Systematic Comparison of Molecular Conformations of H+,K+-ATPase Reveals an Important Contribution of the A-M2 Linker for the Luminal Gating

Gastric H⁺,K⁺-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K⁺-competitive antagonist {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080 at its luminal cavity, based on observations of the structure in the {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H⁺,K⁺-ATPase is consistent with that observed in sarcoplasmic reticulum Ca²⁺-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.     

E2P State Stabilization by the N-terminal Tail of the H,K-ATPase ��-Subunit Is Critical for Efficient Proton Pumping under in Vivo Conditions

The catalytic α-subunits of Na,K- and H,K-ATPase require an accessory β-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the β-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated β-variants were expressed in Xenopus oocytes, together with the S806C α-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E₁P/E₂P states. Whereas the E₁P/E₂P conformational equilibrium was unaffected for the shorter N-terminal deletions βΔ4 and βΔ8, we observed significant shifts toward E₁P for the two larger deletions βΔ13 and βΔ29. Moreover, the reduced ΔF/F ratios of βΔ13 and βΔ29 indicated an increased reverse reaction via E₂P → E₁P + ADP → E₁ + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E₂P-specific inhibitor {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080, which becomes especially apparent at high concentrations (100 μm). Despite unaltered apparent Rb⁺ affinities, the maximal Rb⁺ uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the β-N terminus and α-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase β-subunit may stabilize the pump in the E₂P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the β-N terminus of the closely related Na,K-ATPase does not affect the E₁P/E₂P distribution or pump activity, indicating that the E₂P-stabilizing effect by the β-N terminus is apparently a unique property of the H,K-ATPase.The gastric H,K-ATPase fulfills the remarkable task of pumping protons against a more than 10⁶-fold concentration gradient. H⁺ extrusion is coupled to countertransport of an equal number of K⁺ ions for each ATP molecule hydrolyzed, resulting in an electroneutral overall process (1). Characteristic for all P-type ATPases, the enzyme cycles between the two principal conformational states (E₁ and E₂) and the corresponding phosphointermediates (E₁P and E₂P), which are formed by reversible phosphorylation of an aspartate residue in the highly conserved DKTGTLT motif. According to a Post-Albers-like reaction scheme (see Fig. 1A), the conformational E₁P → E₂P transition converts the high H⁺/low K⁺ affinity of the cation binding pocket into a low H⁺/high K⁺ affinity binding site, hence enabling proton release into the stomach lumen and subsequent binding of extracellular K⁺. Because the pump faces a lumenal proton concentration of ∼150 mm (2), proton release is probably the energetically most demanding step in the reaction cycle. Thus, during the conformational E₁P → E₂P transition, enormous pK_a changes of the H⁺-coordinating residues have to occur that most likely involve the rearrangement of a positively charged lysine side chain (Lys-791 in rat H,K-ATPase) (3).Open in a separate windowFIGURE 1.Post-Albers scheme (A) and cryo-EM structural representation of pig gastric H,K-ATPase in the fluoroaluminate-bound pseudo-E₂P state (B). A, Post-Albers scheme of the proposed reaction cycle of the gastric H,K-ATPase. E₁P/E₂P conformational states giving rise to voltage jump-induced fluorescence changes of TMRM-labeled H,K-ATPase molecules are highlighted (gray box). B, structural representation based on the cryo-EM structure of the pig gastric H,K-ATPase (surface or mesh, contoured at 1 σ; EM Data Bank code 5104) and the corresponding homology model (schematic; Protein Data Bank code 3IXZ). Inset, a close-up view (from the right side of the molecule) showing the putative interaction sites of the β-subunit N terminus with the P-domain (red arrow) and αTM3 (black arrow), respectively. Color coding is indicated in the figure.All P₂-type ATPases share a common catalytic α-subunit, composed of 10 transmembrane domains harboring the ion-binding sites and a large cytoplasmic loop with the nucleotide-binding domain, the phosphorylation domain (P-domain),² and the actuator domain (A-domain) (4). However, a unique feature of K⁺-transporting Na,K- and H,K-ATPase enzymes is the requirement for an accessory β-subunit, which is indispensable for proper folding, maturation, and plasma membrane delivery (5, 6). Despite only 20–30% overall sequence identity between the H,K-ATPase β-subunit and the Na,K β-isoforms, the topogenic structure is similar: a short N-terminal cytoplasmic tail, followed by a single transmembrane segment and a large extracellular C-terminal domain with glycosylation sites and disulfide-bridging cysteines. Numerous studies have demonstrated that the β-subunit of the Na,K-ATPase is more than just a chaperone for the α-subunit, being also required for proper ion transport activity of the holoenzyme. In fact, it has been discovered that different cell- and tissue-specific β-isoforms have distinct effects on the cation affinities (7–9). Furthermore, it was shown that mutational changes in all three topogenic domains of the Na,K-ATPase β-subunit (10–19) as well as chemical interference with disulfide-forming cysteines in the Na,K-ATPase β-subunit ectodomain (20–22) affect the cation transport properties of the sodium pump. Finally, conformational changes in the β-subunit during the Na,K-ATPase reaction cycle were demonstrated by proteolytic digestion studies (23) and voltage clamp fluorometry (24).Far less is known about the functional significance of the single H,K-ATPase β-isoform, especially about its potential impact on cation transport (reviewed in Refs. 25 and 26). We have proven recently that E₂P state-specific transmembrane interactions between residues in αTM7 and two highly conserved tyrosines in the βTM of both Na,K- and H,K-ATPase significantly stabilize the E₂P conformation (19). Mutational disruptions of this interaction resulted in substantial shifts toward E₁P and severely affected H⁺ secretion, which highlighted the physiological relevance of this E₂P state stabilization. Notably, according to the recently published cryo-EM structure of pig gastric H,K-ATPase in the pseudo-E₂P state (27), the N-terminal tail of the β-subunit makes direct contact with the phosphorylation domain of the α-subunit (see Fig. 1B), thus indicating an additional E₂P state stabilization mediated by the β-N terminus. Although this idea was further supported by biochemical studies on N-terminally truncated mutants, direct evidence for this putative E₂P-stabilizing interaction and its potential significance for ion transport in intact cells is still lacking.Here, we demonstrate for the first time the functional importance of the gastric H,K-ATPase β-subunit N terminus in living cells under in vivo conditions: voltage clamp fluorometry, Rb⁺ flux, and {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080 sensitivity measurements revealed E₁P-shifted, ion transport-impaired phenotypes for two N-terminally truncated H,K β-variants, thus substantiating the E₂P-stabilizing effect of the β-N terminus suggested by the recent cryo-EM structure.     

