The influence of cortisol co-secretion on clinical characteristics and postoperative outcomes in unilateral primary aldosteronism.

Context: The prevalence of unilateral primary aldosteronism (UPA) with cortisol co-secretion varies geographically. Objective: To investigate the prevalence and clinical characteristics of UPA with cortisol co-secretion in a Chinese population. Design: Retrospective cohort study. Methods: We recruited 580 patients with UPA who underwent cosyntropin stimulation test (CST) after the 1-mg dexamethasone suppression test (DST) and retrospectively analyzed the clinical characteristics and postoperative outcomes of UPA with and without cortisol co-secretion. Results: UPA with cortisol co-secretion (1 mg DST>1.8 ug/dL) was identified in 65 of 580 (11.2%) patients. These patients were characterized by older age, longer duration of hypertension, higher concentration of plasma aldosterone and midnight cortisol, lower adrenocorticotropic hormone (ACTH) and dehydroepiandrosterone sulfate (DHEAS), larger tumor diameter, and more history of diabetes mellitus. Cortisol and aldosterone levels were higher and DHEAS level was lower in UPA with cortisol co-secretion at 0-120 min after CST. Among 342 UPA patients with KCNJ5 gene sequencing and follow-up results, the complete clinical success rate was lower in UPA with cortisol co-secretion (33.3% vs. 56.4%, P<0.05); the complete biochemical success rate and KCNJ5 mutation did not differ between the two groups. Age, tumor size, and ACTH were independent predictors of UPA with cortisol co-secretion. Sex, BMI, duration of hypertension, KCNJ5 mutation, and cortisol co-secretion were independent predictors for complete clinical success in UPA after surgery. Conclusions: UPA with cortisol co-secretion is not uncommon in China, but the clinical features were distinctly different from those without co-secretion. Cortisol co-secretion is an independent risk factor for incomplete clinical success after surgery in UPA.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism.

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

Identification of Potential Modulators of a Pathogenic G Protein-Gated Inwardly Rectifying K+ Channel 4 Mutant: In Silico Investigation in the Context of Drug Discovery for Hypertension.

Genetic abnormalities have been associated with primary aldosteronism, a major cause of secondary hypertension. This includes mutations in the KCNJ5 gene, which encodes G protein-gated inwardly rectifying K+ channel 4 (GIRK4). For example, the substitution of glycine with glutamic acid gives rise to the pathogenic GIRK4G151E mutation, which alters channel selectivity, making it more permeable to Na+ and Ca2+. While tertiapin and tertiapin-Q are well-known peptide inhibitors of the GIRK4WT channel, clinically, there is a need for the development of selective modulators of mutated channels, including GIRK4G151E. Using in silico methods, including homology modeling, protein-peptide docking, ligand-binding site prediction, and molecular docking, we aimed to explore potential modulators of GIRK4WT and GIRK4G151E. Firstly, protein-peptide docking was performed to characterize the binding site of tertiapin and its derivative to the GIRK4 channels. In accordance with previous studies, the peptide inhibitors preferentially bind to the GIRK4WT channel selectivity filter compared to GIRK4G151E. A ligand-binding site analysis was subsequently performed, resulting in the identification of two potential regions of interest: the central cavity and G-loop gate. Utilizing curated chemical libraries, we screened over 700 small molecules against the central cavity of the GIRK4 channels. Flavonoids, including luteolin-7-O-rutinoside and rutin, and the macrolides rapamycin and troleandomycin bound strongly to the GIRK4 channels. Similarly, xanthophylls, particularly luteoxanthin, bound to the central cavity with a strong preference towards the mutated GIRK4G151E channel compared to GIRK4WT. Overall, our findings suggest potential lead compounds for further investigation, particularly luteoxanthin, that may selectively modulate GIRK4 channels.

Somatic SLC30A1 mutations altering zinc transporter ZnT1 cause aldosterone-producing adenomas and primary aldosteronism.

Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

BACKGROUND: Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. METHODS: Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. RESULTS: We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. CONCLUSIONS: Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

Spatial Memory Training Counteracts Hippocampal GIRK Channel Decrease in the Transgenic APPSw,Ind J9 Alzheimer's Disease Mouse Model.

