α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling

Examination of the efficacy of acute L-alanyl-L-glutamine ingestion during hydration stress in endurance exercise

<p>Abstract</p> <p>Background</p> <p>The effect of acute L-alanyl-L-glutamine (AG; Sustamine™) ingestion on performance changes and markers of fluid regulation, immune, inflammatory, oxidative stress, and recovery was examined in response to exhaustive endurance exercise, during and in the absence of dehydration.</p> <p>Methods</p> <p>Ten physically active males (20.8 ± 0.6 y; 176.8 ± 7.2 cm; 77.4 ± 10.5 kg; 12.3 ± 4.6% body fat) volunteered to participate in this study. During the first visit (T1) subjects reported to the laboratory in a euhydrated state to provide a baseline (BL) blood draw and perform a maximal exercise test. In the four subsequent randomly ordered trials, subjects dehydrated to -2.5% of their baseline body mass. For T2, subjects achieved their goal weight and were not rehydrated. During T3 - T5, subjects reached their goal weight and then rehydrated to 1.5% of their baseline body mass by drinking either water (T3) or two different doses (T4 and T5) of the AG supplement (0.05 g·kg^-1and 0.2 g·kg^-1, respectively). Subjects then exercised at a workload that elicited 75% of their VO₂max on a cycle ergometer. During T2 - T5 blood draws occurred once goal body mass was achieved (DHY), immediately prior to the exercise stress (RHY), and immediately following the exercise protocol (IP). Resting 24 hour (24P) blood samples were also obtained. Blood samples were analyzed for glutamine, potassium, sodium, aldosterone, arginine vasopressin (AVP), C-reactive protein (CRP), interleukin-6 (IL-6), malondialdehyde (MDA), testosterone, cortisol, ACTH, growth hormone and creatine kinase. Statistical evaluation of performance, hormonal and biochemical changes was accomplished using a repeated measures analysis of variance.</p> <p>Results</p> <p>Glutamine concentrations for T5 were significantly higher at RHY and IP than T2 - T4. When examining performance changes (difference between T2 - T5 and T1), significantly greater times to exhaustion occurred during T4 (130.2 ± 340.2 sec) and T5 (157.4 ± 263.1 sec) compared to T2 (455.6 ± 245.0 sec). Plasma sodium concentrations were greater (p < 0.05) at RHY and IP for T2 than all other trials. Aldosterone concentrations at RHY and IP were significantly lower than that at BL and DHY. AVP was significantly elevated at DHY, RHY and IP compared to BL measures. No significant differences were observed between trials in CRP, IL-6, MDA, or in any of the other hormonal or biochemical measures.</p> <p>Conclusion</p> <p>Results demonstrate that AG supplementation provided a significant ergogenic benefit by increasing time to exhaustion during a mild hydration stress. This ergogenic effect was likely mediated by an enhanced fluid and electrolyte uptake.</p

Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development

Characterization of purinergic receptors and receptor-channels expressed in anterior pituitary cells

Purinergic G protein-coupled receptors (P2YR) and ion-conducting receptor-channels (P2XR) are present in the pituitary. However, their identification, expression within pituitary cell subpopulations, and the ability to elevate intracellular Ca2+ concentration ([Ca2+](i)) in response to ATP stimulation were incompletely characterized. Here we show that mixed populations of rat anterior pituitary cells express messenger RNA transcripts for P2Y2R, P2X(2a)R, P2X(2b)R, P2X3R, P2X4R, and P2X7R. The transcripts and functional P2Y2R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs, and their activation by ATP led to an extracellular Ca2+-independent rise in [Ca2+](i) in about 40% of cells tested. Lactotrophs and GH3 cells, but not somatotrophs, also express transcripts for P2X7R, P2X3R, and P2X4R. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Coexpression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X(2a)R and P2X(2b)R, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. Consistent with the cell-specific expression of transcripts for P2X2R and P2X3R, the expression of their functional heteromers was not evident in pituitary cells. Receptors differed in their capacities to elevate and sustain Ca2+ influx-dependent rise in [Ca2+](i) during the prolonged ATP stimulation. These results indicate that the purinergic system of anterior pituitary is extremely complex and provides an effective mechanism for generating a cell- and receptor-specific Ca2+ signaling pattern in response to a common agonist.link_to_subscribed_fulltex

Signaling by purinergic receptors and channels in the pituitary gland

Adenosine 5′-triphosphate is frequently released by cells and acts as an agonist for G protein-coupled P2Y receptors and ligand-gated P2X cationic channels in numerous tissues. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors. In the pituitary gland, adenosine 5′-triphosphate is released from the endings of magnocellular hypothalamic neurons and by anterior pituitary cells through pathway(s) that are still not well characterized. This gland also expresses several members of each family of purinergic receptors. P2X and adenosine receptors are co-expressed in the somata and nerve terminals of vasopressin-releasing neurons as well as in some secretory pituitary cells. P2X receptors stimulate electrical activity and modulate InsP3-dependent calcium release from intracellular stores, whereas adenosine receptors terminate electrical activity. Calcium-mobilizing P2Y receptors are expressed in pituicytes, folliculo-stellate cells and some secretory cells of the anterior pituitary.link_to_OA_fulltex