Altered Receptor Function Research Articles

In vivo functional profiling and structural characterisation of the human Glp1r A316T variant.

Glucagon-like peptide-1 receptor (GLP-1R) agonists (GLP-1RAs) are a highly effective therapy class for type 2 diabetes (T2D) and obesity, yet there are variable patient responses. Variation in the human Glp1r gene leading to altered receptor structure, signal transduction, and function might be directly linked to variable therapeutic responses in patients. A naturally occurring, low-frequency, gain-of-function missense variant, rs10305492 G>A (A316T), protects against T2D and cardiovascular disease. Here we employ CRISPR/Cas9 technology to generate a humanised knock-in mouse model bearing the homozygous Glp1r A316T substitution. Human Glp1r A316T/A316T mice displayed lower fasting blood glucose levels and improved glucose tolerance, as well as increased plasma insulin levels and improved insulin secretion compared to human Glp1r +/+ littermates, even under metabolic stress. They also exhibited alterations in islet cytoarchitecture and β-cell identity under a high-fat, high-sucrose (HFHS) diet. This was however associated with blunted responses to pharmacological GLP-1RAs in vivo . Further investigations in several rodent and human β-cell models demonstrated that the human Glp1r A316T variant exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Our results are further supported by the cryo-EM analysis and molecular dynamics (MD) simulations of the GLP-1R A316T structure, collectively demonstrating that the A316T Glp1r variant governs basal receptor activity and pharmacological responses to GLP-1R-targeting anti-diabetic therapies, highlighting the importance of the molecular characterisation of human Glp1r variants to predict individual therapy responses.

Restorative effects of melatonin on bisphenol a-induced interference of gene expression in hypothalamic pituitary axis following early exposure

Bisphenol-A is a standard monomer used in manufacturing plastics and epoxy resins, and it is widely used in food preservation and packaging. It is an endocrine-disrupting chemical miming the endogenous estradiol hormone. Melatonin regulates sleep-wake cycles and plays essential physiological roles in the body through its antioxidative properties. This research aims to ascertain the impact of Bisphenol A on the hypothalamic-pituitary-ovarian axis and determine melatonin's function on possible BPA-induced effects. Six adult male Wistar rats and 12 adult female Wistar rats of proven fertility were bred and organized into groups. These animals were subjected to subcutaneous injections of high and low doses of bisphenol A from postnatal days 0-3, then oral melatonin. The rats were allowed to mature into full-grown adults and euthanized at 120 ±4 days. The serum and hypothalamic-pituitary-ovarian tissues were collected for various assays. Compared to the control groups, groups administered varying doses of bisphenol A showed significant overexpression of estrogen and androgen receptors. Administration of Melatonin showed some reversal and reparative effects on damage of the hypothalamic pituitary ovarian axis. Elevated estrogen receptor levels induced by Bisphenol A altered receptor function. Melatonin showed some promising reparative effects.

Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function or gain-of-function properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12) and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for loss-of-function and gain-of-function variants. Gain-of-function variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age: 1 month), hypertonic and more often had movement disorders, including hyperekplexia. Patients with loss-of-function variants were older at the time of seizure onset (median age: 16 months), hypotonic and had sleeping disturbances. Loss-of-function and gain-of-function variants were disease-causing in both sexes but affected males often carried de novo or hemizygous loss-of-function variants inherited from healthy mothers, whereas affected females had mostly de novo heterozygous gain-of-function variants.

Altered GABAA receptor function in women with endometriosis: a possible pain-related mechanism.

The mechanism underlying endometriosis-related pain remains poorly understood. Previous studies have indicated that γ-aminobutyric acid (GABA) type A (GABAA ) receptors and GABAergic substances (eg endogenous neurosteroids) play important mechanistic roles in various pain conditions. Our primary objective was to compare GABAA receptor function between women with endometriosis and healthy controls by performing a challenge test with diazepam, a GABAA receptor agonist, using the saccadic eye velocity as the main outcome. The secondary objective was to investigate the relation between GABAA receptor function and serum levels of allopregnanolone, an endogenous positive modulator of the GABAA receptor, in the participating women. 15 women with pelvic pain and laparoscopically confirmed endometriosis and 10 healthy, symptom-free, control women, aged 18-40 years, underwent the diazepam challenge test during the follicular phase of the menstrual cycle. Basal serum allopregnanolone levels were measured prior to diazepam injection. Compared with healthy controls, women with pelvic pain and confirmed endometriosis had a significantly smaller change in saccadic eye velocity after GABAA receptor stimulation with diazepam, indicating lower sensitivity to diazepam. The saccadic eye velocity response was not correlated with the serum allopregnanolone levels. Women with painful endometriosis show altered GABAA receptor function, depicted as a muted response to an exogenous GABAA receptor agonist.

