecture of NOX and p22 has four transmembrane helices. M3, M4, and M5 of NOX2, and M1 and M4 helices of p22 are involved in the heterodimer formation. Dehydrogenase (DH) domain of NOX2 in the resting state is not optimally docked onto the transmembrane domain, leading to inefficient electron transfer and NADPH binding. Structural analysis suggests that the cytosolic factors might activate the NOX2-p22 complex by stabilizing the DH in a productive docked conformation., Competing Interests: RL, KS, JW, XG, LZ, XG, HP, LC No competing interests declared, (© 2022, Liu, Song et al.)

ATP-sensitive potassium channels (K ATP ) are energy sensors that participate in a range of physiologic processes. These channels are also clinically validated drug targets. For decades, K ATP inhibitors have been prescribed for diabetes and K ATP activators have been used for the treatment of hypoglycemia, hypertension, and hair loss. In this Emerging Concepts article, we highlight our current knowledge about the drug binding modes observed using cryogenic electron microscopy techniques. The inhibitors and activators bind to two distinct sites in the transmembrane domain of the sulfonylurea receptor (SUR) subunit. We also discuss the possible mechanism of how these drugs allosterically modulate the dimerization of SUR nucleotide-binding domains (NBDs) and thus K ATP channel activity. SIGNIFICANCE STATEMENT: ATP-sensitive potassium channels (K ATP ) are fundamental to energy homeostasis, and they participate in many vital physiological processes. K ATP channels are important drug targets. Both K ATP inhibitors (insulin secretagogues) and K ATP activators are broadly used clinically for the treatment of related diseases. Recent cryogenic electron microscopy studies allow us to understand the emerging concept of K ATP structural pharmacology., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Mitiglinide is a highly selective fast-acting anti-diabetic drug that induces insulin secretion by inhibiting pancreatic K ATP channels. However, how mitiglinide binds K ATP channels remains unknown. Here, we show the cryo-EM structure of the SUR1 subunit complexed with mitiglinide. The structure reveals that mitiglinide binds inside the common insulin secretagogue-binding site of SUR1, which is surrounded by TM7, TM8, TM16, and TM17. Mitiglinide locks SUR1 in the NBD-separated inward-facing conformation. The detailed structural analysis of the mitiglinide-binding site uncovers the molecular basis of its high selectivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Wu and Chen.)

ATP-sensitive potassium channels (K ATP ) are metabolic sensors that convert the intracellular ATP/ADP ratio to the excitability of cells. They are involved in many physiological processes and implicated in several human diseases. Here we present the cryo-EM structures of the pancreatic K ATP channel in both the closed state and the pre-open state, resolved in the same sample. We observe the binding of nucleotides at the inhibitory sites of the Kir6.2 channel in the closed but not in the pre-open state. Structural comparisons reveal the mechanism for ATP inhibition and Mg-ADP activation, two fundamental properties of K ATP channels. Moreover, the structures also uncover the activation mechanism of diazoxide-type K ATP openers., (© 2022. The Author(s).)

ATP-sensitive potassium channels (K ATP ), composed of Kir6 and SUR subunits, convert the metabolic status of the cell into electrical signals. Pharmacological activation of SUR2- containing K ATP channels by class of small molecule drugs known as K ATP openers leads to hyperpolarization of excitable cells and to vasodilation. Thus, K ATP openers could be used to treat cardiovascular diseases. However, where these vasodilators bind to K ATP and how they activate the channel remains elusive. Here, we present cryo-EM structures of SUR2A and SUR2B subunits in complex with Mg-nucleotides and P1075 or levcromakalim, two chemically distinct K ATP openers that are specific to SUR2. Both P1075 and levcromakalim bind to a common site in the transmembrane domain (TMD) of the SUR2 subunit, which is between TMD1 and TMD2 and is embraced by TM10, TM11, TM12, TM14, and TM17. These K ATP openers synergize with Mg-nucleotides to stabilize SUR2 in the NBD-dimerized occluded state to activate the channel., (© 2022. The Author(s).)

