Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

Background

Agonistic autoantibodies directed at the α₁-adrenergic receptor (α₁-AAB) have been described in patients with hypertension. We implied earlier that α₁-AAB might have a mechanistic role and could represent a therapeutic target.

Methodology/Principal Findings

To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had α₁-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α₁-adrenergic receptor antibodies (α₁-AB). Patient α₁-AAB and rabbit α₁-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human α_1A-adrenergic receptor were incubated with patient α₁-AAB and rabbit α₁-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient α₁-AAB and rabbit α₁-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca²⁺ in cardiomyocytes and induce mesentery artery segment contraction.

Conclusions/Significance

Patient α₁-AAB and rabbit α₁-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α₁-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.     

Activation of Mu Opioid Receptors Sensitizes Transient Receptor Potential Vanilloid Type 1 (TRPV1) via β-Arrestin-2-Mediated Cross-Talk

The transient receptor potential family V1 channel (TRPV1) is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C). Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs) on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.     

β1-Adrenergic Receptor Signaling Activates the Epithelial Calcium Channel,Transient Receptor Potential Vanilloid Type 5 (TRPV5), via the Protein Kinase A Pathway

Epinephrine and norepinephrine are present in the pro-urine. β-Adrenergic receptor (β-AR) blockers administered to counteract sympathetic overstimulation in patients with congestive heart failure have a negative inotropic effect, resulting in reduced cardiac contractility. Positive inotropes, β1-AR agonists, are used to improve cardiac functions. Active Ca²⁺ reabsorption in the late distal convoluted and connecting tubules (DCT2/CNT) is initiated by Ca²⁺ influx through the transient receptor potential vanilloid type 5 (TRPV5) Ca²⁺ channel. Although it was reported that β-ARs are present in the DCT2/CNT region, their role in active Ca²⁺ reabsorption remains elusive. Here we revealed that β1-AR, but not β2-AR, is localized with TRPV5 in DCT2/CNT. Subsequently, treatment of TRPV5-expressing mouse DCT2/CNT primary cell cultures with the β1-AR agonist dobutamine showed enhanced apical-to-basolateral transepithelial Ca²⁺ transport. In human embryonic kidney (HEK293) cells, dobutamine was shown to stimulate cAMP production, signifying functional β1-AR expression. Fura-2 experiments demonstrated increased activity of TRPV5 in response to dobutamine, which could be prevented by the PKA inhibitor H89. Moreover, nonphosphorylable T709A-TRPV5 and phosphorylation-mimicking T709D-TRPV5 mutants were unresponsive to dobutamine. Surface biotinylation showed that dobutamine did not affect plasma membrane abundance of TRPV5. In conclusion, activation of β1-AR stimulates active Ca²⁺ reabsorption in DCT2/CNT; an increase in TRPV5 activity via PKA phosphorylation of residue Thr-709 possibly plays an important role. These data explicate a calciotropic role in addition to the inotropic property of β1-AR.     

Arresting a Transient Receptor Potential (TRP) Channel: β-ARRESTIN 1 MEDIATES UBIQUITINATION AND FUNCTIONAL DOWN-REGULATION OF TRPV4*?

β-Arrestins, originally discovered to desensitize activated G protein-coupled receptors, (aka seven-transmembrane receptors, 7TMRs) also mediate 7TMR internalization and G protein-independent signaling via these receptors. More recently, several regulatory roles of β-arrestins for atypical 7TMRs and non-7TM receptors have emerged. Here, we uncover an entirely novel regulatory role of β-arrestins in cross-talk between the angiotensin receptor (AT1aR) and a member of the transient receptor potential (TRP) ion channel family, TRPV4. AT1aR and TRPV4 form a constitutive complex in the plasma membrane, and angiotensin stimulation leads to recruitment of β-arrestin 1 to this complex. Surprisingly, angiotensin stimulation results in ubiquitination of TRPV4, a process that requires β-arrestin 1, and subsequently to internalization and functional down-regulation of TRPV4. β-Arrestin 1 interacts with, and acts as an adaptor for AIP4, an E3 ubiquitin ligase responsible for TRPV4 ubiquitination. Thus, our data provide the first evidence of a functional link between β-arrestins and TRPV4 and uncovers an entirely novel mechanism to maintain appropriate intracellular Ca²⁺ concentration to avoid excessive Ca²⁺ signaling.     