Lack or Inhibition of Dopaminergic Stimulation Induces a Development Increase of Striatal Tyrosine Hydroxylase-Positive Interneurons

We examined the role of endogenous dopamine (DA) in regulating the number of intrinsic tyrosine hydroxylase-positive (TH⁺) striatal neurons using mice at postnatal day (PND) 4 to 8, a period that corresponds to the developmental peak in the number of these neurons. We adopted the strategy of depleting endogenous DA by a 2-day treatment with α-methyl-p-tyrosine (αMpT, 150 mg/kg, i.p.). This treatment markedly increased the number of striatal TH⁺ neurons, assessed by stereological counting, and the increase was highly correlated to the extent of DA loss. Interestingly, TH⁺ neurons were found closer to the clusters of DA fibers after DA depletion, indicating that the concentration gradient of extracellular DA critically regulates the distribution of striatal TH⁺ neurons. A single i.p. injection of the D1 receptor antagonist, {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (0.1 mg/kg), the D2/D3 receptor antagonist, raclopride (0.1 mg/kg), or the D4 receptor antagonist, L-745,870 (5 mg/kg) in mice at PND4 also increased the number of TH⁺ neurons after 4 days. Treatment with the D1-like receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 (10 mg/kg) or with the D2-like receptor agonist, quinpirole (1 mg/kg) did not change the number of TH⁺ neurons. At least the effects of {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 were prevented by a combined treatment with {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393. Immunohistochemical analysis indicated that striatal TH⁺ neurons expressed D2 and D4 receptors, but not D1 receptors. Moreover, treatment with the α4β2 receptor antagonist dihydro-β-erythroidine (DHβE) (3.2 mg/kg) also increased the number of TH⁺ neurons. The evidence that DHβE mimicked the action of {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 in increasing the number of TH⁺ neurons supports the hypothesis that activation of D1 receptors controls the number of striatal TH⁺ neurons by enhancing the release of acetylcholine. These data demonstrate for the first time that endogenous DA negatively regulates the number of striatal TH⁺ neurons by direct and indirect mechanisms mediated by multiple DA receptor subtypes.     