TRPC3 and TRPC6 channels are calcium-permeable non-selective cation channels that are involved in many physiological processes. The gain-of-function (GOF) mutations of TRPC6 lead to familial focal segmental glomerulosclerosis (FSGS) in humans, but their pathogenic mechanism remains elusive. Here, we report the cryo-EM structures of human TRPC3 in both high-calcium and low-calcium conditions. Based on these structures and accompanying electrophysiological studies, we identified both inhibitory and activating calcium-binding sites in TRPC3 that couple intracellular calcium concentrations to the basal channel activity. These calcium sensors are also structurally and functionally conserved in TRPC6. We uncovered that the GOF mutations of TRPC6 activate the channel by allosterically abolishing the inhibitory effects of intracellular calcium. Furthermore, structures of human TRPC6 in complex with two chemically distinct inhibitors bound at different ligand-binding pockets reveal different conformations of the transmembrane domain, providing templates for further structure-based drug design targeting TRPC6-related diseases such as FSGS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

TRPC5 channel is a nonselective cation channel that participates in diverse physiological processes. TRPC5 inhibitors show promise in the treatment of anxiety disorder, depression, and kidney disease. However, the binding sites and inhibitory mechanism of TRPC5 inhibitors remain elusive. Here, we present the cryo-EM structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and HC-070, to the resolution of 2.7 Å. The structures reveal that clemizole binds inside the voltage sensor-like domain of each subunit. In contrast, HC-070 is wedged between adjacent subunits and replaces the glycerol group of a putative diacylglycerol molecule near the extracellular side. Moreover, we found mutations in the inhibitor binding pockets altered the potency of inhibitors. These structures suggest that both clemizole and HC-070 exert the inhibitory functions by stabilizing the ion channel in a nonconductive closed state. These results pave the way for further design and optimization of inhibitors targeting human TRPC5., Competing Interests: KS, MW, WG, LQ, YK, JW, LC No competing interests declared, (© 2021, Song et al.)

Dual oxidases (DUOXs) produce hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen. They are involved in many crucial biological processes and human diseases, especially in thyroid diseases. DUOXs are protein complexes co-assembled from the catalytic DUOX subunits and the auxiliary DUOXA subunits and their activities are regulated by intracellular calcium concentrations. Here, we report the cryo-EM structures of human DUOX1-DUOXA1 complex in both high-calcium and low-calcium states. These structures reveal the DUOX1 complex is a symmetric 2:2 hetero-tetramer stabilized by extensive inter-subunit interactions. Substrate NADPH and cofactor FAD are sandwiched between transmembrane domain and the cytosolic dehydrogenase domain of DUOX. In the presence of calcium ions, intracellular EF-hand modules might enhance the catalytic activity of DUOX by stabilizing the dehydrogenase domain in a conformation that allows electron transfer.

Resting membrane potential determines the excitability of the cell and is essential for the cellular electrical activities. The NALCN channel mediates sodium leak currents, which positively adjust resting membrane potential towards depolarization. The NALCN channel is involved in several neurological processes and has been implicated in a spectrum of neurodevelopmental diseases. Here, we report the cryo-EM structure of rat NALCN and mouse FAM155A complex to 2.7 Å resolution. The structure reveals detailed interactions between NALCN and the extracellular cysteine-rich domain of FAM155A. We find that the non-canonical architecture of NALCN selectivity filter dictates its sodium selectivity and calcium block, and that the asymmetric arrangement of two functional voltage sensors confers the modulation by membrane potential. Moreover, mutations associated with human diseases map to the domain-domain interfaces or the pore domain of NALCN, intuitively suggesting their pathological mechanisms.

Sterol O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage. SOAT1 is a target to treat several human diseases. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 Å resolution reveals that a small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design targeting hSOAT1 and other mammalian MBOAT family members.

Sulfonylureas and glinides are commonly used oral insulin secretagogues (ISs) that act on the pancreatic ATP-sensitive potassium (K ATP ) channel to promote insulin secretion in order to lower the blood glucose level. Physiologically, K ATP channels are inhibited by intracellular ATP and activated by Mg-ADP. Therefore, they sense the cellular energy status to regulate the permeability of potassium ions across the plasma membrane. The pancreatic K ATP channel is composed of the pore-forming Kir6.2 subunits and the regulatory SUR1 subunits. Previous electrophysiological studies have established that ISs bind to the SUR1 subunit and inhibit the channel activity primarily by two mechanisms. First, ISs prevent Mg-ADP activation. Second, ISs inhibit the channel activity of Kir6.2 directly. Several cryo-EM structures of the pancreatic K ATP channel determined recently have provided remarkable structural insights into these two mechanisms.

