Expression of endogenous epitope-tagged GPR4 in the mouse brain.

GPR4 is a proton-sensing G protein-coupled receptor implicated in many peripheral and central physiological processes. GPR4 expression has previously been assessed only via detection of the cognate transcript or indirectly, by use of fluorescent reporters. In this work, CRISPR/Cas9 knock-in technology was used to encode a hemagglutinin (HA) epitope tag within the endogenous locus of Gpr4 and visualize GPR4-HA in the mouse central nervous system using a specific, well characterized HA antibody; GPR4 expression was further verified by complementary Gpr4 mRNA detection. HA immunoreactivity was found in a limited set of brain regions, including in the retrotrapezoid nucleus (RTN), serotonergic raphe nuclei, medial habenula, lateral septum, and several thalamic nuclei. GPR4 expression was not restricted to cells of a specific neurochemical identity as it was observed in excitatory, inhibitory, and aminergic neuronal cell groups. HA immunoreactivity was not detected in brain vascular endothelium, despite clear expression of Gpr4 mRNA in endothelial cells. In the RTN, GPR4 expression was detected at the soma and in proximal dendrites along blood vessels and the ventral surface of the brainstem; HA immunoreactivity was not detected in RTN projections to two known target regions. This localization of GPR4 protein in mouse brain neurons corroborates putative sites of expression where its function has been previously implicated (e.g., CO2-regulated breathing by RTN), and provides a guide for where GPR4 could contribute to other CO2/H+ modulated brain functions. Finally, GPR4-HA animals provide a useful reagent for further study of GPR4 in other physiological processes outside of the brain.Significance Statement GPR4 is a proton-sensing G-protein coupled receptor whose expression is necessary for a number of diverse physiological processes including acid-base sensing in the kidney, immune function, and cancer progression. In the brain, GPR4 has been implicated in the hypercapnic ventilatory response mediated by brainstem neurons. While knockout studies in animals have clearly demonstrated its necessity for normal physiology, descriptions of GPR4 expression have been limited due to a lack of specific antibodies for use in mouse models. In this paper, we implemented a CRISPR/Cas9 knock-in approach to incorporate the coding sequence for a small epitope tag into the locus of GPR4. Using these mice, we were able to describe GPR4 protein expression directly for the first time.

Liver-Selective Imidazolopyrazine Mitochondrial Uncoupler SHD865 Reverses Adiposity and Glucose Intolerance in Mice.

Excess body fat is a risk factor for metabolic diseases and is a leading preventable cause of morbidity and mortality worldwide. There is a strong need to find new treatments that decrease the burden of obesity and lower the risk of obesity-related comorbidities, including cardiovascular disease and type 2 diabetes. Pharmacologic mitochondrial uncouplers represent a potential treatment for obesity through their ability to increase nutrient oxidation. Herein, we report the in vitro and in vivo characterization of compound SHD865, the first compound to be studied in vivo in a newly discovered class of imidazolopyrazine mitochondrial uncouplers. SHD865 is a derivative of the furazanopyrazine uncoupler BAM15. SHD865 is a milder mitochondrial uncoupler than BAM15 that results in a lower maximal respiration rate. In a mouse model of diet-induced adiposity, 6-week treatment with SHD865 completely restored normal body composition and glucose tolerance to levels like those of chow-fed controls, without altering food intake. SHD865 treatment also corrected liver steatosis and plasma hyperlipidemia to normal levels comparable with chow-fed controls. SHD865 has maximal oral bioavailability in rats and slow clearance in human microsomes and hepatocytes. Collectively, these data identify the potential of imidazolopyrazine mitochondrial uncouplers as drug candidates for the treatment of obesity-related disorders.

Criteria for central respiratory chemoreceptors: experimental evidence supporting current candidate cell groups.