Allosteric Modulation of a Cannabinoid G Protein-coupled Receptor: BINDING SITE ELUCIDATION AND RELATIONSHIP TO G PROTEIN SIGNALING*

The cannabinoid 1 (CB₁) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) ({"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569), has the paradoxical effect of increasing the equilibrium binding of [³H](−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 can displace the CB₁ antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 at CB₁. To this end, we used computation, synthesis, mutation, and functional studies to identify the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-binding site in the CB₁ TMH3-6-7 region. This site is consistent with the results of K3.28¹⁹²A, F3.36²⁰⁰A, W5.43²⁷⁹A, W6.48³⁵⁶A, and F3.25¹⁸⁹A mutation studies, which revealed the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 piperidine ring nitrogen and K3.28¹⁹² that is important for {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 to act as an inverse agonist. At this allosteric site, {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 promotes an intermediate conformation of the CB₁ receptor, explaining {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569''s ability to increase equilibrium binding of CP55,940. This site also explains {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569''s ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63¹⁷⁶. 3) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.     

Arylbenzazepines Are Potent Modulators for the Delayed Rectifier K+ Channel: A Potential Mechanism for Their Neuroprotective Effects

(±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D₁-like dopamine receptor agonistic activity. The precise mechanism for the (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-mediated neuroprotection remains elusive. We report here that (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is a potent blocker for delayed rectifier K⁺ channel. (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 inhibited the delayed rectifier K⁺ current (I _K) dose-dependently in rat hippocampal neurons. The IC ₅₀ value for inhibition of I _K was 41.9±2.3 µM (Hill coefficient = 1.81±0.13, n = 6), whereas that for inhibition of I _A was 307.9±38.5 µM (Hill coefficient = 1.37±0.08, n = 6). Thus, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is 7.3-fold more potent in suppressing I _K than I _A. Moreover, the inhibition of I _K by (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 with the K⁺ channel. The intracellular application of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 had no effects of on I _K, indicating that the compound most likely acts at the outer mouth of the pore of K⁺ channel. We also tested the enantiomers of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, R-(+) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-201), and S-(−) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-202), as well as {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393; all these compounds inhibited I _K. However, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of I _K , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959.     

KLK8 promotes the proliferation and metastasis of colorectal cancer via the activation of EMT associated with PAR1

Kallikrein-related peptidase 8 (KLK8) acts as an oncogene or anti-oncogene in various tumours, and the abnormal expression of KLK8 is involved in the carcinogenesis of several tumours. However, the role of KLK8 in colorectal cancer (CRC) and the underlying mechanism remain largely unclear. In this study, the carcinogenic effect of KLK8 was determined via CCK-8 and colony formation assays in vitro and a xenograft model in nude mice in vivo. The metastasis-promoting effect of KLK8 was investigated with transwell migration and invasion assays and wound-healing assay in vitro and a metastasis model in nude mice in vivo. Bioinformatics analyses and mechanistic experiments were conducted to elucidate the molecular mechanism. Herein, we reported that KLK8 had a promotive effect on the proliferation, migration and invasion of RKO and SW480 cells. Epithelial−mesenchymal transition (EMT) played an important role in the promotive effects of KLK8 on CRC. In addition, protease-activated receptor-1 (PAR-1) antagonist {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797 but not protease-activated receptor-2 (PAR-2) antagonist FSLLRY-NH2 attenuated the proliferation, migration and invasion of KLK8-upregulated RKO and SW480 cells. PAR-1 antagonist {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797 reduced the tumour volume of xenograft model and decreased the metastatic nodules in the livers of metastasis model. Furthermore, {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797 could reverse the positive impact of KLK8 on the EMT process in CRC both in vitro and in vivo. Taken together, these findings demonstrated for the first time that KLK8 promoted EMT and CRC progression, and this effect might be, at least partly mediated by PAR1-dependent pathway.Subject terms: Colorectal cancer, Oncogenes, Tumour biomarkers     

Pharmacological profiles of the murine gastric and colonic H,K-ATPases

Background

The H,K-ATPase, consisting of α and β subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα₁ and HKα₂ encoded by different genes. The ouabain-resistant gastric HKα₁-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα₂-H,K-ATPase from different species shows poor primary structure conservation of the HKα₂ subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα₁, HKα₂, or HKβ genes.