Soluble guanylate cyclase (sGC) is the primary sensor of nitric oxide. It has a central role in nitric oxide signalling and has been implicated in many essential physiological processes and disease conditions. The binding of nitric oxide boosts the enzymatic activity of sGC. However, the mechanism by which nitric oxide activates the enzyme is unclear. Here we report the cryo-electron microscopy structures of the human sGCα1β1 heterodimer in different functional states. These structures revealed that the transducer module bridges the nitric oxide sensor module and the catalytic module. Binding of nitric oxide to the β1 haem-nitric oxide and oxygen binding (H-NOX) domain triggers the structural rearrangement of the sensor module and a conformational switch of the transducer module from bending to straightening. The resulting movement of the N termini of the catalytic domains drives structural changes within the catalytic module, which in turn boost the enzymatic activity of sGC.

Repaglinide (RPG) is a short-acting insulin secretagogue widely prescribed for the treatment of type 2 diabetes. It boosts insulin secretion by inhibiting the pancreatic ATP-sensitive potassium channel (K ATP ). However, the mechanisms by which RPG binds to the K ATP channel are poorly understood. Here, we describe two cryo-EM structures: the pancreatic K ATP channel in complex with inhibitory RPG and adenosine-5'-(γ-thio)-triphosphate (ATPγS) at 3.3 Å and a medium-resolution structure of a RPG-bound mini SUR1 protein in which the N terminus of the inward-rectifying potassium channel 6.1 (Kir6.1) is fused to the ABC transporter module of the sulfonylurea receptor 1 (SUR1). These structures reveal the binding site of RPG in the SUR1 subunit. Furthermore, the high-resolution structure reveals the complex architecture of the ATP binding site, which is formed by both Kir6.2 and SUR1 subunits, and the domain-domain interaction interfaces., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Laparoscopic surgery typically requires deep neuromuscular blockade (NMB), but whether deep or moderate NMB is superior for thoracoscopic surgery remains controversial., Methods: Patients scheduled for thoracoscopic lobectomy under intravenous anesthesia were randomly assigned to receive moderate [train of four (TOF) 1-2] or deep NMB [TOF 0, post-tetanic count (PTC) 1-5]. Depth of anesthesia was controlled at a Narcotrend rating of 30 ± 5 in both groups. The primary outcome was the need to use an additional muscle relaxant (cisatracurium) during surgery. Secondary outcomes included surgeon satisfaction, recovery time of each stage after drug withdrawal [time from withdrawal until TOF recovery to 20% (antagonists administration), 25, 75, 90, 100%], blood gas data, VAS pain grade after extubation, the time it takes for patients to begin walking after surgery, postoperative complications and hospitalization time. Results were analyzed on an intention-to-treat basis., Results: Thirty patients were enrolled per arm, and all but one patient in each arm was included in the final analysis. Among patients undergoing moderate NMB, surgeons applied additional cisatracurium in 8 patients because of body movement and 5 because of coughing (13/29, 44.8%). Additional cisatracurium was not applied to any of the patients undergoing deep NMB (p < 0.001). Surgeons reported significantly higher satisfaction for patients undergoing deep NMB (p < 0.001, Wilcoxon rank sum test). The mean difference between the two groups in the time from withdrawal until TOF recovery of 25% or 90% was 10 min (p < 0.001). The two groups were similar in other recovery data, blood gas analysis, VAS pain grade, days for beginning to walk and mean hospitalization time., Conclusions: Deep NMB can reduce the use of additional muscle relaxant and increase surgeon satisfaction during thoracoscopic lobectomy., Trial Registration: Chinese Clinical Trial Registry, ChiCTR-IOR-15007117 , 22 September 2015.

Mechanosensitive ion channels convert mechanical stimuli into a flow of ions. These channels are widely distributed from bacteria to higher plants and humans, and are involved in many crucial physiological processes. Here we show that two members of the OSCA protein family in Arabidopsis thaliana, namely AtOSCA1.1 and AtOSCA3.1, belong to a new class of mechanosensitive ion channels. We solve the structure of the AtOSCA1.1 channel at 3.5-Å resolution and AtOSCA3.1 at 4.8-Å resolution by cryo-electron microscopy. OSCA channels are symmetric dimers that are mediated by cytosolic inter-subunit interactions. Strikingly, they have structural similarity to the mammalian TMEM16 family proteins. Our structural analysis accompanied with electrophysiological studies identifies the ion permeation pathway within each subunit and suggests a conformational change model for activation.