Correlation of age at seizure onset with GABAA receptor subunit and chloride Co-transporter configuration in Focal cortical dysplasia (FCD)

Focal cortical dysplasia (FCD) represents a group of malformations of cortical development, which are speculated to be related to early developmental defects in the cerebral cortex. According to dysmature cerebral development hypothesis of FCD altered GABAA receptor function is known to contribute to abnormal neuronal network. Here, we studied the possible association between age at seizure onset in FCD with the subunit configuration of GABAA receptors in resected brain specimens obtained from patients with FCD. We observed a significantly higher ratio of α4/α1 subunit-containing GABAA receptors in patients with early onset (EO) FCD as compared to those with late onset (LO) FCD as is seen during the course of development where α4-containing GABAA receptors expression is high as compared to α1-containing GABAA receptors expression. Likewise, the influx to efflux chloride co-transporter expression of NKCC1/KCC2 was also increased in patients with EO FCD as seen during brain development. In addition, we observed that the ratio of GABA/Glutamate neurotransmitters was lower in patients with EO FCD as compared to that in patients with LO FCD. Our findings suggest altered configuration of GABAA receptors in FCD which could be contributing to aberrant depolarizing GABAergic activity. In particular, we observed a correlation of age at seizure onset in FCD with subunit configuration of GABAA receptors, levels of NKCC1/KCC2 and the ratio of GABA/Glutamate neurotransmitters such that the patients with EO FCD exhibited a more critically modulated GABAergic network.

Genetic Variants of Adrenoceptors.

Adrenoceptors are class A G-protein-coupled receptors grouped into three families (α1-, α2-, and β-adrenoceptors), each one including three members. All nine corresponding adrenoceptor genes display genetic variation in their coding and adjacent non-coding genomic region. Coding variants, i.e., nucleotide exchanges within the transcribed and translated receptor sequence, may result in a difference in amino acid sequence thus altering receptor function and signaling. Such variants have been intensely studied in vitro in overexpression systems and addressed in candidate-gene studies for distinct clinical parameters. In recent years, large cohorts were analyzed in genome-wide association studies (GWAS), where variants are detected as significant in context with specific traits. These studies identified two of the in-depth characterized 18 coding variants in adrenoceptors as repeatedly statistically significant genetic risk factors - p.Arg389Gly in the β1- and p.Thr164Ile in the β2-adrenoceptor, along with 56 variants in the non-coding regions adjacent to the adrenoceptor gene loci, the functional role of which is largely unknown at present. This chapter summarizes current knowledge on the two coding variants in adrenoceptors that have been consistently validated in GWAS and provides a prospective overview on the numerous non-coding variants more recently attributed to adrenoceptor gene loci.

Molecular Insights into Epigenetics and Cannabinoid Receptors.

The actions of cannabis are mediated by G protein-coupled receptors that are part of an endogenous cannabinoid system (ECS). ECS consists of the naturally occurring ligands N-arachidonylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), their biosynthetic and degradative enzymes, and the CB1 and CB2 cannabinoid receptors. Epigenetics are heritable changes that affect gene expression without changing the DNA sequence, transducing external stimuli in stable alterations of the DNA or chromatin structure. Cannabinoid receptors are crucial candidates for exploring their functions through epigenetic approaches due to their significant roles in health and diseases. Epigenetic changes usually promote alterations in the expression of genes and proteins that can be evaluated by various transcriptomic and proteomic analyses. Despite the exponential growth of new evidence on the critical functions of cannabinoid receptors, much is still unknown regarding the contribution of various genetic and epigenetic factors that regulate cannabinoid receptor gene expression. Recent studies have identified several immediate and long-lasting epigenetic changes, such as DNA methylation, DNA-associated histone proteins, and RNA regulatory networks, in cannabinoid receptor function. Thus, they can offer solutions to many cellular, molecular, and behavioral impairments found after modulation of cannabinoid receptor activities. In this review, we discuss the significant research advances in different epigenetic factors contributing to the regulation of cannabinoid receptors and their functions under both physiological and pathological conditions. Increasing our understanding of the epigenetics of cannabinoid receptors will significantly advance our knowledge and could lead to the identification of novel therapeutic targets and innovative treatment strategies for diseases associated with altered cannabinoid receptor functions.