Transient Receptor Potential Channel 1 Deficiency Impairs Host Defense and Proinflammatory Responses to Bacterial Infection by Regulating Protein Kinase Cα Signaling

Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca²⁺ homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1^−/− mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca²⁺ entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca²⁺ entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca²⁺ entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit.     

A Potential Link between the C5a Receptor 1 and the β1-Adrenoreceptor in the Mouse Heart

Purpose

Inflammation may contribute to the pathogenesis of specific cardiovascular diseases, but it is uncertain if mediators released during the inflammatory process will affect the continued efficacy of drugs used to treat clinical signs of the cardiac disease. We investigated the role of the complement 5a receptor 1 (C5aR1/CD88) in the cardiac response to inflammation or atenolol, and the effect of C5aR1 deletion in control of baseline heart rate in an anesthetized mouse model.

Methods

An initial study showed that PMX53, an antagonist of C5aR1 in normal C57BL6/J (wild type, WT) mice reduced heart rate (HR) and appeared to have a protective effect on the heart following induced sepsis. C5aR1 knockout (CD88-/-) mice had a lower HR than wild type mice, even during sham surgery. A model to assess heart rate variability (HRV) in anesthetized mice was developed to assess the effects of inhibiting the β₁-adrenoreceptor (β₁-AR) in a randomized crossover study design.

Results

HR and LF Norm were constitutively lower and SDNN and HF Norm constitutively higher in the CD88-/- compared with WT mice (P< 0.001 for all outcomes). Administration of atenolol (2.5 mg/kg) reduced the HR and increased HRV (P< 0.05, respectively) in the wild type but not in the CD88-/- mice. There was no shift of the sympathovagal balance post-atenolol in either strains of mice (P> 0.05), except for the reduced LF/HF (Lower frequency/High frequency) ratio (P< 0.05) at 60 min post-atenolol, suggesting increased parasympathetic tone of the heart due to the effect of atenolol administration. The HR of the WT mice were lower post atenolol compared to the CD88-/- mice (P = 0.001) but the HRV of CD88-/- mice were significantly increased (P< 0.05), compared with WT mice.

Conclusion

Knockout of the C5aR1 attenuated the effect of β₁-AR in the heart, suggesting an association between the β₁-AR and C5aR1, although further investigation is required to determine if this is a direct or causal association.     

Decreased Autophagy in Rat Heart Induced by Anti-β1-Adrenergic Receptor Autoantibodies Contributes to the Decline in Mitochondrial Membrane Potential

It has been recognized that changes in mitochondrial structure plays a key role in development of cardiac dysfunction, and autophagy has been shown to exert maintenance of mitochondrial homeostasis effects. Our previous study found that anti-β₁-adrenergic receptor autoantibodies (β₁-AABs) could lead to cardiac dysfunction along with abnormalities in mitochondrial structure. The present study tested the hypothesis that β₁-AABs may induce the decline in mitochondrial membrane potential (ΔΨm) by suppression of cardiac autophagy, which contributed to cardiac dysfunction. Male adult rats were randomized to receive a vehicle or peptide corresponding to the second extracellular loop of the β₁ adrenergic receptor (β₁-AAB group, 0.4 μg/g every two weeks for 12 weeks) and treated with rapamycin (RAPA, an autophagy agonist) at 5 mg/kg/day for two days before detection. At the 4th week, 8th week and 12th week of active immunization, the rats were sacrificed and cardiac function and the levels of cardiac LC3 and Beclin-1 were detected. ΔΨm in cardiac myocytes was determined by myocardial radionuclide imaging technology and JC-1 staining. In the present study, β₁-AABs caused cardiac dysfunction, reduced ΔΨm and decreased cardiac autophagy. Treatment with RAPA markedly attenuated β₁-AABs-induced cardiac injury evidenced by recovered ΔΨm. Taken together, these results suggested that β₁-AABs exerted significant decreased ΔΨm, which may contribute to cardiac dysfunction, most likely by decreasing cardiac autophagy in vivo. Moreover, myocardial radionuclide imaging technology may be needed to assess the risk in developing cardiac dysfunction for the people who have β₁-AABs in their blood.     