Methods

Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα₁, HKα₂, or HKβ knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined.

Results

The colonic H,K-ATPase demonstrated slightly greater affinity for K⁺ than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα₂ KO but was observed in HKβ KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα₁-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα₂-H,K-ATPase was not inhibited by these compounds.

Conclusions

These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride.

General Significance

Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.     

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

Background

Dopamine signaling is mediated by G_s protein-coupled “D₁-like” receptors (D₁ and D₅) and G_i-coupled “D₂-like” receptors (D_2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the G_i-coupled dopamine D₂ receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the G_s-coupled dopamine D₁-like receptor subtypes have never been identified on these cells. Activation of G_s-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D₁-like receptor is expressed on ASM, and modulates its function through G_s-coupling.

Methods

The mRNA and protein expression of dopamine D₁-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D₁ receptor, HASM cells were treated with dopamine or the dopamine D₁-like receptor agonists ({"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 or {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D₁ receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BK_Ca) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.

Results

Messenger RNA encoding the dopamine D₁ and D₅ receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D₁ receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D₁ receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D₁-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D₁-like receptor antagonists {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 or {"type":"entrez-protein","attrs":{"text":"SCH39166","term_id":"1052842517","term_text":"SCH39166"}}SCH39166. {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.

Conclusions

These results demonstrate that the dopamine D₁ receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.     

Adenosine A2A Receptor Deficiency Up-Regulates Cystatin F Expression in White Matter Lesions Induced by Chronic Cerebral Hypoperfusion

In previous studies, we have shown that the inactivation of the adenosine A2A receptor exacerbates chronic cerebral hypoperfusion-induced white matter lesions (WMLs) by enhancing neuroinflammatory responses. However, the molecular mechanism underlying the effect of the adenosine A2A receptor remains unknown. Recent studies have demonstrated that cystatin F, a potent endogenous cysteine protease inhibitor, is selectively expressed in immune cells in association with inflammatory demyelination in central nervous system diseases. To understand the expression of cystatin F and its potential role in the effect of A2A receptor on WMLs induced through chronic cerebral hypoperfusion, we investigated cystatin F expression in the WMLs of A2A receptor gene knockout mice, the littermate wild-type mice and wild-type mice treated daily with the A2A receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 or both {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and A2A receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 after chronic cerebral hypoperfusion. The results of quantitative-PCR and western blot analysis revealed that cystatin F mRNA and protein expression were significantly up-regulated in the WMLs after chronic cerebral hypoperfusion. In addition, cystatin F expression in the corpus callosum was significantly increased in A2A receptor gene knockout mice and markedly decreased in mice treated with {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 on both the mRNA and protein levels. Additionally, {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 counteracted the attenuation of cystatin F expression produced by {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 after chronic cerebral hypoperfusion. Moreover, double immunofluorescence staining revealed that cystatin F was co-localized with the activated microglia marker CD11b. In conclusion, the cystatin F expression in the activated microglia is closely associated with the effect of the A2A receptors, which may be related to the neuroinflammatory responses occurring during the pathological process.     

Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase

Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca²⁺ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Moreover, Jujuboside B increased intracellular Ca²⁺ concentration dose-dependently, which was inhibited by EGTA and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Besides, Jujuboside B induced a rapid Ca²⁺ influx instantaneously after depleting intracellular Ca²⁺ store, which was significantly inhibited by {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca²⁺ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved endothelium-dependent hyperpolarizaiton through endothelial potassium channels. Jujuboside B is a natural compound with new pharmacological effects on improving endothelial dysfunction and treating vascular diseases.     

CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger,protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels

Inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca²⁺ homeostasis. However, the Ca²⁺ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca²⁺ levels are modulated by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 on neuronal Ca²⁺ homeostasis using cameleon-based mitochondrial Ca²⁺ and cytosolic Ca²⁺ ([Ca²⁺]_i) live imaging. We observed that NCLX-driven mitochondrial Ca²⁺ exchange occurs in cortical neurons under basal conditions as {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 induced a decrease in [Ca²]_i concomitant with a Ca²⁺ accumulation inside the mitochondria. In turn, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 also inhibited mitochondrial Ca²⁺ efflux after the stimulation of acetylcholine receptors. In contrast, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 strongly prevented depolarization-induced [Ca²⁺]_i increase by blocking voltage-gated Ca²⁺ channels (VGCCs), whereas it did not induce depletion of ER Ca²⁺ stores. Moreover, mitochondrial Ca²⁺ overload was reduced as a consequence of diminished Ca²⁺ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca²⁺ overload by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 modulates cytosolic and mitochondrial Ca²⁺ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.     

Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 were blocked by {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.     

Electrophysiological Effects of SKF83959 on Hippocampal CA1 Pyramidal Neurons: Potential Mechanisms for the Drug's Neuroprotective Effects

Although the potent anti-parkinsonian action of the atypical D₁-like receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D₁ receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D₁, D₂, 5-HT_2A/2C receptors and α₁-adrenoceptor, nor by intracellular dialysis of GDP-β-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959. In voltage-clamp experiments, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D₁ receptor activation. Moreover, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 increased Ih current through a D₁ receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug''s neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson''s disease.     

Methamphetamine and Dopamine Receptor D1 Regulate Entrainment of Murine Circadian Oscillators

We investigated the effect of methamphetamine (MA) injections on the circadian organization of behavior and individual tissues in the mouse. Scheduled, daily injections of MA resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. Daily MA also shifted the peak time of PER2 expression in the liver, pituitary, and salivary glands. It has been suggested that reward pathways, and dopamine signaling in particular, may underlie the effects of MA on the circadian system. To test this hypothesis, we examined the effect of the D1 receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (SCH) on circadian rhythms. The MA-induced shift in the phase of pituitary and salivary glands was attenuated by pretreatment with the D1 antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (SCH). Interestingly, daily SCH, administered alone, also affected some circadian oscillators. The livers and lungs (but not pituitaries or salivary glands) of mice treated with daily injections of SCH displayed disrupted rhythms of PER2 expression, suggesting that D1 receptor signaling is important for entrainment of these organs. From these results, we conclude that MA has widespread effects within the circadian system, and that these effects are mediated, at least in part, by the dopaminergic system. This study also identifies a role for dopamine signaling in normal entrainment of circadian oscillators.     

Requirements for membrane attack complex formation and anaphylatoxins binding to collagen-activated platelets

Background

The activation of complement during platelet activation is incompletely understood. Objectives: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets.

Methods

C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation.

Results

C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg²⁺-EGTA and significantly reduced by the thrombin inhibitor hirudin (−37%, p<0.05), but was unaffected by chondroitinase, compstatin, {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg²⁺-EGTA, but not hirudin, {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (−71% and −44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface.

Conclusions

C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the β₁ subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH₄⁺ as activating cation. The enzyme was also stimulated by Na⁺, but in contrast to the rat enzyme, hardly by K⁺. SCH 28080 inhibited the NH₄⁺-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K⁺], and [Na⁺] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E₁P to E₂P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H⁺/Na⁺ and/or H⁺/NH₄⁺ transport but not in Na⁺/K⁺ transport.     

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb+

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K₂)Mg–MgF₄, an E2∙Pi-like state capable to occlude K⁺ (or Rb⁺). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb⁺ with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1–E2 conformational transition and the kinetics of Rb⁺ exchange between the medium and the E2(Rb₂)Mg–MgF₄ state. Our results show that both in the absence and in the presence of Rb⁺, simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg–MgF₄ complex, that is unable to shift to E1 upon addition of Na⁺. The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg–MgF₄ formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb⁺ caused by Mg^2 +. Na⁺-induced release of Rb⁺ from E2(Rb₂)Mg–MgF₄ occurs at the same rate as from E2(Rb₂) but is insensitive to ADP. The rate of Rb⁺ occlusion into the E2Mg–MgF₄ state is 5–8 times lower than that described for the E2Mg–vanadate complex. Since the E2Mg–MgF₄ and E2Mg–vanadate complexes represent different intermediates in the E2-P → E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.