Function (Oxford, England)最新文献

We recently reported¹¹ that fentanyl activates carotid body (CB) afferents via kappa opioid receptors (KOR), while CB denervation exacerbates, co-administration of fentanyl with a KOR agonist attenuates opioid-induced respiratory depression (OIRD). These findings indicated that CB chemoreflex activation by fentanyl may counteract OIRD. The present study investigated the cellular mechanisms underlying CB afferent activation by fentanyl. We hypothesized that Ca²⁺ signaling in glomus cells mediates CB activation by fentanyl. Using Fura-2 calcium imaging in rat glomus cells, we observed that fentanyl increased intracellular Ca²⁺ even in the absence of extracellular calcium. Pretreatment with thapsigargin, which depletes internal Ca²⁺ stores, abolished Ca2+ response, suggesting that fentanyl releases Ca²⁺ from intracellular stores. In HEK cells expressing KOR and Gαq, fentanyl promoted KOR-Gαq complex formation and stimulated phospholipase C (PLC), elevating inositol trisphosphate (IP₃) levels in the CB. Pharmacological blockade of KOR, Gαq, PLC, or IP₃ receptors prevented both the rise in [Ca²⁺]ᵢ and CB afferent activation. Collectively, these results identify a previously uncharacterized KOR-Gαq-PLC- IP₃R-Ca²⁺ signaling pathway in glomus cells that mediates CB afferent activation by fentanyl, providing new mechanistic insight into how CB chemoreflex activation by fentanyl may mitigate OIRD.

Projections from the paraventricular nucleus of the hypothalamus (PVN) to the nucleus of the solitary tract (nTS) facilitate the peripheral chemoreflex. A significant proportion of this projection is comprised of corticotropin-releasing hormone (CRH) neurons. Orexin neurons in the perifornical hypothalamus augment the peripheral chemoreflex, project to the PVN, and facilitate the hypoxia-induced activation of nTS-projecting CRH neurons. We hypothesized that nTS- projecting CRH neurons are necessary for the full reflex, and that orexin facilitates the reflex via the CRH-nTS pathway. We chemogenetically silenced or activated nTS-projecting CRH neurons during normoxia and acute hypoxia. For each rat, reflex strength was tested in both inactive and active phases as the activity of orexin neurons is phase dependent. Testing was done following vehicle, Compound 21 (1 mg/kg) to activate Gi- or Gq-DREADDs, and after systemic Ox1R blockade (SB-334867;1 mg/kg). We performed immunohistochemistry to assess how chemogenetic manipulation of nTS-projecting CRH neurons influenced their activation by hypoxia (via cFos). Activating the CRH-nTS pathway had no effect on the chemoreflex in either phase. Silencing the pathway in the active phase, but not inactive phase, reduced the strength of the reflex by ~50% and prevented further inhibition by Ox1R blockade, suggesting orexin acts via Ox1R on CRH neurons. Pathway silencing reduced the proportion of nTS-projecting CRH neurons activated by hypoxia, consistent with the effects of pathway silencing on the reflex. These data suggest that orexin augments the peripheral chemoreflex in the active phase via the CRH- nTS pathway.

The quantity of physiological data has grown exponentially, yielding insights into mechanisms of phenotypic and disease pathways. Among the powerful tools for physiological omics is the study of RNA, where broad sequencing of RNA leads to hypothesis generation and testing while providing observational discovery. Emphasis has been placed on RNA molecules that code for proteins, even though they represent a minority of total RNA. Diverse sequencing methods have rapidly expanded the identification of non-protein-coding molecules, including nonsensemediated decay (NMD) and long non-coding RNAs (lncRNA), which now represent the most diverse class of RNA. Increasing attention needs to be paid to the data processing of RNA sequencing to interpret transcript-level mapping data in the context of protein biology, as many protein-coding genes have diverse noncoding transcripts. Over the past several years, single-cell and spatial transcriptomics have yielded unprecedented insights into cellular, tissue, and organ physiology. Building on these advancements, bulk RNA sequencing tools have begun producing robust deconvolution methods that enhance the analysis of human genes, the detection of foreign RNA from bacteria and viruses, and provide deep insights into complex immunological events, such as B- and T-cell recombination. Over a million RNA sequencing datasets have been generated, providing resources for data scientists to reprocess data and expand larger databases. From model organisms to complex human diseases, RNA sequencing resources continue to transform our knowledge of the complexity of personalized disease insights. Observational science is at the core of physiology, and growth of RNA sequencing represents a significant tool for physiologists.

Baroreflex responsiveness and orthostatic stability in humans can be assessed by a variety of approaches including exposure to graded levels of lower body negative pressure (LBNP). However, such approaches have limited applicability in animal studies owing to the need to anesthetize or sedate the animal. We recently reported a novel approach for the assessment of baroreceptor responsiveness in the awake rat using LBNP, and presented preliminary findings that 3% isoflurane anesthesia completely blocked the normally robust baroreflex. In the present study, we sought to extend these findings by studying the effects of several common anesthetics on LBNP responsiveness. Blood pressure (BP) and heart rate (HR) responses to progressive levels of LBNP were first made in awake rats (male and female), followed by measurements under various anesthetics regimens: 1) pentobarbital; 2) ketamine plus xylazine; 3) isoflurane at 3%, 2%, and 1.5%; 4) urethane delivered as an i.p. bolus, slow i.p. infusion, and slow i.v. infusion. As previously reported, BP in awake rats was well maintained up to -15 mmHg LBNP, accompanied by a robust baroreflex tachycardia. Despite varying effects on steady-state BP and HR, all of the anesthetics tested severely or completely blocked the ability to maintain BP during LBNP and completely blocked reflex tachycardia. BP, but not reflex tachycardia, during LBNP was partially preserved only in those rats treated with i.v. urethane. These data confirm that the functional baroreflexes that normally maintain BP during orthostatic challenge are blocked by commonly used anesthetics.