An interoceptive homeostatic system monitors levels of CO2/H+ and provides a proportionate drive to respiratory control networks that adjust lung ventilation to maintain physiologically appropriate levels of CO2 and rapidly regulate tissue acid-base balance. It has long been suspected that the sensory cells responsible for the major CNS contribution to this so-called respiratory CO2/H+ chemoreception are located in the brainstem-but there is still substantial debate in the field as to which specific cells subserve the sensory function. Indeed, at the present time, several cell types have been championed as potential respiratory chemoreceptors, including neurons and astrocytes. In this review, we advance a set of criteria that are necessary and sufficient for definitive acceptance of any cell type as a respiratory chemoreceptor. We examine the extant evidence supporting consideration of the different putative chemoreceptor candidate cell types in the context of these criteria and also note for each where the criteria have not yet been fulfilled. By enumerating these specific criteria we hope to provide a useful heuristic that can be employed both to evaluate the various existing respiratory chemoreceptor candidates, and also to focus effort on specific experimental tests that can satisfy the remaining requirements for definitive acceptance.

The astrocytic Na+ -HCO3 - cotransporter, NBCe1, is dispensable for respiratory chemosensitivity.

The interoceptive homeostatic mechanism that controls breathing, blood gases and acid-base balance in response to changes in CO2 /H+ is exquisitely sensitive, with convergent roles proposed for chemosensory brainstem neurons in the retrotrapezoid nucleus (RTN) and their supporting glial cells. For astrocytes, a central role for NBCe1, a Na+ -HCO3 - cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO2 -induced local extracellular acidification or purinergic signalling). We tested these NBCe1-centric models by using conditional knockout mice in which Slc4a4 was deleted from astrocytes. In GFAP-Cre;Slc4a4fl/fl mice we found diminished expression of Slc4a4 in RTN astrocytes by comparison to control littermates, and a concomitant reduction in NBCe1-mediated current. Despite disrupted NBCe1 function in RTN-adjacent astrocytes from these conditional knockout mice, CO2 -induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO2 -stimulated breathing, were indistinguishable from NBCe1-intact littermates; hypoxia-stimulated breathing and sighs were likewise unaffected. We obtained a more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen-treated Aldh1l1-Cre/ERT2;Slc4a4fl/fl mice. Again, there was no difference in effects of CO2 or hypoxia on breathing or on neuron/astrocyte activation in NBCe1-deleted mice. These data indicate that astrocytic NBCe1 is not required for the respiratory responses to these chemoreceptor stimuli in mice, and that any physiologically relevant astrocytic contributions must involve NBCe1-independent mechanisms. KEY POINTS: The electrogenic NBCe1 transporter is proposed to mediate local astrocytic CO2 /H+ sensing that enables excitatory modulation of nearby retrotrapezoid nucleus (RTN) neurons to support chemosensory control of breathing. We used two different Cre mouse lines for cell-specific and/or temporally regulated deletion of the NBCe1 gene (Slc4a4) in astrocytes to test this hypothesis. In both mouse lines, Slc4a4 was depleted from RTN-associated astrocytes but CO2 -induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact. Likewise, respiratory chemoreflexes evoked by changes in CO2 or O2 were unaffected by loss of astrocytic Slc4a4. These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes.

Neuromedin B-Expressing Neurons in the Retrotrapezoid Nucleus Regulate Respiratory Homeostasis and Promote Stable Breathing in Adult Mice.

Respiratory chemoreceptor activity encoding arterial Pco2 and Po2 is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomic evidence suggests that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN Nmb neurons are necessary for the CO2-dependent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN Nmb neurons causes compensated respiratory acidosis because of alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN Nmb neurons. Interestingly, ventilation following RTN Nmb -lesion was unresponsive to hypercapnia, but behavioral responses to CO2 (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests that RTN Nmb neurons are dedicated to the respiratory effects of arterial Pco2/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.SIGNIFICANCE STATEMENT Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial Pco2 and Po2, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO2 on breathing. Our functional and anatomic data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO2-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO2- and O2-sensing mechanisms in respiratory homeostasis of mammals.