Identification of voltage-gated K+ channel beta 2 (Kvβ2) subunit as a novel interaction partner of the pain transducer Transient Receptor Potential Vanilloid 1 channel (TRPV1)

The Transient Receptor Potential Vanilloid 1 (TRPV1, vanilloid receptor 1) ion channel plays a key role in the perception of thermal and inflammatory pain, however, its molecular environment in dorsal root ganglia (DRG) is largely unexplored. Utilizing a panel of sequence-directed antibodies against TRPV1 protein and mouse DRG membranes, the channel complex from mouse DRG was detergent-solubilized, isolated by immunoprecipitation and subsequently analyzed by mass spectrometry. A number of potential TRPV1 interaction partners were identified, among them cytoskeletal proteins, signal transduction molecules, and established ion channel subunits. Based on stringent specificity criteria, the voltage-gated K⁺ channel beta 2 subunit (Kvβ2), an accessory subunit of voltage-gated K⁺ channels, was identified of being associated with native TRPV1 channels. Reverse co-immunoprecipitation and antibody co-staining experiments confirmed TRPV1/Kvβ2 association. Biotinylation assays in the presence of Kvβ2 demonstrated increased cell surface expression levels of TRPV1, while patch-clamp experiments resulted in a significant increase of TRPV1 sensitivity to capsaicin. Our work shows, for the first time, the association of a Kvβ subunit with TRPV1 channels, and suggests that such interaction may play a role in TRPV1 channel trafficking to the plasma membrane.     

Ligand-specific Conformational Changes in the ��1 Glycine Receptor Ligand-binding Domain

Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer β-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both β-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on α1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner β-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner β-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.Glycine receptor (GlyR)3 chloride channels are pentameric Cys loop receptors that mediate fast synaptic transmission in the nervous system (1, 2). This family also includes nicotinic acetylcholine receptors (nAChRs), γ-aminobutyric acid type A and type C receptors, and serotonin type 3 receptors. Individual subunits comprise a large ligand-binding domain (LBD) and a transmembrane domain consisting of four α-helices (M1–M4). The LBD consists of a 10-strand β-sandwich made of an inner β-sheet with six strands and an outer β-sheet with four strands (3). The ligand-binding site is situated at the interface of adjacent subunits and is formed by loops A–C from one subunit and loops D–F from the neighboring subunit (3).The activation mechanism of Cys loop receptors is currently the subject of intense investigation because it is key to understanding receptor function under normal and pathological conditions (4, 5). Based on structural analysis of Torpedo nAChRs, Unwin and colleagues (6, 7) originally proposed that agonist binding induced the inner β-sheet to rotate, whereas the outer β-sheet tilted slightly upwards with loop C clasping around the agonist. These movements were thought to be transmitted to the transmembrane domain via a differential movement of loop 2 (β1-β2) and loop 7 (β6-β7) (both part of the inner β-sheet) and the pre-M1 domain (which is linked via a β-strand to the loop C in the outer sheet). The idea of large loop C movements accompanying agonist binding is supported by structural and functional data (3, 8–13). However, a direct link between loop C movements and channel gating has proved more difficult to establish. Although computational modeling studies have suggested that this loop may be a major component of the channel opening mechanism (14–18), experimental support for this model is not definitive. Similarly, loop F is also thought to move upon ligand binding, although there is as yet no consensus as to whether these changes represent local or global conformational changes (11, 19–21). Recently, a comparison of crystal structures of bacterial Cys loop receptors in the closed and open states revealed that although both the inner and outer β-sheets exhibit different conformations in closed and open states, the pre-M1 domain remains virtually stationary (22, 23). It is therefore relevant to question whether loop C, loop F, and pre-M1 movements are essential for Cys loop receptor activation.Strychnine is a classical competitive antagonist of GlyRs (24, 25), and to date there is no evidence that it can produce LBD structural changes. In this study we use voltage-clamp fluorometry (VCF) to compare glycine- and strychnine-induced conformational changes in the GlyR loops 2, C, D, E, and F and the pre-M1 domain in an attempt to determine whether they signal ligand-binding events, local conformational changes, or conformational changes associated with receptor activation.In a typical VCF experiment, a domain of interest is labeled with an environmentally sensitive fluorophore, and current and fluorescence are monitored simultaneously during ligand application. VCF is ideally suited for identifying ligand-specific conformational changes because it can report on electrophysiologically silent conformational changes (26), such as those induced by antagonists. Indeed, VCF has recently provided valuable insights into the conformational rearrangements of various Cys loop receptors (19, 21, 27–33).     