TRPC6 and TRPC3 are receptor-activated nonselective cation channels that belong to the family of canonical transient receptor potential (TRPC) channels. They are activated by diacylglycerol, a lipid second messenger. TRPC6 and TRPC3 are involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high-affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 Å resolution. We also present the structure of human TRPC3 at 4.4 Å resolution. These structures show two-layer architectures in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domains, including the N-terminal ankyrin repeats and the C-terminal coiled-coil, contribute to the tetramer assembly. The high-affinity inhibitor BTDM wedges between the S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structures uncover the molecular architecture of TRPC channels and provide a structural basis for understanding the mechanism of these channels.

ATP-sensitive potassium channels (K ATP ) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K ATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K ATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.

ATP-sensitive potassium channels (K ATP ) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of K ATP , we elucidated the structure of a hetero-octameric pancreatic K ATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closed conformation. In another structural population, a putative co-purified phosphatidylinositol 4,5-bisphosphate (PIP 2 ) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for K ATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP 2 lipid., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The SAD (synapses of amphids defective) kinases, including SAD-A and SAD-B, play important roles in the regulation of neuronal development, cell cycle, and energy metabolism. Our recent study of mouse SAD-A identified a unique autoinhibitory sequence (AIS), which binds at the junction of the kinase domain (KD) and the ubiquitin-associated (UBA) domain and exerts autoregulation in cooperation with UBA. Here, we report the crystal structure of the mouse SAD-B C-terminal fragment including the AIS and the kinase-associated domain 1 (KA1) at 2.8 Å resolution. The KA1 domain is structurally conserved, while the isolated AIS sequence is highly flexible and solvent-accessible. Our biochemical studies indicated that the SAD-B AIS exerts the same autoinhibitory role as that in SAD-A. We believe that the flexible isolated AIS sequence is readily available for interaction with KD-UBA and thus inhibits SAD-B activity.

Background: Lysophosphatidic acid receptor 1 and Rho/ROCK signaling is implicated in bone cancer pain development. However, it remains unknown whether the two signaling pathways function together in P2X3 receptor-mediated bone cancer pain., Results: In this study, using a rat model of bone cancer, we examined the expression of P2X3 and lysophosphatidic acid receptor 1 in rat dorsal root ganglion neurons and further dissected whether lysophosphatidic acid receptor 1 and Rho/ROCK-mediated pathways interacted in modulating rat pain behavior. Bone cancer was established by inoculating Walker 256 cells into the left tibia of female Wistar rats. We observed a gradual and yet significant decline in mean paw withdrawal threshold in rats with bone cancer, but not in control rats. Our immunohistochemical staining revealed that the number of P2X3- and lysophosphatidic acid receptor 1-positive dorsal root ganglion neurons was significantly greater in rats with bone cancer than control rats. Lysophosphatidic acid receptor 1 blockade with VPC32183 significantly attenuated decline in mean paw withdrawal threshold. Flinching behavior test further showed that lysophosphatidic acid receptor 1 inhibition with VPC32183 transiently but significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Rho inhibition by intrathecal BoTXC3 caused a rapid reversal in decline in mean paw withdrawal threshold of rats with bone cancer. Flinching behavior test showed that BoTXC3 transiently and significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Similar findings were observed with ROCK inhibition by intrathecal Y27632. Furthermore, VPC32183 and BoTXC3 effectively aborted the appearance of lysophosphatidic acid-induced calcium influx peak., Conclusions: Lysophosphatidic acid and its receptor LPAR1, acting through the Rho-ROCK pathway, regulate P2X3 receptor in the development of both mechanical and spontaneous pain in bone cancer., (© The Author(s) 2016.)