Central respiratory chemoreception.

Brain PCO2 is sensed primarily via changes in [H+]. Small pH changes are detected in the medulla oblongata and trigger breathing adjustments that help maintain arterial PCO2 constant. Larger perturbations of brain CO2/H+, possibly also sensed elsewhere in the CNS, elicit arousal, dyspnea, and stress, and cause additional breathing modifications. The retrotrapezoid nucleus (RTN), a rostral medullary cluster of glutamatergic neurons identified by coexpression of Phoxb and Nmb transcripts, is the lynchpin of the central respiratory chemoreflex. RTN regulates breathing frequency, inspiratory amplitude, and active expiration. It is exquisitely responsive to acidosis in vivo and maintains breathing autorhythmicity during quiet waking, slow-wave sleep, and anesthesia. The RTN response to [H+] is partly an intrinsic neuronal property mediated by proton sensors TASK-2 and GPR4 and partly a paracrine effect mediated by astrocytes and the vasculature. The RTN also receives myriad excitatory or inhibitory synaptic inputs including from [H+]-responsive neurons (e.g., serotonergic). RTN is silenced by moderate hypoxia. RTN inactivity (periodic or sustained) contributes to periodic breathing and, likely, to central sleep apnea. RTN development relies on transcription factors Egr2, Phox2b, Lbx1, and Atoh1. PHOX2B mutations cause congenital central hypoventilation syndrome; they impair RTN development and consequently the central respiratory chemoreflex.

5-HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity.

A brainstem homeostatic system senses CO2 /H+ to regulate ventilation, blood gases and acid-base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO2 /H+ sensitivity of RTN neurons is mediated indirectly, by raphe-derived serotonin acting on 5-HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT-PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb+ /Phox2b+ ) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These dorsolateral Nmb+ /Htr7+ neurons tended to express Nmb at high levels and the intrinsic RTN proton detectors Gpr4 and Kcnk5 at low levels. In mouse brainstem slices, CO2 -stimulated firing in RTN neurons was mostly unaffected by a 5-HT7 receptor antagonist, SB269970 (n = 11/13). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co-injected LP-44, a 5-HT7 receptor agonist, but had no effect on CO2 -stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5-HT7 receptors have negligible effects on CO2 -evoked firing activity in RTN neurons or on CO2 -stimulated breathing in mice. KEY POINTS: Neurons of the retrotrapezoid nucleus (RTN) are intrinsic CO2 /H+ chemosensors and serve as an integrative excitatory hub for control of breathing. Serotonin can activate RTN neurons, in part via 5-HT7 receptors, and those effects have been implicated in conferring an indirect CO2  sensitivity. Multiple single cell molecular approaches revealed low levels of 5-HT7 receptor transcript expression restricted to a limited population of RTN neurons. Pharmacological experiments showed that 5-HT7 receptors in RTN are not required for CO2 /H+ -stimulation of RTN neuronal activity or CO2 -stimulated breathing. These data do not support a role for 5-HT7 receptors in respiratory chemosensitivity mediated by RTN neurons.

TRPM4 Contributes to Subthreshold Membrane Potential Oscillations in Multiple Mouse Pacemaker Neurons.

Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons of the locus coeruleus (LC), circadian neurons of the suprachiasmatic nucleus (SCN), and CO2/H+-activated neurons of the brainstem retrotrapezoid nucleus (RTN), large subthreshold membrane potential oscillations contribute to the pacemaker-like action potential discharge. The oscillations and firing in LC and SCN involve contributions from leak sodium (NALCN) and L-type calcium channels while recent work from RTN suggested an additional pivotal role for a secondary calcium-activated and voltage-gated cationic current sensitive to TRPM4 channel blockers. Here, we tested whether TRPM4 contributes to subthreshold oscillations in mouse LC and SCN. By RNAscope in situ hybridization, Trpm4 transcripts were detected in both cell groups. In whole-cell recordings from acute slice preparations, prominent voltage-dependent membrane potential oscillations were revealed in LC and SCN after blocking action potentials. These oscillations were inhibited by two chemically-distinct blockers of TRPM4, 9-phenanthrol (9-pt) and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). Under whole-cell voltage clamp, inward currents evoked by oscillation voltage waveforms were inhibited in LC by blocking L-type calcium channels and TRPM4. These data implicate TRPM4 in the large subthreshold membrane potential oscillations that underlie tonic action potential discharge in LC and SCN, providing a voltage-dependent and calcium-dependent cationic current to augment the depolarizing inward Na+ and Ca2+ currents previously associated with this distinctive electroresponsive property.

Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation.

Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.

Deacetylation as a receptor-regulated direct activation switch for pannexin channels.

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.

TRPM4 mediates a subthreshold membrane potential oscillation in respiratory chemoreceptor neurons that drives pacemaker firing and breathing.

Brainstem networks that control regular tidal breathing depend on excitatory drive, including from tonically active, CO2/H+-sensitive neurons of the retrotrapezoid nucleus (RTN). Here, we examine intrinsic ionic mechanisms underlying the metronomic firing activity characteristic of RTN neurons. In mouse brainstem slices, large-amplitude membrane potential oscillations are evident in synaptically isolated RTN neurons after blocking action potentials. The voltage-dependent oscillations are abolished by sodium replacement; blocking calcium channels (primarily L-type); chelating intracellular Ca2+; and inhibiting TRPM4, a Ca2+-dependent cationic channel. Likewise, oscillation voltage waveform currents are sensitive to calcium and TRPM4 channel blockers. Extracellular acidification and serotonin (5-HT) evoke membrane depolarization that augments TRPM4-dependent oscillatory activity and action potential discharge. Finally, inhibition of TRPM4 channels in the RTN of anesthetized mice reduces central respiratory output. These data implicate TRPM4 in a subthreshold oscillation that supports the pacemaker-like firing of RTN neurons required for basal, CO2-stimulated, and state-dependent breathing.

ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels.

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

A brainstem peptide system activated at birth protects postnatal breathing.

Among numerous challenges encountered at the beginning of extrauterine life, the most celebrated is the first breath that initiates a life-sustaining motor activity1. The neural systems that regulate breathing are fragile early in development, and it is not clear how they adjust to support breathing at birth. Here we identify a neuropeptide system that becomes activated immediately after birth and supports breathing. Mice that lack PACAP selectively in neurons of the retrotrapezoid nucleus (RTN) displayed increased apnoeas and blunted CO2-stimulated breathing; re-expression of PACAP in RTN neurons corrected these breathing deficits. Deletion of the PACAP receptor PAC1 from the pre-Bötzinger complex-an RTN target region responsible for generating the respiratory rhythm-phenocopied the breathing deficits observed after RTN deletion of PACAP, and suppressed PACAP-evoked respiratory stimulation in the pre-Bötzinger complex. Notably, a postnatal burst of PACAP expression occurred in RTN neurons precisely at the time of birth, coinciding with exposure to the external environment. Neonatal mice with deletion of PACAP in RTN neurons displayed increased apnoeas that were further exacerbated by changes in ambient temperature. Our findings demonstrate that well-timed PACAP expression by RTN neurons provides an important supplementary respiratory drive immediately after birth and reveal key molecular components of a peptidergic neural circuit that supports breathing at a particularly vulnerable period in life.

Ion Channel Function and Electrical Excitability in the Zona Glomerulosa: A Network Perspective on Aldosterone Regulation.

