Tissue injury generates endogenous factors that heighten our sense of pain by increasing the response of sensory nerve endings to noxious stimuli. Bradykinin and nerve growth factor (NGF) are two such pro-algesic agents that activate G-protein-coupled (BK2) and tyrosine kinase (TrkA) receptors, respectively, to stimulate phospholipase C (PLC) signalling pathways in primary afferent neurons. How these actions produce sensitization to physical or chemical stimuli has not been elucidated at the molecular level. Here, we show that bradykinin- or NGF-mediated potentiation of thermal sensitivity in vivo requires expression of VR1, a heat-activated ion channel on sensory neurons. Diminution of plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) levels through antibody sequestration or PLC-mediated hydrolysis mimics the potentiating effects of bradykinin or NGF at the cellular level. Moreover, recruitment of PLC-gamma to TrkA is essential for NGF-mediated potentiation of channel activity, and biochemical studies suggest that VR1 associates with this complex. These studies delineate a biochemical mechanism through which bradykinin and NGF produce hypersensitivity and might explain how the activation of PLC signalling systems regulates other members of the TRP channel family.

While trying to identify new members of the somatostatin receptor family of G protein-coupled receptors, we isolated cDNAs from a mouse brain library encoding two related receptor-like proteins, designated msl-l and msl-2, of 380 and 372 amino acids, respectively. There was 61% identity and 71% similarity between the sequences of msl-1 and msl-2.Among members of the G protein-coupled receptor superfamily, the sequences of both msl-1 and msl-1 were most closely related to those of the somatostatin receptors (SSTRs), having -35% identity with the sequence of SSTR1. Transient expression in COS-1 cells showed that msl-1 and msl-2 did not bind somatostatin. Rather they bound opioids selectively and with high affnity and had the pharmacological properties of K and 8 opioid receptors, respectively. Indeed, the sequence of msl-2 was identical to that of a 8 opioid receptor recently doned by other workers. Functional characterization of K/msl-l and 8/msl-2 opioid receptors showed that they were coupled to G proteins and mediated opioid receptor class-specific agonist inhibition of forskolin-stimulated cAMP formation. RNA blotting studies and in situ hybridization histochemistry showed that K opioid receptor mRNA was expressed at high levels in brain in the neocortex, hippocampus, amygdala, medial habenula, hypothalamus (arcuate and paraventricular nuclei), locus ceruleus, and parabrachial nucleus, suggesting that this receptor may play a role in arousal and regulation ofautonomic and neuroendocrine functions.

Appropriate development of nervous system connectivity involves a variety of processes, including neuronal life-and-death decisions, differentiation, axon guidance and migration, and synaptogenesis. Although these activities likely require specialized signaling events, few substrates unique to these neurotrophic functions have been identified. Here we describe the cloning of ankyrin repeat-rich membrane spanning (ARMS), which encodes a novel downstream target of neurotrophin and ephrin receptor tyrosine kinases, Trk and Eph, respectively. The amino acid sequence of ARMS is highly conserved from nematode to human, suggesting an evolutionarily conserved role for this protein. The ARMS protein consists of 1715 amino acids containing four putative transmembrane domains, multiple ankyrin repeats, a sterile alpha motif domain, and a potential PDZ-binding motif. In the rat, ARMS is specifically expressed in the developing nervous system and in highly plastic areas of the adult brain, regions enriched in Trks and Eph receptors. ARMS can physically associate with TrkA and p75 neurotrophin receptors. Moreover, endogenous ARMS protein is tyrosine phosphorylated after neurotrophin treatment of pheochromocytoma 12 cells and primary hippocampal neurons or ephrin B treatment of NG108-15 cells, demonstrating that ARMS is a downstream target for both neurotrophin and ephrin receptors.

