Hypoxia-inducible factor 1 recruits FACT and RNF20/40 to mediate histone ubiquitination and transcriptional activation of target genes.

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.

Targeting hypoxia-inducible factors with 32-134D safely and effectively treats diabetic eye disease in mice.

Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.

Plexin-B3 expression stimulates MET signaling, breast cancer stem cell specification, and lung metastasis.

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.

NARF is a hypoxia-induced coactivator for OCT4-mediated breast cancer stem cell specification.

Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor (NARF) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.

Disruption of the HIF-1 pathway in individuals with Ollier disease and Maffucci syndrome.

Ollier disease (OD) and Maffucci Syndrome (MS) are rare disorders characterized by multiple enchondromas, commonly causing bone deformities, limb length discrepancies, and pathological fractures. MS is distinguished from OD by the development of vascular anomalies. Both disorders are cancer predisposition syndromes with malignancies developing in ~50% of the individuals with OD or MS. Somatic gain-of-function variants in IDH1 and IDH2 have been described in the enchondromas, vascular anomalies and chondrosarcomas of approximately 80% of the individuals with OD and MS. To date, however, no investigation of germline causative variants for these diseases has been comprehensively performed. To search for germline causative variants, we performed whole exome sequencing or whole genome sequencing of blood or saliva DNA in 94 unrelated probands (68 trios). We found that 7 had rare germline missense variants in HIF1A, 6 had rare germline missense variants in VHL, and 3 had IDH1 variants including 2 with mosaic IDH1-p.Arg132His variant. A burden analysis using 94 probands assigned as cases and 2,054 unrelated individuals presenting no OD- or MS-related features as controls, found that variants in HIF1A, VHL, and IDH1 were all significantly enriched in cases compared to controls. To further investigate the role of HIF-1 pathway in the pathogenesis of OD and MS, we performed RNA sequencing of fibroblasts from 4 probands with OD or MS at normoxia and at hypoxia. When cultured in hypoxic conditions, both proband and control cells showed altered expression of a subset of HIF-1 regulated genes. However, the set of differentially expressed genes in proband fibroblasts included a significantly reduced number of HIF-1 regulated genes compared to controls. Our findings suggest that germline or early post-zygotic variants identified in HIF1A, VHL, and IDH1 in probands with OD and MS underlie the development of the phenotypic abnormalities in a subset of individuals with OD and MS, but extensive functional studies are needed to further confirm it.

HIF inhibitor 32-134D eradicates murine hepatocellular carcinoma in combination with anti-PD1 therapy.

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide and available therapies, including immunotherapies, are ineffective for many patients. HCC is characterized by intratumoral hypoxia, and increased expression of hypoxia-inducible factor 1α (HIF-1α) in diagnostic biopsies is associated with patient mortality. Here we report the development of 32-134D, a low-molecular-weight compound that effectively inhibits gene expression mediated by HIF-1 and HIF-2 in HCC cells, and blocks human and mouse HCC tumor growth. In immunocompetent mice bearing Hepa1-6 HCC tumors, addition of 32-134D to anti-PD1 therapy increased the rate of tumor eradication from 25% to 67%. Treated mice showed no changes in appearance, behavior, body weight, hemoglobin, or hematocrit. Compound 32-134D altered the expression of a large battery of genes encoding proteins that mediate angiogenesis, glycolytic metabolism, and responses to innate and adaptive immunity. This altered gene expression led to significant changes in the tumor immune microenvironment, including a decreased percentage of tumor-associated macrophages and myeloid-derived suppressor cells, which mediate immune evasion, and an increased percentage of CD8+ T cells and natural killer cells, which mediate antitumor immunity. Taken together, these preclinical findings suggest that combining 32-134D with immune checkpoint blockade may represent a breakthrough therapy for HCC.

Histone citrullination by PADI4 is required for HIF-dependent transcriptional responses to hypoxia and tumor vascularization.

Hypoxia-inducible factors (HIFs) activate transcription of target genes by recruiting coactivators and chromatin-modifying enzymes. Peptidylarginine deiminase 4 (PADI4) catalyzes the deimination of histone arginine residues to citrulline. Here, we demonstrate that PADI4 expression is induced by hypoxia in a HIF-dependent manner in breast cancer and hepatocellular carcinoma cells. PADI4, in turn, is recruited by HIFs to hypoxia response elements (HREs) and is required for HIF target gene transcription. Hypoxia induces histone citrullination at HREs that is PADI4 and HIF dependent. RNA sequencing revealed that almost all HIF target genes in breast cancer cells are PADI4 dependent. PADI4 is required for breast and liver tumor growth and angiogenesis in mice. PADI4 expression is correlated with HIF-1α expression and vascularization in human breast cancer biopsies. Thus, HIF-dependent recruitment of PADI4 to target genes and local histone citrullination are required for transcriptional responses to hypoxia.