G-protein-gated inwardly rectifying potassium (GIRK) channels are critical determinants of neuronal excitability. They have been proposed as potential targets to restore excitatory/inhibitory balance in acute amyloidosis models, where hyperexcitability is a hallmark. However, the role of GIRK signaling in transgenic mice models of Alzheimer's disease (AD) is largely unknown. Here, we study whether progressive amyloid-ß (Aß) accumulation in the hippocampus during aging alters GIRK channel expression in mutant ß-amyloid precursor protein (APPSw,Ind J9) transgenic AD mice. Additionally, we examine the impact of spatial memory training in a hippocampal-dependent task, on protein expression of GIRK subunits and Regulator of G-protein signaling 7 (RGS7) in the hippocampus of APPSw,Ind J9 mice. Firstly, we found a reduction in GIRK2 expression (the main neuronal GIRK channels subunit) in the hippocampus of 6-month-old APPSw,Ind J9 mice. Moreover, we found an aging effect on GIRK2 and GIRK3 subunits in both wild type (WT) and APPSw,Ind J9 mice. Finally, when 6-month-old animals were challenged to a spatial memory training, GIRK2 expression in the APPSw,Ind J9 mice were normalized to WT levels. Together, our results support the evidence that GIRK2 could account for the excitatory/inhibitory neurotransmission imbalance found in AD models, and training in a cognitive hippocampal dependent task may have therapeutic benefits of reversing this effect and lessen early AD deficits.

Pathogenesis of Primary Aldosteronism: Impact on Clinical Outcome.

Primary aldosteronism (PA) is the most common form of secondary arterial hypertension, with a prevalence of approximately 20% in patients with resistant hypertension. In the last decade, somatic pathogenic variants in KCNJ5, CACNA1D, ATP1A1 and ATP2B3 genes, which are involved in maintaining intracellular ionic homeostasis and cell membrane potential, were described in aldosterone-producing adenomas (aldosteronomas). All variants in these genes lead to the activation of calcium signaling, the major trigger for aldosterone production. Genetic causes of familial hyperaldosteronism have been expanded through the report of germline pathogenic variants in KCNJ5, CACNA1H and CLCN2 genes. Moreover, PDE2A and PDE3B variants were associated with bilateral PA and increased the spectrum of genetic etiologies of PA. Of great importance, the genetic investigation of adrenal lesions guided by the CYP11B2 staining strongly changed the landscape of somatic genetic findings of PA. Furthermore, CYP11B2 staining allowed the better characterization of the aldosterone-producing adrenal lesions in unilateral PA. Aldosterone production may occur from multiple sources, such as solitary aldosteronoma or aldosterone-producing nodule (classical histopathology) or clusters of autonomous aldosterone-producing cells without apparent neoplasia denominated aldosterone-producing micronodules (non-classical histopathology). Interestingly, KCNJ5 mutational status and classical histopathology of unilateral PA (aldosteronoma) have emerged as relevant predictors of clinical and biochemical outcome, respectively. In this review, we summarize the most recent advances in the pathogenesis of PA and discuss their impact on clinical outcome.

Update on the Genetics of Primary Aldosteronism and Aldosterone-Producing Adenomas.

PURPOSE OF THE REVIEW: Primary aldosteronism (PA) is the leading cause of secondary hypertension, accounting for over 10% of patients with high blood pressure. It is characterized by autonomous production of aldosterone from the adrenal glands leading to low-renin levels. The two most common forms arise from bilateral adrenocortical hyperplasia (BAH) and aldosterone-producing adenoma (APA). We discuss recent discoveries in the genetics of PA. RECENT FINDINGS: Most APAs harbor variants in the KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1 genes. With the exception of ß-catenin (CTNNB1), all other causative genes encode ion channels; pathogenic variants found in PA lead to altered ion transportation, cell membrane depolarization, and consequently aldosterone overproduction. Some of these genes are found mutated in the germline state (CYP11B2, CLCN2, KCNJ5, CACNA1H, and CACNA1D), leading then to familial hyperaldosteronism, and often BAH rather than single APAs. Several genetic defects in the germline or somatic state have been identified in PA. Understanding how these molecular abnormalities lead to excess aldosterone contributes significantly to the elucidation of the pathophysiology of low-renin hypertension. It may also lead to new and more effective therapies for this disease acting at the molecular level.

Familial forms and molecular profile of primary hyperaldosteronism.