Arterial stiffness, endothelial dysfunction and impaired fibrinolysis are pathogenic mechanisms contributing to cardiovascular risk in ANCA-associated vasculitis

Cardiovascular disease is a complication of systemic inflammatory diseases including anti-neutrophil cytoplasm antibody-associated vasculitis (AAV). The mechanisms of cardiovascular morbidity in AAV are poorly understood, and risk-reduction strategies are lacking. Therefore, in a series of double-blind, randomized case-control forearm plethysmography and crossover systemic interventional studies, we examined arterial stiffness and endothelial function in patients with AAV in long-term disease remission and in matched healthy volunteers (32 each group). The primary outcome for the case-control study was the difference in endothelium-dependent vasodilation between health and AAV, and for the crossover study was the difference in pulse wave velocity (PWV) between treatment with placebo and selective endothelin-A receptor antagonism. Parallel invitro studies of circulating monocytes and platelets explored mechanisms. Compared to healthy volunteers, patients with AAV had 30% reduced endothelium-dependent vasodilation and 50% reduced acute release of endothelial active tissue plasminogen activator (tPA), both significant in the case-control study. Patients with AAV had significantly increased arterial stiffness (PWV: 7.3 versus 6.4 m/s). Plasma endothelin-1 was two-fold higher in AAV and independently predicted PWV and tPA release. Compared to placebo, both selective endothelin-A and dual endothelin-A/B receptor blockade reduced PWV and increased tPA release in AAV in the crossover study. Mechanistically, patients with AAV had increased platelet activation, more platelet-monocyte aggregates, and altered monocyte endothelin receptor function, reflecting reduced endothelin-1 clearance. Patients with AAV in long-term remission have elevated cardiovascular risk and endothelin-1 contributes to this. Thus, our data support a role for endothelin-blockers to reduce cardiovascular risk by reducing arterial stiffness and increasing circulating tPA activity.

Partial ablation of frontal cortical subplate leads to developmental abnormalities in KCC2 in the prefrontal cortex

During early brain development, the subplate relays thalamocortical afferents to the overlying cortex. Disconnection of thalamic inputs to the prefrontal cortex by lesions of the subplate of the developing prefrontal cortex at early neonatal periods result in adult-onset behavioral abnormalities reminiscent of positive, negative, and cognitive symptoms of schizophrenia. Delayed maturation of γ-amino butyric acid (GABA) function may contribute to certain abnormalities of the prefrontal cortex and clinical manifestations of schizophrenia. Lesions to the subplate have also been implicated in developmental abnormalities of GABA neurotransmission in somatosensory and visual cortices. Therefore, we sought to examine the effects of subplate lesions in the developing prefrontal cortex of rats on the expression of GABA markers [parvalbumin and glutamic acid decarboxylase (GAD67)] and proteins responsible for GABAergic synaptic maturation [potassium-chloride cotransporter (KCC2) and sodium‑potassium-chloride cotransporter (NKCC1)]. Lesioned and control rats were sacrificed between postnatal days (P) 5 and 90 and immunolabeled for parvalbumin, GAD67, KCC2, and NKCC1 in the prelimbic area of the prefrontal cortex. We found decreased immunoreactivity of KCC2 on neuronal cell membranes at P11 compared to control rats. However, the overall immunoreactivity of KCC2 and NKCC1 did not differ between lesion and control animals at all time points studied. Lesioned rats also showed decreased expression of parvalbumin, but not GAD67. Our results indicate that mechanisms underlying trafficking and membrane binding of KCC2 may contribute to altered GABA receptor function during development in schizophrenia.

A de novo missense variant in GABRA4 alters receptor function in an epileptic and neurodevelopmental phenotype.

Variants in γ‐aminobutyric acid A (GABAA) receptor genes cause different forms of epilepsy and neurodevelopmental disorders. To date, GABRA4, encoding the α4‐subunit, has not been associated with a monogenic condition. However, preclinical evidence points toward seizure susceptibility. Here, we report a de novo missense variant in GABRA4 (c.899C>T, p.Thr300Ile) in an individual with early‐onset drug‐resistant epilepsy and neurodevelopmental abnormalities. An electrophysiological characterization of the variant, which is located in the pore‐forming domain, shows accelerated desensitization and a lack of seizure‐protective neurosteroid function. In conclusion, our findings strongly suggest an association between de novo variation in GABRA4 and a neurodevelopmental disorder with epilepsy.