The tetrodotoxin-resistant sodium channel Na(V)1.8/SNS is expressed exclusively in sensory neurons and appears to have an important role in pain pathways. Unlike other sodium channels, Na(V)1.8 is poorly expressed in cell lines even in the presence of accessory beta-subunits. Here we identify annexin II light chain (p11) as a regulatory factor that facilitates the expression of Na(V)1.8. p11 binds directly to the amino terminus of Na(V)1.8 and promotes the translocation of Na(V)1.8 to the plasma membrane, producing functional channels. The endogenous Na(V)1.8 current in sensory neurons is inhibited by antisense downregulation of p11 expression. Because direct association with p11 is required for functional expression of Na(V)1.8, disrupting this interaction may be a useful new approach to downregulating Na(V)1.8 and effecting analgesia.

Background: After heart transplantation, Endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive and its conventional histologic interpretation has limitations. This is a validation study to assesses the performance of a sensitive blood biomarker— percent donor-derived cell-free DNA (%ddcfDNA) — for detection of AR in cardiac transplant recipients. Methods: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data was collected to define AR, its two phenotypes (acute cellular rejection, ACR, and antibody-mediated rejection, AMR) and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range - IQR) for AR, AMR and ACR to controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis. Results: The study included 171 subjects with median post-transplant follow-up of 17.7 months (IQR: 12.1-23.6), with 1,392 EMBx, and 1,834 ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (0.03-0.21) by 28 days. %ddcfDNA increased again with AR compared to controls values (0.38, IQR=0.31-0.83, vs. 0.03, IQR=0.01-0.14 p<0.001). The rise was detected 0.5 and 3.2 months before histopathological diagnosis of ACR and AMR. The area-under-the- receiver-operator characteristics curve (AUROC) for AR was 0.92. A 0.25 %ddcfDNA threshold had a negative predictive value (NPV) for AR of 99% and would have safely eliminated 81% of EMBx. %ddcfDNA showed distinctive characteristics comparing AMR to ACR, included 5-fold higher levels (pAMR ≥2 1.68, IQR=0.49-2.79 vs. ACR grade ≥2R 0.34, IQR=0.28-0.72), higher AUROC (0.95 vs. 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments. Conclusions: %ddcfDNA detected AR with a high AUROC and NPV. Monitoring with ddcfDNA, demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in heart transplant patients and paves the way for a clinical utility study. Clinical Trial Registration: URL: http://clinicaltrials.gov Unique Identifier: NCT02423070

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The mu (mu) opioid receptors, which mediate the effects of morphine, are widely distributed in brain. We have examined the distribution of mRNA encoding a mu opioid receptor in rat brain with in situ hybridization histochemistry at the single-cell level to obtain information about the cell types synthesizing this receptor. Only neurons, not glia, were labeled in discrete brain regions. High levels of labeling were detected in the thalamus, striosomes of the caudate-putamen, globus pallidus, and brain regions involved in nociception, arousal, respiratory control, and, possibly, addiction. The general distribution of the receptor mRNA paralleled that of mu opioid binding sites with some notable exceptions. These include the cerebral cortex, which contains binding sites, but very few labeled neurons. No labeling was observed in the cerebellum, a region devoid of mu binding sites. Three main findings emerged from these experiments: 1) the mRNA was present in regions mediating both the therapeutic (analgesia) and the unwanted (respiratory depression, addiction) effects of morphine, 2) the mRNA was very densely expressed by neurons known to receive dense enkephalin-containing inputs, and 3) the dissociation between the presence of binding sites and absence of mRNA in some brain regions supports a presynaptic localization of mu opioid receptors in these areas. Alternatively, other subtypes of mu opioid receptors may be encoded by a different mRNA. These results provide new insights into the receptor types and neuronal circuits involved in the effects of endogenous opioids and morphine.

INTRODUCTION The clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories. METHODS We conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza. RESULTS We found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist. CONCLUSION cfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19–induced tissue injury. FUNDING Intramural Targeted Anti–COVID-19 grant, NIH.