The SAD/BRSK kinases participate in various important life processes, including neural development, cell cycle and energy metabolism. Like other members of the AMPK family, SAD contains an N-terminal kinase domain followed by the characteristic UBA and KA1 domains. Here we identify a unique autoinhibitory sequence (AIS) in SAD kinases, which exerts autoregulation in cooperation with UBA. Structural studies of mouse SAD-A revealed that UBA binds to the kinase domain in a distinct mode and, more importantly, AIS nestles specifically into the KD-UBA junction. The cooperative action of AIS and UBA results in an 'αC-out' inactive kinase, which is conserved across species and essential for presynaptic vesicle clustering in C. elegans. In addition, the AIS, along with the KA1 domain, is indispensable for phospholipid binding. Taken together, these data suggest a model for synergistic autoinhibition and membrane activation of SAD kinases.

Autophagy is a highly conserved pathway that permits recycling of nutrients within the cell and is rapidly upregulated during starvation or cell stress. Autophagy has been implicated in the pathophysiological process of warm ischemia‑reperfusion injury in the rat lung. Cold ischemia (CI) preservation for lung transplantation also exhibits cell stress and nutrient deprivation, however, little is known with regard to the involvement of autophagy in this process. In the present study, CI preservation‑induced autophagy and apoptosis was investigated in the lungs of Sprague Dawley rats. Sprague Dawley rat lungs were flushed and preserved at 4˚C (i.e. CI) for various durations (0, 3, 6, 12 and 24 h). The levels of autophagy, autophagic cell death and apoptosis were measured at each time point following CI. The results revealed that autophagy was induced by CI preservation, which was initiated at 3 h, peaked at 6 h after CI and declined thereafter. Additionally, a coexistence of autophagic cell death and apoptosis was observed in rat lung tissues following prolonged CI. These findings demonstrate that autophagy is involved in the pathophysiological process of lung CI. Furthermore, autophagic cell death in addition to necrosis and apoptosis occurs following CI in the lung. CI preservation may therefore be a potential mechanism of lung injury during organ preservation prior to lung transplantation.

Background: Ischemia-reperfusion injury (IRI) after lung transplantation remains a significant cause of morbidity and mortality. Lung IRI induces nitric oxide synthesis (iNOS) and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that ischemia-induced iNOS intensifies with reperfusion and contributes to IRI-induced pulmonary arterial regulatory dysfunction, which may lead to early graft failure and cause pulmonary edema. The aim of this study was to determine whether ischemia-reperfusion alters inducible and endothelial nitric oxide synthase expression, potentially affecting pulmonary perfusion. We further evaluated the role of iNOS in post-transplantation pulmonary arterial disorder., Methods: We randomized 32 Sprague-Dawley rats into two groups. The control group was given a sham operation whilst the experimental group received orthotropic lung transplants with a modified three-cuff technique. Changes in lung iNOS, and endothelial nitric oxide synthase expression were measured after lung transplantation by enzyme-linked immunosorbent assay (ELISA). Vasoconstriction in response to exogenous phenylephrine and vasodilation in response to exogenous acetylcholine of pulmonary arterial rings were measured in vitro as a measure of vascular dysfunction. To elucidate the roles of iNOS in regulating vascular function, an iNOS activity inhibitor (N6-(1-iminoethyl)-L-lysine, L-NIL) was used to treat isolated arterial rings. In order to test whether iNOS inhibition has a therapeutic effect, we further used L-NIL to pre-treat transplanted lungs and then measured post-transplantation arterial responses., Results: Lung transplantation caused upregulation of iNOS expression. This was also accompanied by suppression of both vasoconstriction and vasodilation of arterial rings from transplanted lungs. Removal of endothelium did not interfere with the contraction of pulmonary arterial rings from transplanted lungs. In contrast, iNOS inhibition rescued the vasoconstriction response to exogenous phenylephrine of pulmonary arterial rings from transplanted lungs. In addition, lung transplantation led to suppression of PaO2/FiO2 ratio, increased intrapulmonary shunt (Q s/Q t), and increase of lung wet to dry ratio (W/D), malondialdehyde and myeloperoxidase levels, all of which were reversed upon iNOS inhibition. Furthermore, inhibition of iNOS significantly rescued vascular function and alleviated edema and inflammatory cell infiltration in the transplanted lung., Conclusions: Our data suggest that lung transplantation causes upregulation of iNOS expression, and pulmonary vascular dysfunction. iNOS inhibition reverses the post-transplantational pulmonary vascular dysfunction.