HIF-1α and HIF-2α redundantly promote retinal neovascularization in patients with ischemic retinal disease.

Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2-specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.

Hypoxia-inducible factor-dependent ADAM12 expression mediates breast cancer invasion and metastasis.

Breast cancer patients with increased expression of hypoxia-inducible factors (HIFs) in primary tumor biopsies are at increased risk of metastasis, which is the major cause of breast cancer-related mortality. The mechanisms by which intratumoral hypoxia and HIFs regulate metastasis are not fully elucidated. In this paper, we report that exposure of human breast cancer cells to hypoxia activates epidermal growth factor receptor (EGFR) signaling that is mediated by the HIF-dependent expression of a disintegrin and metalloprotease 12 (ADAM12), which mediates increased ectodomain shedding of heparin-binding EGF-like growth factor, an EGFR ligand, leading to EGFR-dependent phosphorylation of focal adhesion kinase. Inhibition of ADAM12 expression or activity decreased hypoxia-induced breast cancer cell migration and invasion in vitro, and dramatically impaired lung metastasis after orthotopic implantation of MDA-MB-231 human breast cancer cells into the mammary fat pad of immunodeficient mice.

Autophagic cell death in viral infection: Do TAM receptors play a role?

Containment and clearance of invading pathogens, such as viruses, by suppression of viral replication through antiviral mechanisms (e.g. CRISPR, interferon response or programmed cell death) provide examples of evolutionary developed responses by hosts to limit the establishment of infection. Degradation of the cytoplasm en masse provides an ideal cellular response against intruding pathogens. Degradation of such scale is achieved by a process called (macro)autophagy, where double membrane vacuoles, autophagosomes, engulf cytoplasm and organelles for lysosomal degradation. However, chronic and unrestrained autophagy poses catastrophic consequences to a cell especially when vital organelles (e.g. mitochondria or nucleus) are engulfed and destroyed. Recent findings in the field of autophagy and cell death regulation describe mechanisms that distinguish whether autophagy takes a moderate or excess route. This review aims to present new perspectives and re-examines current assumptions related to cell death regulation by autophagy. The emerging role of TAM receptors in the modulation of autophagy (i.e. both homeostatic and lethal) in the context of virus infections is also discussed in addition to chemical strategies for studying autophagy.

Molecular Characterization of Equilibrative Nucleoside Transporters in the Rat Carotid Body and Their Regulation by Chronic Hypoxia.

The mammalian carotid body (CB) is the main peripheral arterial chemoreceptor organ that is excited by decreases in blood PO2 (hypoxia) and increases in blood PCO2/H+. An increase in CB afferent carotid sinus nerve (CSN) discharge results in respiratory and cardiovascular reflex responses that help maintain homeostasis. The CB consists mainly of innervated clusters of the chemoreceptive type I (glomus) cells that are associated with the processes of glial-like type II cells. Extracellular ATP and adenosine (ADO) levels increase in response to acute hypoxia and there is evidence that during chronic sustained hypoxia ADO elevation plays a major role in regulating CB chemosensitivity and CSN discharge. We recently characterized the molecular identities of ectonucleotidase enzymes involved in regulating extracellular ATP hydrolysis to produce ADO in the rat CB. In the present study, we focus on a molecular characterization of the equilibrative nucleoside transporter (ENT) system that is known to regulate extracellular ADO concentrations in the rat CB based on pharmacological studies. Examination of ENT expression using quantitative PCR (qPCR) analysis revealed the expression of both ENT1 and ENT2 mRNAs in whole CB extracts from ~2-week-old juvenile rats. In dissociated rat CB cultures, both ENT1 and ENT2 immunoreactivity was localized to type I cell clusters. Furthermore, we show that ENT1 and ENT2 mRNA expression is downregulated in CBs isolated from rat pups exposed to chronic hypobaric hypoxia (~1 week). These findings reveal the molecular identities of the ENT system expressed in the rat CB and are consistent with the proposed shift to ADO signaling during chronic hypoxia.