CXCR4 Mediates Enhanced Cell Migration in CALM-AF10 Leukemia.

Leukemia transformed by the CALM-AF10 chromosomal translocation is characterized by a high incidence of extramedullary disease, central nervous system (CNS) relapse, and a poor prognosis. Invasion of the extramedullary compartment and CNS requires leukemia cell migration out of the marrow and adherence to the cells of the local tissue. Cell adhesion and migration are increasingly recognized as contributors to leukemia development and therapeutic response. These processes are mediated by a variety of cytokines, chemokines, and their receptors, forming networks of both secreted and cell surface factors. The cytokines and cytokine receptors that play key roles in CALM-AF10 driven leukemia are unknown. We find high cell surface expression of the cytokine receptor CXCR4 on leukemia cells expressing the CALM-AF10 oncogenic protein, contributing to the migratory nature of this leukemia. Our discovery of altered cytokine receptor expression and function provides valuable insight into the propagation and persistence of CALM-AF10 driven leukemia.

Structure-function relationships of the disease-linked A218T oxytocin receptor variant

Various single nucleotide polymorphisms (SNPs) in the oxytocin receptor (OXTR) gene have been associated with behavioral traits, autism spectrum disorder (ASD) and other diseases. The non-synonymous SNP rs4686302 results in the OXTR variant A218T and has been linked to core characteristics of ASD, trait empathy and preterm birth. However, the molecular and intracellular mechanisms underlying those associations are still elusive. Here, we uncovered the molecular and intracellular consequences of this mutation that may affect the psychological or behavioral outcome of oxytocin (OXT)-treatment regimens in clinical studies, and provide a mechanistic explanation for an altered receptor function. We created two monoclonal HEK293 cell lines, stably expressing either the wild-type or A218T OXTR. We detected an increased OXTR protein stability, accompanied by a shift in Ca2+ dynamics and reduced MAPK pathway activation in the A218T cells. Combined whole-genome and RNA sequencing analyses in OXT-treated cells revealed 7823 differentially regulated genes in A218T compared to wild-type cells, including 429 genes being associated with ASD. Furthermore, computational modeling provided a molecular basis for the observed change in OXTR stability suggesting that the OXTR mutation affects downstream events by altering receptor activation and signaling, in agreement with our in vitro results. In summary, our study provides the cellular mechanism that links the OXTR rs4686302 SNP with genetic dysregulations associated with aspects of ASD.

Functional Changes to the Modulation of α4β2* Nicotinic Acetylcholine Receptors during Postnatal Maturation

The neurotransmitter acetylcholine (ACh) acting at its α4β2* subtype of nicotinic acetylcholine receptor (nAChR) within the cerebral cortex plays an important role to support cognitive functions. ACh normally initiates a robust excitatory response at α4β2* nAChRs, although this activity may be modulated by a number of mechanisms that alter receptor function. Negative receptor modulation may occur via desensitization by the competitive agonist nicotine or via presumed negative allosteric modulation by the endogenous progesterone-derived neurosteroid 3α-hydroxy-5α-prenan-20-one (allopregnanolone; ALLO). Conversely, positive modulation may occur via positive allosteric modulation by the alkaloid galantamine. Although each form of modulation has been demonstrated to occur in α4β2* nAChRs located on pyramidal neurons within layer VI of the medial prefrontal cortex (mPFC) in young postnatal male mice, the presence and relative strength of modulation in young postnatal female mice, and at maturity for both sexes, is unclear. Using whole-cell electrophysiology in mFPC layer VI neurons, we measured the ability of nicotine (300 nM), ALLO (10 μM), and galantamine (0.1 μM) to modulate ACh activation of α4β2* nAChRs in male and female mice at postnatal day (P)15-20 (young postnatal age) and P80-120 (adulthood). For nicotine, nAChR desensitization at P15-20 was greater than at P80-120 and this result was driven by an effect of postnatal age in male mice only. In contrast, ALLO inhibited nAChR function to a similar degree at both P15-20 and P80-120. Galantamine potentiated nAChR function to a similar degree at both P15 and P80-120, although this potentiation was significantly greater in females than in males at each age. Results from this study suggest that distinct factors are involved in each type of receptor modulation, which are differentially altered during postnatal maturation and across biological sex. Future studies will focus on mechanisms by which these factors influence the modulation of α4β2* nAChRs during postnatal maturation.