Primary hyperaldosteronism (PAH) is the most frequent cause of secondary arterial hypertension. Most PAHs occur sporadically, but 5% of cases have a hereditary origin (familial PAH). Four forms of familial PAH have been described. Type I familial PAH is produced by a fusion of the CYP11B2 and CYP11B1 genes, in this way the synthesis of aldosterone becomes to be regulated by ACTH instead of by angiotensin II. In type II, III and IV familial PAH there is an increase in the transcription and expression of CYP11B2 responsible for aldosterone synthesis due to a germinal mutation in CLCN2, KCNJ5 and CACNA1H, respectively. On the other hand, somatic mutations have been identified in 50% of sporadic PAHs, with gain-of-function mutations at the level of KCNJ5, ATP1A1, ATP2B3 and CACNA1D being the most common. This review provides a detailed description of the different forms of familial PAH and the molecular profile of patients with sporadic PAH.

Neuronal G protein-gated K+ channels.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels exert a critical inhibitory influence on neurons. Neuronal GIRK channels mediate the G protein-dependent, direct/postsynaptic inhibitory effect of many neurotransmitters including γ-aminobutyric acid (GABA), serotonin, dopamine, adenosine, somatostatin, and enkephalin. In addition to their complex regulation by G proteins, neuronal GIRK channel activity is sensitive to phosphatidylinositol 4,5-bisphosphate (PIP2), phosphorylation, regulator of G protein signaling (RGS) proteins, intracellular Na+ and Ca2+, and cholesterol. The application of genetic and viral manipulations in rodent models, together with recent progress in the development of GIRK channel modulators, has increased our understanding of the physiological and behavioral impact of neuronal GIRK channels. Work in rodent models has also revealed that neuronal GIRK channel activity is modified, transiently or persistently, by various stimuli including exposure drugs of abuse, changes in neuronal activity patterns, and aversive experience. A growing body of preclinical and clinical evidence suggests that dysregulation of GIRK channel activity contributes to neurological diseases and disorders. The primary goals of this review are to highlight fundamental principles of neuronal GIRK channel biology, mechanisms of GIRK channel regulation and plasticity, the nascent landscape of GIRK channel pharmacology, and the potential relevance of GIRK channels to the pathophysiology and treatment of neurological diseases and disorders.

The Role of Urinary Extracellular Vesicles Sodium Chloride Cotransporter in Subtyping Primary Aldosteronism.

Background: Adrenal venous sampling (AVS) is recognized as the gold standard for subtyping primary aldosteronism (PA), but its invasive nature and technical challenges limit its availability. A recent study reported that sodium chloride cotransporter (NCC) in urinary extracellular vesicles (uEVs) is a promising marker for assessing the biological activity of aldosterone and can be treated as a potential biomarker of PA. The current study was conducted to verify the hypothesis that the expression of NCC and its phosphorylated form (pNCC) in uEVs are different in various subtypes and genotypes of PA and can be used to select AVS candidates. Methods: A total of 50 patients with PA were enrolled in the study. Urinary extracellular vesicles (uEVs) were isolated from spot urine samples using ultracentrifugation. NCC and pNCC expressions were tested in patients diagnosed with PA who underwent AVS. Sanger sequencing of KCNJ5 was performed on DNA extracted from adrenal adenoma. Results: pNCC (1.89 folds, P<.0001) and NCC (1.82 folds, P=0.0002) was more abundant in the uEVs in the high lateralization index (h-LI, ≥ 4) group than in the low LI (l-LI, < 4) group. Carriers of the somatic KCNJ5 mutations, compared with non-carriers, had more abundant pNCC expression (2.16 folds, P=0.0039). Positive correlation between pNCC abundance and plasma aldosterone level was found in this study (R = 0.1220, P = 0.0129). Conclusions: The expression of pNCC in uEVs in patients with PA with various subtypes and genotypes was different. It can be used as biomarker of AVS for PA subtyping.

AsKC11, a Kunitz Peptide from Anemonia sulcata, Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels.