Hypoxia-inducible factor 1-dependent expression of adenosine receptor 2B promotes breast cancer stem cell enrichment.

Breast cancer stem cells (BCSCs), which are characterized by a capacity for unlimited self-renewal and for generation of the bulk cancer cell population, play a critical role in cancer relapse and metastasis. Hypoxia is a common feature of the cancer microenvironment that stimulates the specification and maintenance of BCSCs. In this study, we found that hypoxia increased expression of adenosine receptor 2B (A2BR) in human breast cancer cells through the transcriptional activity of hypoxia-inducible factor 1. The binding of adenosine to A2BR promoted BCSC enrichment by activating protein kinase C-δ, which phosphorylated and activated the transcription factor STAT3, leading to increased expression of interleukin 6 and NANOG, two key mediators of the BCSC phenotype. Genetic or pharmacological inhibition of A2BR expression or activity decreased hypoxia- or adenosine-induced BCSC enrichment in vitro, and dramatically impaired tumor initiation and lung metastasis after implantation of MDA-MB-231 human breast cancer cells into the mammary fat pad of immunodeficient mice. These data provide evidence that targeting A2BR might be an effective strategy to eradicate BCSCs.

Sensory Processing and Integration at the Carotid Body Tripartite Synapse: Neurotransmitter Functions and Effects of Chronic Hypoxia.

Maintenance of homeostasis in the respiratory and cardiovascular systems depends on reflexes that are initiated at specialized peripheral chemoreceptors that sense changes in the chemical composition of arterial blood. In mammals, the bilaterally-paired carotid bodies (CBs) are the main peripheral chemoreceptor organs that are richly vascularized and are strategically located at the carotid bifurcation. The CBs contribute to the maintenance of O2, CO2/H+, and glucose homeostasis and have attracted much clinical interest because hyperactivity in these organs is associated with several pathophysiological conditions including sleep apnea, obstructive lung disease, heart failure, hypertension, and diabetes. In response to a decrease in O2 availability (hypoxia) and elevated CO2/H+ (acid hypercapnia), CB receptor type I (glomus) cells depolarize and release neurotransmitters that stimulate apposed chemoafferent nerve fibers. The central projections of those fibers in turn activate cardiorespiratory centers in the brainstem, leading to an increase in ventilation and sympathetic drive that helps restore blood PO2 and protect vital organs, e.g., the brain. Significant progress has been made in understanding how neurochemicals released from type I cells such as ATP, adenosine, dopamine, 5-HT, ACh, and angiotensin II help shape the CB afferent discharge during both normal and pathophysiological conditions. However, type I cells typically occur in clusters and in addition to their sensory innervation are ensheathed by the processes of neighboring glial-like, sustentacular type II cells. This morphological arrangement is reminiscent of a "tripartite synapse" and emerging evidence suggests that paracrine stimulation of type II cells by a variety of CB neurochemicals may trigger the release of "gliotransmitters" such as ATP via pannexin-1 channels. Further, recent data suggest novel mechanisms by which dopamine, acting via D2 receptors (D2R), may inhibit action potential firing at petrosal nerve endings. This review will update current ideas concerning the presynaptic and postsynaptic mechanisms that underlie chemosensory processing in the CB. Paracrine signaling pathways will be highlighted, and particularly those that allow the glial-like type II cells to participate in the integrated sensory response during exposures to chemostimuli, including acute and chronic hypoxia.

Role of glial-like type II cells as paracrine modulators of carotid body chemoreception.

Mammalian carotid bodies (CB) are chemosensory organs that mediate compensatory cardiorespiratory reflexes in response to low blood PO2 (hypoxemia) and elevated CO2/H+ (acid hypercapnia). The chemoreceptors are glomus or type I cells that occur in clusters enveloped by neighboring glial-like type II cells. During chemoexcitation type I cells depolarize, leading to Ca2+-dependent release of several neurotransmitters, some excitatory and others inhibitory, that help shape the afferent carotid sinus nerve (CSN) discharge. Among the predominantly excitatory neurotransmitters are the purines ATP and adenosine, whereas dopamine (DA) is inhibitory in most species. There is a consensus that ATP and adenosine, acting via postsynaptic ionotropic P2X2/3 receptors and pre- and/or postsynaptic A2 receptors respectively, are major contributors to the increased CSN discharge during chemoexcitation. However, it has been proposed that the CB sensory output is also tuned by paracrine signaling pathways, involving glial-like type II cells. Indeed, type II cells express functional receptors for several excitatory neurochemicals released by type I cells including ATP, 5-HT, ACh, angiotensin II, and endothelin-1. Stimulation of the corresponding G protein-coupled receptors increases intracellular Ca2+, leading to the further release of ATP through pannexin-1 channels. Recent evidence suggests that other CB neurochemicals, e.g., histamine and DA, may actually inhibit Ca2+ signaling in subpopulations of type II cells. Here, we review evidence supporting neurotransmitter-mediated crosstalk between type I and type II cells of the rat CB. We also consider the potential contribution of paracrine signaling and purinergic catabolic pathways to the integrated sensory output of the CB during chemotransduction.