Reduced visual cortical plasticity in autism spectrum disorder

There is increasing evidence implicating altered NMDA-receptor function in autism spectrum disorder (ASD). To investigate potential alterations in NMDA-dependent cortical plasticity in ASD, we examined the effect of visual high-frequency stimulation (HFS) on changes in plasticity in the visual cortex, measured by persistent changes in visual evoked potentials (VEPs), in individuals with ASD (n = 16) and neurotypical controls (NT; n = 15). VEPs were elicited by a checkerboard circle (0.83 Hz, 2-min blocks) at baseline and at 2, 4, and 20 min following exposure to HFS (8.87 Hz, 2 min), previously shown to induce LTP-like changes in the visual cortex. Difference waves were created by subtracting VEPs measured at baseline from each Post-HFS measure, and group differences assessed. We found that HFS resulted in reduced short-term potentiation of VEPs in ASD compared to NT participants. Thus, whilst ASD participants showed significant potentiation of the VEP immediately after HFS, this enhancement was not maintained, and only persisted into the second post-HFS assessment block in NT participants. Notably, ASD individuals who self-reported being more sensitive to visual stimuli showed greater shorter-term potentiation following visual HFS. Critically, there were no group differences in degree of neural entrainment to the visual HFS, or in attentional vigilance and task performance. These findings suggests that visual cortical plasticity is atypical in ASD, results consistent with reported altered NMDA receptor function in ASD.

Mu-Opioid Receptor Polymorphism Moderates Sensitivity to Parental Behaviors During Characterization of Personality Traits.

PurposeAttachment research shows that attachment experiences with parents in childhood influence the characterization of personality traits. Meanwhile, it is known that mu-opioid receptor function is involved in human attachment. Furthermore, a few studies suggest that the A118G polymorphism of the mu-opioid receptor gene (OPRM1) is associated with altered mu-opioid receptor function. Thus, we examined if the OPRM1 polymorphism moderates the sensitivity to parental behaviors and thereby contributes to the characterization of personality traits.Materials and MethodsParticipants were 725 healthy Japanese. Parenting practices of their parents were evaluated by the Parental Bonding Instrument (PBI) with the care and protection subscales. Personality was evaluated using the Temperament and Character Inventory (TCI). The OPRM1 A118G polymorphism was detected by a PCR method.ResultsMultiple regression analyses revealed significant effects of the interaction between the OPRM1 genotype and maternal protection on scores of the self-directedness and cooperativeness dimensions, while significant main effects of the OPRM1 genotype on scores of the TCI were not found. Further analyses showed that there were significant negative correlations between maternal protection scores and the two dimensional scores in the A/A and A/G genotypes with higher correlation coefficients in the former, but not in the G/G genotype.ConclusionThe present study suggests that the OPRM1 polymorphism contributes to the characterization of personality traits by moderating the sensitivity to parental behaviors, especially maternal protection.

Steroid hormone secretion after stimulation of mineralocorticoid and NMDA receptors and cardiovascular risk in patients with depression

Major depressive disorder (MDD) is associated with altered mineralocorticoid receptor (MR) and glucocorticoid receptor function, and disturbed glutamatergic signaling. Both systems are closely intertwined and likely contribute not only to the pathophysiology of MDD, but also to the increased cardiovascular risk in MDD patients. Less is known about other steroid hormones, such as aldosterone and DHEA-S, and how they affect the glutamatergic system and cardiovascular disease risk in MDD. We examined salivary cortisol, aldosterone, and DHEA-S secretion after stimulation of MR and glutamatergic NMDA receptors in 116 unmedicated depressed patients, and 116 age- and sex-matched healthy controls. Patients (mean age = 34.7 years, SD = ±13.3; 78% women) and controls were randomized to four conditions: (a) control condition (placebo), (b) MR stimulation (0.4 mg fludrocortisone), (c) NMDA stimulation (250 mg D-cycloserine (DCS)), and (d) combined MR/NMDA stimulation (fludrocortisone + DCS). We additionally determined the cardiovascular risk profile in both groups. DCS had no effect on steroid hormone secretion, while cortisol secretion decreased in both fludrocortisone conditions across groups. Independent of condition, MDD patients showed (1) increased cortisol, increased aldosterone, and decreased DHEA-S concentrations, and (2) increased glucose levels and decreased high-density lipoprotein cholesterol levels compared with controls. Depressed patients show profound alterations in several steroid hormone systems that are associated both with MDD pathophysiology and increased cardiovascular risk. Prospective studies should examine whether modulating steroid hormone levels might reduce psychopathology and cardiovascular risk in depressed patients.