Inflammation is a critical immune response to tissue injury or infection, involving a cascade of molecular and cellular events. This review examines acute inflammation, focusing on the key receptors, signaling pathways, mediators, and cellular players involved in the response throughout the body. The latter part of the review narrows its focus to kidney inflammation, a vital organ often affected by both sterile and non-sterile insults. By exploring the roles of immune and non-immune cells, this review highlights general inflammatory mechanisms and their impact on kidney-specific pathophysiology.

Kidneys are central in maintaining acid-base homeostasis by recovering filtered bicarbonate (HCO3-) in the proximal tubule and by secreting H+ in the collecting duct. Here, we demonstrate a critical role of the exchange protein directly activated by cAMP (Epac) signaling, and particularly the Epac2, in governing renal adaptation to dietary acid load. RNAseq analysis of the renal cortical area revealed that Epac1&2 deficiency was associated with changes in gene profile seen in acidosis. Renal expression of Epac2 but not Epac1 was enhanced by acid load. Epac2-/- mice developed a pronounced metabolic acidosis due to the inability to acidify urine in response to dietary acid load. Deletion of Epac2 and Epac1 exerted additive inhibitory actions on expression of the Na+/H+ exchanger (NHE-3, Slc9a3) in the proximal tubule. Using super-resolution STED microscopy, we detected NHE-3 redistribution to the base of the brush border, which led to the impaired recovery after acidification in freshly isolated split-opened proximal tubules from Epac1&2-/- mice. Deletion of Epac2 but not Epac1 diminished H+ secretion in freshly isolated split-opened collecting ducts, compromised apical translocation of V-ATPase, and reduced anion exchanger 1 (AE1, Slc4a1) expression in the A-type intercalated cells, and caused lower levels of titratable acids in urine, whereas ammoniagenesis was not compromised. Overall, we demonstrate a previously unrecognized role of Epac signaling in renal adaptation to dietary acidification. While both Epac1 and Epac2 isoforms control NHE-3-dependent H+ secretion in the proximal tubule, only Epac2 is essential to augment H+ transport in the collecting duct to acidify urine.

Our bone health as an adult is defined by patterns of development in early life, with perturbed growth during fetal and neonatal periods predisposing individuals to poor bone health in adulthood. Studies have identified poor maternal diet during pregnancy as a critical factor in shaping offspring bone development, with significant impacts on adult bone structure and health. However, the association between a father's diet and the bone health of his offspring remains poorly defined. To address this knowledge gap, we fed male C57BL/6 mice either a control normal protein diet (NPD; 18% protein) or an isocaloric low-protein diet (LPD; 9% protein) for a minimum of 8 wk. Using these males, we generated offspring through artificial insemination, in combination with vasectomized male mating. Using this approach, we derived offspring from either NPD or LPD sperm but in the presence of NPD or LPD seminal plasma. Using micro-computed tomography and synchrotron X-ray diffraction, we observed significant changes in offspring femur morphology and hydroxyapatite crystallographic parameters from just 3 wk of age in offspring derived from LPD sperm or seminal plasma. We also observed that differential femur morphology and hydroxyapatite crystallographic parameters were maintained into adulthood and into a second generation. Analysis of paternal sperm identified a down regulation of 26 osteogenic genes associated with extracellular matrix levels and maintenance, transcription and growth factors, and bone ossification. These observations indicate that poor paternal diet at the time of conception affects offspring bone development and morphology in an age and generation specific manner.

The extent of walking impairment varies among individuals with peripheral artery disease (PAD), which may reflect differences in the adaptability of lower extremity muscles to ischemia-reperfusion injury characteristic of the disease. Analyses of gastrocnemius muscle biopsies from 113 individuals with PAD [mean ankle-brachial index (ABI) = 0.65 ± 0.13, 38 (33.6%) women, 76 (67.2%) Black] showed a wide range of myofiber type distributions (9.6%-82.6% type 1 myofibers). The abundance of oxidative type 1 myofibers negatively correlated with ABI (r = -0.22, P = 0.02), a measure of PAD severity. The abundance of type 1 myofibers also negatively correlated to 2a/x myofiber abundance (r = -0.76, P < 0.001). Eighty % of participants had NCAM+ myofibers, a potential indicator of myofiber denervation. Overall, 3.2% of total myofibers were NCAM+. Of 113 muscle biopsies, 86 (76.1%) contained type 1 myofibers with regions lacking intermyofibrillar mitochondria (IMFM-), which may represent formation of target myofibers. In type 1 myofiber IMFM- areas, 77.8% contained 2x myosin heavy chain and/or the autophagy marker LC3. Electron microscopy within one muscle with IMFM- myofibers confirmed sarcomere disruption in IMFM- regions. These analyses support the possibility of type 2 myofibers transitioning to type 1 in PAD and suggest IMFM- target fibers may represent visualization of this process for the first time. Because type 1 myofibers are more resistant to oxidative damage, results suggest the possibility that a higher proportion of type 1 myofibers in PAD with increasing disease severity may be a compensatory mechanism to maintain muscle.