Hypoxia-regulated catecholamine secretion in chromaffin cells.

Adrenal catecholamine (CAT) secretion is a general physiological response of animals to environmental stressors such as hypoxia. This represents an important adaptive mechanism to maintain homeostasis and protect vital organs such as the brain. In adult mammals, CAT secretory responses are triggered by activation of the sympathetic nervous system that supplies cholinergic innervation of adrenomedullary chromaffin cells (AMC) via the splanchnic nerve. In the neonate, the splanchnic innervation of AMC is immature or absent, yet hypoxia stimulates a non-neurogenic CAT secretion that is critical for adaptation to extra-uterine life. This non-neurogenic, hypoxia-sensing mechanism in AMC is gradually lost or suppressed postnatally along a time course that parallels the development of splanchnic innervation. Moreover, denervation of adult AMC results in a gradual return of the direct hypoxia-sensing mechanism. The signaling pathways by which neonatal AMC sense acute hypoxia leading to non-neurogenic CAT secretion and the mechanisms that underlie the re-acquisition of hypoxia-sensing properties by denervated adult AMC, are beginning to be understood. This review will focus on current views concerning the mechanisms responsible for direct acute hypoxia sensing and CAT secretion in perinatal AMC and how they are regulated by innervation during postnatal development. It will also briefly discuss plasticity mechanisms likely to contribute to CAT secretion during exposures to chronic and intermittent hypoxia.

Characterization of ectonucleotidase expression in the rat carotid body: regulation by chronic hypoxia.

The carotid body (CB) chemoreflex maintains blood Po2 and Pco2/H+ homeostasis and displays sensory plasticity during exposure to chronic hypoxia. Purinergic signaling via P1 and P2 receptors plays a pivotal role in shaping the afferent discharge at the sensory synapse containing catecholaminergic chemoreceptor (type I) cells, glial-like type II cells, and sensory (petrosal) nerve endings. However, little is known about the family of ectonucleotidases that control synaptic nucleotide levels. Using quantitative PCR (qPCR), we first compared expression levels of ectonucleoside triphosphate diphosphohydrolases (NTPDases1,2,3,5,6) and ecto-5'-nucleotidase (E5'Nt/CD73) mRNAs in juvenile rat CB vs. brain, petrosal ganglia, sympathetic (superior cervical) ganglia, and a sympathoadrenal chromaffin (MAH) cell line. In whole CB extracts, qPCR revealed a high relative expression of surface-located members NTPDase1,2 and E5'Nt/CD73, compared with low NTPDase3 expression. Immunofluorescence staining of CB sections or dissociated CB cultures localized NTPDase2,3 and E5'Nt/CD73 protein to the periphery of type I clusters, and in association with sensory nerve fibers and/or isolated type II cells. Interestingly, in CBs obtained from rats reared under chronic hypobaric hypoxia (~60 kPa, equivalent to 4,300 m) for 5-7 days, in addition to the expected upregulation of tyrosine hydroxylase and VEGF mRNAs, there was a significant upregulation of NTPDase3 and E5'Nt/CD73 mRNA, but a downregulation of NTPDase1 and NTPDase2 relative to normoxic controls. We conclude that NTPDase1,2,3 and E5'Nt/CD73 are the predominant surface-located ectonucleotidases in the rat CB and suggest that their differential regulation during chronic hypoxia may contribute to CB plasticity via control of synaptic ATP, ADP, and adenosine pools.

Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A.

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K(+) (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved µ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined µ {[d-Arg(2),Ly(4)]dermorphin-(1-4)-amide}- and δ ([d-Pen(2),5,P-Cl-Phe(4)]enkephalin)-opioid agonists (2 µM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.