Background: Rapid and effective treatment of cancer-induced bone pain remains a clinical challenge and patients with bone metastasis are more likely to experience severe pain. The voltage-gated sodium channel Nav1.8 plays a critical role in many aspects of nociceptor function. Therefore, we characterized a rat model of cancer pain and investigated the potential role of Nav1.8., Methods: Adult female Wistar rats were used for the study. Cancer pain was induced by inoculation of Walker 256 breast carcinosarcoma cells into the tibia. After surgery, mechanical and thermal hyperalgesia and ambulation scores were evaluated to identify pain-related behavior. We used real-time RT-PCR to determine Nav1.8 mRNA expression in bilateral L4/L5 dorsal root ganglia (DRG) at 16-19 days after surgery. Western blotting and immunofluorescence were used to compare the expression and distribution of Nav1.8 in L4/L5 DRG between tumor-bearing and sham rats. Antisense oligodeoxynucleotides (ODNs) against Nav1.8 were administered intrathecally at 14-16 days after surgery to knock down Nav1.8 protein expression and changes in pain-related behavior were observed., Results: Tumor-bearing rats exhibited mechanical hyperalgesia and ambulatory-evoked pain from day 7 after inoculation of Walker 256 cells. In the advanced stage of cancer pain (days 16-19 after surgery), normalized Nav1.8 mRNA levels assessed by real-time RT-PCR were significantly lower in ipsilateral L4/L5 DRG of tumor-bearing rats compared with the sham group. Western-blot showed that the total expression of Nav1.8 protein significantly decreased bilaterally in DRG of tumor-bearing rats. Furthermore, as revealed by immunofluorescence, only the expression of Nav1.8 protein in small neurons down regulated significantly in bilateral DRG of cancer pain rats. After administration of antisense ODNs against Nav1.8, Nav1.8 protein expression decreased significantly and tumor-bearing rats showed alleviated mechanical hyperalgesia and ambulatory-evoked pain., Conclusions: These findings suggest that Nav1.8 plays a role in the development and maintenance of bone cancer pain.

It has been reported that proteinase-activated receptor 2 (PAR2) receptor activation enhances the animal's pain response and PAR2 coexpresses with P2X3 in dorsal root ganglion neurons. However, whether PAR2 activation has a direct impact on P2X3 currents is still not clear. In this study, we performed the patch-clamp experiments in cultured dorsal root ganglion neurons and found that when incubated with trypsin or the PAR2 agonist SL-NH2 for a short time (3 min), instead of increasing, P2X3 currents amplitude decreased significantly. Meanwhile, the opening of P2X3 ion channel accelerated. Protein kinase A inhibitor H89 could not reverse above phenomenon, but played a synergistic effect on the contrary. These results suggest that the enhanced pain response caused by PAR2 activation is not through direct increase of the P2X3 current amplitude, and the acceleration of P2X3 opening may participate in the enhanced pain response in a long-time view. Moreover, protein kinase A does not participate in the inhibition of P2X3 currents caused by PAR2 activation.

Aim: To determine the effects of hypertonic solution on myocardial ischemia and reperfusion injury in normal and stroke-prone hypertensive rat hearts in vitro., Methods: Hearts were perfused in an isolated-perfused Langendorff apparatus and perfused with normal or hypertonic solution (360 mOsm/L, by addition of NaCl to the normal perfusate of 300 mOsm/L) before subjected to 30 min ischemia followed by 40 min isotonic reperfusion. Heart function, myocardial creatine kinase leakage, norepinephrine release, and ventricular calcium content were determined., Results: Normal rat hearts with hypertonic perfusion showed higher recovery rate of spontaneous beating than control hearts after ischemia. Hypertensive rat hearts perfused with hypertonic solution also had better recovery in diastolic function and less creatine kinase leakage than hypertensive controls. Concomitantly, myocardial release of norepinephrine was also reduced from hypertensive hearts perfused with hypertonic solution. There was no significant difference in myocardial calcium content between normal and hypertonic perfused hypertensive hearts., Conclusion: Hypertonic perfusion may precondition the hearts and protect them from ischemia and reperfusion injury in both normal and hypertensive rats. The modulation of hypertonic perfusion on myocardial norepinephrine release and its role in cardioprotection needs further investigation.