(1) Background: G protein-coupled inward-rectifier potassium (GIRK) channels, especially neuronal GIRK1/2 channels, have been the focus of intense research interest for developing drugs against brain diseases. In this context, venom peptides that selectively activate GIRK channels can be seen as a new source for drug development. Here, we report on the identification and electrophysiological characterization of a novel activator of GIRK1/2 channels, AsKC11, found in the venom of the sea anemone Anemonia sulcata. (2) Methods: AsKC11 was purified from the sea anemone venom by reverse-phase chromatography and the sequence was identified by mass spectrometry. Using the two-electrode voltage-clamp technique, the activity of AsKC11 on GIRK1/2 channels was studied and its selectivity for other potassium channels was investigated. (3) Results: AsKC11, a Kunitz peptide found in the venom of A. sulcata, is the first peptide shown to directly activate neuronal GIRK1/2 channels independent from Gi/o protein activity, without affecting the inward-rectifier potassium channel (IRK1) and with only a minor effect on KV1.6 channels. Thus, AsKC11 is a novel activator of GIRK channels resulting in larger K+ currents because of an increased chord conductance. (4) Conclusions: These discoveries provide new insights into a novel class of GIRK activators.

Genetics of Primary Aldosteronism.

Primary aldosteronism is considered the commonest cause of secondary hypertension. In affected individuals, aldosterone is produced in an at least partially autonomous fashion in adrenal lesions (adenomas, [micro]nodules or diffuse hyperplasia). Over the past decade, next-generation sequencing studies have led to the insight that primary aldosteronism is largely a genetic disorder. Sporadic cases are due to somatic mutations, mostly in ion channels and pumps, and rare cases of familial hyperaldosteronism are caused by germline mutations in an overlapping set of genes. More than 90% of aldosterone-producing adenomas carry somatic mutations in K+ channel Kir3.4 (KCNJ5), Ca2+ channel CaV1.3 (CACNA1D), alpha-1 subunit of the Na+/K+ ATPase (ATP1A1), plasma membrane Ca2+ transporting ATPase 3 (ATP2B3), Ca2+ channel CaV3.2 (CACNA1H), Cl- channel ClC-2 (CLCN2), ß-catenin (CTNNB1), and/or G-protein subunits alpha q/11 (GNAQ/11). Mutations in some of these genes have also been identified in aldosterone-producing (micro)nodules, suggesting a disease continuum from a single cell, acquiring a somatic mutation, via a nodule to adenoma formation, and from a healthy state to subclinical to overt primary aldosteronism. Individual glands can have multiple such lesions, and they can occur on both glands in bilateral disease. Familial hyperaldosteronism, typically with early onset, is caused by germline mutations in steroid 11-beta hydroxylase/ aldosterone synthase (CYP11B1/2), CLCN2, KCNJ5, CACNA1H, and CACNA1D.

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.

Characterization of a mutated KCNJ5 gene, G387R, in unilateral primary aldosteronism.

Somatic mutation in the KCNJ5 gene is a common driver of autonomous aldosterone overproduction in aldosterone-producing adenomas (APA). KCNJ5 mutations contribute to a loss of potassium selectivity, and an inward Na+ current could be detected in cells transfected with mutated KCNJ5. Among 223 unilateral primary aldosteronism (uPA) individuals with a KCNJ5 mutation, we identified 6 adenomas with a KCNJ5 p.Gly387Arg (G387R) mutation, previously unreported in uPA patients. The six uPA patients harboring mutant KCNJ5-G387R were older, had a longer hypertensive history, and had milder elevated preoperative plasma aldosterone levels than those APA patients with more frequently detected KCNJ5 mutations. CYP11B2 immunohistochemical staining was only positive in three adenomas, while the other three had co-existing multiple aldosterone-producing micronodules. The bioinformatics analysis predicted that function of the KCNJ5-G387R mutant channel could be pathological. However, the electrophysiological experiment demonstrated that transfected G387R mutant cells did not have an aberrantly stimulated ion current, with lower CYP11B2 synthesis and aldosterone production, when compared to that of the more frequently detected mutant KCNJ5-L168R transfected cells. In conclusion, mutant KCNJ5-G387R is not a functional KCNJ5 mutation in unilateral PA. Compared with other KCNJ5 mutations, the observed mildly elevated aldosterone expression actually hindered the clinical identification of clinical unilateral PA. The KCNJ5-G387R mutation needs to be distinguished from functional KCNJ5 mutations during genomic analysis in APA evaluation because of its functional silence.