From bedside-to-bench: What disease-associated variants are teaching us about the NMDA receptor.

NMDA receptors (NMDARs) are glutamate-gated ion channels that contribute to nearly all brain processes. Not surprisingly then, genetic variations in the genes encoding NMDAR subunits can be associated with neurodevelopmental, neurological and psychiatric disorders. These disease-associated variants (DAVs) present challenges, such as defining how DAV-induced alterations in receptor function contribute to disease progression and how to treat the affected individual clinically. As a starting point to overcome these challenges, we need to refine our understanding of the complexity of NMDAR structure function. In this regard, DAVs have expanded our knowledge of NMDARs because they do not just target well-known structure-function motifs, but rather give an unbiased view of structural elements that are important to the biology of NMDARs. Indeed, established NMDAR structure-function motifs have been validated by the appearance of disorders in patients where these motifs have been altered, and DAVs have identified novel structural features in NMDARs such as gating triads and hinges in the gating machinery. Still, the majority of DAVs remain unexplored and occur at sites in the protein with unidentified function or alter receptor properties in multiple and unanticipated ways. Detailed mechanistic and structural investigations are required of both established and novel motifs to develop a highly refined pathomechanistic model that accounts for the complex machinery that regulates NMDARs. Such a model would provide a template for rational drug design and a starting point for personalized medicine.

Altered NK cell receptor repertoire and function of natural killer cells in patients with acute myocardial infarction: A three-month follow-up study.

NK cells are important in the onset of acute myocardial infarction (AMI) by their ability to secrete IFN-γ and other inflammatory cytokines. They also participate in regulating pathological cardiac remodeling after myocardial infarction. Mechanisms of regulation, however, are incompletely understood. Herein, the aim of this study is to explore the possible association between the expression pattern of different NK cell receptors (phenotype), as well as the cytotoxic function of NK cells from AMI patients with their myocardial function after three months follow-up. We analyzed the phenotype and function of both CD56dimCD16+ and CD56brightCD16- NK cells from twenty-one patients within the first 72 h after ST-elevation AMI and three-month follow-up, as well as fifteen healthy controls. Clinical characteristics and ventricular function determined by echocardiography were also evaluated. NK cells from AMI patients showed an activated phenotype, characterized by high TNF-α production and low percentages of the activating receptor NKG2D. Interestingly, AMI patients display higher levels of circulating IL-10+ NK cells. Three-month follow-up showed that NK cells exhibit a diminished cytotoxic function. These data show that NK cells may have a role mediating myocardial remodeling by regulating the inflammatory response, mainly by the production of IL-10. We also propose that NKG2D may have a role in the onset of the inflammatory response immediately after AMI. The precise regulation of NK cells function may represent an important step in recovery of myocardial function.

A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function.

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

Targetable purinergic receptors P2Y12 and A2b antagonistically regulate bladder function.

Abnormalities in purine availability or purinergic receptor density are commonly seen in patients with lower urinary tract symptoms (LUTS), but the underlying mechanisms relating altered receptor function to LUTS are unknown. Here we provide extensive evidence for the reciprocal interplay of multiple receptors responding to ATP, ADP (adenosine diphosphate), and adenosine, agonists that regulate bladder function significantly. ADP stimulated P2Y12 receptors, causing bladder smooth muscle (BSM) contraction, whereas adenosine signaling through potentially newly defined A2b receptors, actively inhibited BSM purinergic contractility. The modulation of adenylyl cyclase-cAMP signaling via A2b and P2Y12 interaction actively regulated bladder contractility by modulating intracellular calcium levels. KO mice lacking the receptors display diametrically opposed bladder phenotypes, with P2Y12-KO mice exhibiting an underactive bladder (UAB) phenotype with increased bladder capacity and reduced voiding frequency, whereas A2b-KO mice have an overactive bladder (OAB), with decreased capacity and increased voiding frequency. The opposing phenotypes in P2Y12-KO and A2b-KO mice not only resulted from dysregulated BSM contractility, but also from abnormal BSM cell growth. Finally, we demonstrate that i.p. administration of drugs targeting P2Y12 or A2b receptor rescues these abnormal phenotypes in both KO mice. These findings strongly indicate that P2Y12 and A2b receptors are attractive therapeutic targets for human patients with LUTS.