Ontogeny of O2 and CO2//H+ chemosensitivity in adrenal chromaffin cells: role of innervation.

The adrenal medulla plays a key role in the physiological responses of developing and mature mammals by releasing catecholamines (CAT) during stress. In rodents and humans, the innervation of CAT-producing, adrenomedullary chromaffin cells (AMCs) is immature or absent during early postnatal life, when these cells possess 'direct' hypoxia- and CO2/H(+)-chemosensing mechanisms. During asphyxial stressors at birth, these mechanisms contribute to a CAT surge that is critical for adaptation to extra-uterine life. These direct chemosensing mechanisms regress postnatally, in parallel with maturation of splanchnic innervation. Here, we review the evidence that neurotransmitters released from the splanchnic nerve during innervation activate signaling cascades that ultimately cause regression of direct AMC chemosensitivity to hypoxia and hypercapnia. In particular, we consider the roles of cholinergic and opioid receptor signaling, given that splanchnic nerves release acetylcholine and opiate peptides onto their respective postsynaptic nicotinic and opioid receptors on AMCs. Recent in vivo and in vitro studies in the rat suggest that interactions involving α7 nicotinic acetylcholine receptors (nAChRs), the hypoxia inducible factor (HIF)-2α signaling pathway, protein kinases and ATP-sensitive K(+) (KATP) channels contribute to the selective suppression of hypoxic chemosensitivity. In contrast, interactions involving µ- and/or δ-opiod receptor signaling pathways contribute to the suppression of both hypoxic and hypercapnic chemosensitivity, via regulation of the expression of KATP channels and carbonic anhydrase (CA I and II), respectively. These data suggest that the ontogeny of O2 and CO2/H(+) chemosensitivity in chromaffin cells can be regulated by the tonic release of presynaptic neurotransmitters.

Chronic exposure of neonatal rat adrenomedullary chromaffin cells to opioids in vitro blunts both hypoxia and hypercapnia chemosensitivity.

At birth, rat adrenomedullary chromaffin cells (AMCs) respond directly to asphyxial stressors such as hypoxia and hypercapnia by triggering catecholamine secretion, which is critical for proper transition to extrauterine life. These non-neurogenic responses are suppressed postnatally in parallel with the development of splanchnic innervation, and reappear following denervation of the adult adrenal gland. To test whether neural factors released from the splanchnic nerve may regulate AMC chemosensitivity, we previously showed that nicotinic agonists in utero and in vitro suppressed hypoxia, but not hypercapnia, sensitivity. Here, we considered the potential role of opiate peptides which are also released from the splanchnic nerve and act via postsynaptic µ-, δ- and -opioid receptors. Treatment of neonatal rat AMC cultures for ∼1 week with µ- and/or δ- (but not ) opioid agonists (2 µm) led to a marked suppression of both hypoxia and hypercapnia sensitivity, as measured by K(+) current inhibition and membrane depolarization; co-incubation with naloxone prevented the effects of combined opioids. The suppression of hypoxia sensitivity was attributable to upregulation of K(ATP) current density and the K(ATP) channel subunit Kir6.2, and was reversed by the K(ATP) channel blocker, glibenclamide. By contrast, suppression of hypercapnia sensitivity was associated with down-regulation of two key mediators of CO(2) sensing, i.e. carbonic anhydrase I and II. Collectively, these studies point to a novel role for opioid receptor signalling in the developmental regulation of chromaffin cell chemosensitivity, and suggest that prenatal exposure to opioid drugs could lead to impaired arousal responses in the neonate.

Developmental regulation of glucosensing in rat adrenomedullary chromaffin cells: potential role of the K(ATP) channel.

During birth, when the maternal supply of glucose is occluded, there is a drastic fall in blood glucose in the newborn. This stimulus triggers the non-neurogenic release of catecholamines from adrenomedullary chromaffin cells, which restores blood glucose homeostasis. In this report we present preliminary data showing that glucosensing is present in neonatal chromaffin cells from adrenal slices but absent in chromaffin cells from juvenile slices. Moreover, we show that the aglycemia-evoked rise in intracellular Ca2+ is robust in neonatal chromaffin cells but blunted in juvenile chromaffin cells. Lastly, we show that the Kir6.2 subunit of the KATP channel, is upregulated in the adrenal medulla in juvenile animals providing a potential mechanism for the developmental regulation of glucosensing.