Aldosterone excess is a pathogenic factor in many hypertensive disorders. The discovery of numerous somatic and germline mutations in ion channels in primary hyperaldosteronism underscores the importance of plasma membrane conductances in determining the activation state of zona glomerulosa (zG) cells. Electrophysiological recordings describe an electrically quiescent behavior for dispersed zG cells. Yet, emerging data indicate that in native rosette structures in situ, zG cells are electrically excitable, generating slow periodic voltage spikes and coordinated bursts of Ca2+ oscillations. We revisit data to understand how a multitude of conductances may underlie voltage/Ca2+ oscillations, recognizing that zG layer self-renewal and cell heterogeneity may complicate this task. We review recent data to understand rosette architecture and apply maxims derived from computational network modeling to understand rosette function. The challenge going forward is to uncover how the rosette orchestrates the behavior of a functional network of conditional oscillators to control zG layer performance and aldosterone secretion.

Pannexin 1 channels in renin-expressing cells influence renin secretion and blood pressure homeostasis.

Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear. Pannexin 1 (Panx1) channels localize to both afferent arterioles and juxtaglomerular cells and provide a transmembrane conduit for ATP release and ion permeability in the kidney and the vasculature. We hypothesized that Panx1 channels in renin-expressing cells regulate renin secretion in vivo. Using a renin cell-specific Panx1 knockout model, we found that male Panx1 deficient mice exhibiting a heightened activation of the renin-angiotensin-aldosterone system have markedly increased plasma renin and aldosterone concentrations, and elevated mean arterial pressure with altered peripheral hemodynamics. Following ovariectomy, female mice mirrored the male phenotype. Furthermore, constitutive Panx1 channel activity was observed in As4.1 renin-secreting cells, whereby Panx1 knockdown reduced extracellular ATP accumulation, lowered basal intracellular calcium concentrations and recapitulated a hyper-secretory renin phenotype. Moreover, in response to stress stimuli that lower blood pressure, Panx1-deficient mice exhibited aberrant "renin recruitment" as evidenced by reactivation of renin expression in pre-glomerular arteriolar smooth muscle cells. Thus, renin-cell Panx1 channels suppress renin secretion and influence adaptive renin responses when blood pressure homeostasis is threatened.

The Retrotrapezoid Nucleus: Central Chemoreceptor and Regulator of Breathing Automaticity.

The ventral surface of the rostral medulla oblongata has been suspected since the 1960s to harbor central respiratory chemoreceptors [i.e., acid-activated neurons that regulate breathing to maintain a constant arterial PCO2 (PaCO2)]. The key neurons, a.k.a. the retrotrapezoid nucleus (RTN), have now been identified. In this review we describe their transcriptome, developmental lineage, and anatomical projections. We also review their contribution to CO2 homeostasis and to the regulation of breathing automaticity during sleep and wake. Finally, we discuss several mechanisms that contribute to the activation of RTN neurons by CO2in vivo: cell-autonomous effects of protons; paracrine effects of pH mediated by surrounding astrocytes and blood vessels; and excitatory inputs from other CO2-responsive CNS neurons.

Adrenal Tissue-Specific Deletion of TASK Channels Causes Aldosterone-Driven Angiotensin II-Independent Hypertension.

The renin-angiotensin system tightly controls aldosterone synthesis. Dysregulation is evident in hypertension (primary aldosteronism), low renin, and resistant hypertension) but also can exist in normotension. Whether chronic, mild aldosterone autonomy can elicit hypertension remains untested. Previously, we reported that global genetic deletion of 2 pore-domain TWIK-relative acid-sensitive potassium channels, TASK-1 and TASK-3, from mice produces striking aldosterone excess, low renin, and hypertension. Here, we deleted TASK-1 and TASK-3 channels selectively from zona glomerulosa cells and generated a model of mild aldosterone autonomy with attendant hypertension that is aldosterone-driven and Ang II (angiotensin II)-independent. This study shows that a zona glomerulosa-specific channel defect can produce mild autonomous hyperaldosteronism sufficient to cause chronic blood pressure elevation.

Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-CreERT2+/Panx1fl/fl mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.