GRIN1 Regulates ��-Opioid Receptor Activities by Tethering the Receptor and G Protein in the Lipid Raft

The lipid raft location of μ-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein α-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence ²⁶⁷GSKEK²⁷¹ within the MOR third intracellular loop that is not involved in Gα interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Gα interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Gα. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-β-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Gα with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.     

Role of the Ryanodine Receptor in Ischemic Brain Damage—Localized Reduction of Ryanodine Receptor Binding During Ischemia in Hippocampus CA1

1. The ryanodine receptor has recently been shown to play a pivotal role in the regulation of intracellular Ca²⁺ concentration via Ca²⁺-induced Ca²⁺ release (CICR). Effects of ischemia on CICR in the brain tissue, however, remain largely unknown since only a few reports have been published on this subject. In this paper we report on work in this area by our group and review related progress in this field.2. We examined alterations of ryanodine receptor binding and local cerebral blood flow (LCBF) at 15 min, 30 min, and 2 hr after occlusion of the right common carotid artery in the gerbil brain. A quantitative autoradiographic method permitted simultaneous measurement of these parameters in the same brain. The LCBF was significantly reduced in most of the cerebral regions on the occluded side during each time period of ischemia. In contrast, only in the hippocampus CA1 on the occluded side was a significant reduction in ryanodine binding found at 15 min, 30 min and 2 hr after the occlusion.3. These findings suggest that suppression of ryanodine binding in the hippocampus CA1 may be attributable to a regionally specific perturbation of CICR and that this perturbation may be closely associated with the pathophysiological mechanism that leads to the selective ischemic vulnerability of this region.4. Other recent studies have also reported an important role for ryanodine receptors in neuronal injury such as the delayed neuronal death in the hippocampus CA1. These data suggest that derangement of CICR is likely to be involved in acute neuronal necrosis as well as in delayed neuronal death in ischemia.5. Further studies on clarifying the role of CICR in ischemic brain damage are needed in order to develop new therapeutic strategies for stroke patients.     

Synthesis of 3′-Acetamidoadenosine Derivatives as Potential A3 Adenosine Receptor Agonists

On the basis of high binding affinity of 3′-aminoadenosine derivatives 2b at the human A₃ adenosine receptor (AR), 3′-acetamidoadenosine derivatives 3a–e were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A₃AR, our results revealed that 3′-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A₃AR.     

Transient global cerebral ischemia induces up-regulation of MLTKα in hippocampal CA1 neurons

MLTK (mixed-lineage kinase-like mitogen-activated protein triple kinase) is a member of the mitogen-activated protein kinase family and functioned as a mitogen activated kinase kinase kinase. MLTKα, one of the alternatively spliced forms of MLTK, could activate the c-Jun N-terminal kinase pathway, which involved in cellular stress responses and apoptosis. But the role of MLTKα in neural apoptosis was still unclear. Here, we performed a transient global cerebral ischemia model (TGCI) in adult rats and detected the dynamic changes of MLTKα in hippocampal CA1 neurons and brain cortex. We found the MLTKα expression was increased shortly after TGCI and peaked after 8 h. In spatial distribution, MLTKα was widely located in neurons rather than astrocytes and microglia. Moreover, there was a concomitant up-regulation of active caspase-3. Taken together, we hypothesized the up-regulation of MLTKα played an essential role in the apoptosis of hippocampal CA1 neurons.