Ivabradine augments high-frequency dynamic gain of the heart rate response to low- and moderate-intensity vagal nerve stimulation under ß-blockade.

Our previous study indicated that intravenously administered ivabradine (IVA) augmented the dynamic heart rate (HR) response to moderate-intensity vagal nerve stimulation (VNS). Considering an accentuated antagonism, the results were somewhat paradoxical; i.e., the accentuated antagonism indicates that an activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels via the accumulation of intracellular cyclic adenosine monophosphate (cAMP) augments the HR response to VNS, whereas the inhibition of HCN channels by IVA also augmented the HR response to VNS. To remove the possible influence from the accentuated antagonism, we examined the effects of IVA on the dynamic vagal control of HR under ß-blockade. In anesthetized rats (n = 7), the right vagal nerve was stimulated for 10 min according to binary white noise signals between 0 and 10 Hz (V0-10), between 0 and 20 Hz (V0-20), and between 0 and 40 Hz (V0-40). The transfer function from VNS to HR was estimated. Under ß-blockade (propranolol, 2 mg/kg iv), IVA (2 mg/kg iv) did not augment the asymptotic low-frequency gain but increased the asymptotic high-frequency gain in V0-10 (0.53 ± 0.10 vs. 1.74 ± 0.40 beats/min/Hz, P < 0.01) and V0-20 (0.79 ± 0.14 vs. 2.06 ± 0.47 beats/min/Hz, P < 0.001). These changes, which were observed under a minimal influence from sympathetic background tone, may reflect an increased contribution of the acetylcholine-sensitive potassium channel (IK,ACh) pathway after IVA, because the HR control via the IK,ACh pathway is faster and acts in the frequency range higher than the cAMP-mediated pathway.NEW & NOTEWORTHY Since ivabradine (IVA) inhibits hyperpolarization-activated cyclic nucleotide-gated channels, interactions among the sympathetic effect, vagal effect, and IVA can occur in the control of heart rate (HR). To remove the sympathetic effect, we estimated the transfer function from vagal nerve stimulation to HR under ß-blockade in anesthetized rats. IVA augmented the high-frequency dynamic gain during low- and moderate-intensity vagal nerve stimulation. Untethering the hyperpolarizing effect of acetylcholine-sensitive potassium channels after IVA may be a possible underlying mechanism.

Rapid assessment of G protein signaling of four opioid receptors using a real-time fluorescence-based membrane potential assay.

Opioids are the most powerful analgesics used clinically; however, severe side effects limit their long-term use. Various concepts involving biased intracellular signaling, partial agonism or multi-receptor targeting have been proposed to identify novel opioids with increased analgesic efficacy but reduced side effects. The search for such 'better opioids' implies screening of huge compound libraries and requires highly reliable, easy to perform and high throughput screening (HTS) assays. Here, we utilize an established membrane potential assay to monitor activation of G protein-coupled inwardly rectifying potassium (GIRK) channels, one of the main effectors of opioid receptor signaling, as readout to determine pharmacological profiles of opioids in a non-invasive manner. Specifically, in this study, we optimize assay conditions and extend the application of this assay to screen all four members of the opioid receptor family, stably expressed in AtT-20 and HEK293 cells. This ultra-sensitive system yielded EC50 values in the nano-molar range. We further validate this system for screening cells stably co-expressing two opioid receptors, which could be a valuable tool for investigating bi-functional ligands and studying interactions between receptors. Additionally, we demonstrate the utility of this assay to study antagonists as well as ligands with varying efficacies. Our results suggest that this assay could easily be up-scaled to HTS assay in order to efficiently study receptor activation and screen for novel opioids.

Ion channels as lipid sensors: from structures to mechanisms.

Ion channels play critical roles in cellular function by facilitating the flow of ions across the membrane in response to chemical or mechanical stimuli. Ion channels operate in a lipid bilayer, which can modulate or define their function. Recent technical advancements have led to the solution of numerous ion channel structures solubilized in detergent and/or reconstituted into lipid bilayers, thus providing unprecedented insight into the mechanisms underlying ion channel-lipid interactions. Here, we describe how ion channel structures have evolved to respond to both lipid modulators and lipid activators to control the electrical activities of cells, highlighting diverse mechanisms and common themes.

Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles.

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (P=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (P<0.001) and an almost 3-fold upregulation of CYP11B (P<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.