ß-endorphin at the intersection of pain and cancer progression: Preclinical evidence.

We examined the association between endogenous opioid ß-endorphin, cancer progression and pain in a transgenic mouse model of breast cancer, with a rat C3(1) simian virus 40 large tumor antigen fusion gene (C3TAg). C3TAg mice develop ductal epithelial atypia at 8 weeks, progression to intra-epithelial neoplasia at 12 weeks, and invasive carcinoma with palpable tumors at 16 weeks. Consistent with invasive carcinoma at 4 months of age, C3TAg mice demonstrate a significant increase in hyperalgesia compared to younger C3TAg or control FVBN mice without tumors. Our data show that the growing tumor contributes to circulating ß-endorphin. As an endogenous ligand of mu opioid receptor, ß-endorphin has analgesic activity. Paradoxically, we observed an increase in pain in transgenic breast cancer mice with significantly high circulating and tumor-associated ß-endorphin. Increased circulating ß-endorphin correlates with increasing tumor burden. ß-endorphin induced the activation of mitogenic and survival-promoting signaling pathways, MAPK/ERK 1/2, STAT3 and Akt, observed by us in human MDA-MB-231 cells suggesting a role for ß-endorphin in breast cancer progression and associated pain.

Heme Causes Pain in Sickle Mice via Toll-Like Receptor 4-Mediated Reactive Oxygen Species- and Endoplasmic Reticulum Stress-Induced Glial Activation.

Aims: Lifelong pain is a hallmark feature of sickle cell disease (SCD). How sickle pathobiology evokes pain remains unknown. We hypothesize that increased cell-free heme due to ongoing hemolysis activates toll-like receptor 4 (TLR4), leading to the formation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Together, these processes lead to spinal microglial activation and neuroinflammation, culminating in acute and chronic pain. Results: Spinal heme levels, TLR4 transcripts, oxidative stress, and ER stress were significantly higher in sickle mice than controls. In vitro, TLR4 inhibition in spinal cord microglial cells attenuated heme-induced ROS and ER stress. Heme treatment led to a time-dependent increase in the characteristic features of sickle pain (mechanical and thermal hyperalgesia) in both sickle and control mice; this effect was absent in TLR4-knockout sickle and control mice. TLR4 deletion in sickle mice attenuated chronic and hypoxia/reoxygenation (H/R)-evoked acute hyperalgesia. Sickle mice treated with the TLR4 inhibitor resatorvid; selective small-molecule inhibitor of TLR4 (TAK242) had significantly reduced chronic hyperalgesia and had less severe H/R-evoked acute pain with quicker recovery. Notably, reducing ER stress with salubrinal ameliorated chronic hyperalgesia in sickle mice. Innovation: Our findings demonstrate the causal role of free heme in the genesis of acute and chronic sickle pain and suggest that TLR4 and/or ER stress are novel therapeutic targets for treating pain in SCD. Conclusion: Heme-induced microglial activation via TLR4 in the central nervous system contributes to the initiation and maintenance of sickle pain via ER stress in SCD. Antioxid. Redox Signal. 34, 279-293.

Spatiotemporal Alterations in Gait in Humanized Transgenic Sickle Mice.

Sickle cell disease (SCD) is a hemoglobinopathy affecting multiple organs and featuring acute and chronic pain. Purkinje cell damage and hyperalgesia have been demonstrated in transgenic sickle mice. Purkinje cells are associated with movement and neural function which may influence pain. We hypothesized that Purkinje cell damage and/or chronic pain burden provoke compensatory gait changes in sickle mice. We found that Purkinje cells undergoe increased apoptosis as shown by caspase-3 activation. Using an automated gait measurement system, MouseWalker, we characterized spatiotemporal gait characteristics of humanized transgenic BERK sickle mice in comparison to control mice. Sickle mice showed alteration in stance instability and dynamic gait parameters (walking speed, stance duration, swing duration and specific swing indices). Differences in stance instability may reflect motor dysfunction due to damaged Purkinje cells. Alterations in diagonal and all stance indices indicative of hesitation during walking may originate from motor dysfunction and/or arise from fear and/or anticipation of movement-evoked pain. We also demonstrate that stance duration, diagonal swing indices and all stance indices correlate with both mechanical and deep tissue hyperalgesia, while stance instability correlates with only deep tissue hyperalgesia. Therefore, objective analysis of gait in SCD may provide insights into neurological impairment and pain states.

Substance P modulates electroacupuncture analgesia in humanized mice with sickle cell disease.

Purpose: Chronic pain is a major comorbidity of sickle cell disease (SCD). Acupuncture, a non-opioid and non-addictive therapy to treat pain, was found to reduce pain in the majority (80%) of SCD patients in an earlier retrospective review. We observed that electroacupuncture (EA) decreased hyperalgesia in transgenic mice with SCD with varied analgesia from high to moderate to no response. Interestingly, poor responders exhibited high levels of substance P (SP), a mediator of chronic pain, as well as active p38 MAPK in spinal cords. The present study aimed to investigate the roles of inhibition of SP and SP-activated p38 MAPK in chronic pain in sickle mice that are poorly responsive to EA intervention (moderate/non-responders). Materials and methods: Humanized mouse model with SCD defined as moderate- and non-responders to EA were intraperitoneally administered with antagonist of SP receptor NK1R (netupitant, 10 mg/kg/day, i.p.) or p38 MAPK inhibitor (SB203580, 10 mg/kg/day, i.p.) alone or in combination with EA (acupoint GB30, every 3rd day until day 12). Hyperalgesia to mechanical, thermal and cold stimuli, as well as deep tissue were measured. Phosphorylated p38 MAPK (phospho-p38 MAPK) in the lumbar spinal cord was quantified using western blotting. Phospho-p38 MAPK nuclear translocation in spinal dorsal horn was examined using immunohistochemical staining and confocal microscopy. Results: In EA poor-responders, combined treatment with EA and netupitant significantly enhanced the analgesic effects of EA in poor-responders on mechanical, heat, cold, and deep tissue pain, and decreased phosphorylation of p38 MAPK in lumbar spinal cords and its nuclear translocation in the spinal dorsal horn. Furthermore, combined treatment with EA and SB203580 significantly improved analgesic effects of EA on mechanical and heat hyperalgesia, but not cold or deep tissue hyperalgesia. However, additional EA treatment only, or administration of either netupitant or SB203580 alone did not lead to analgesic effects. Conclusions: These results suggest a pivotal role of SP in maintaining the chronic pain in SCD via spinal phospho-p38 MAPK signaling, which may hinder the effect of EA in poor responders. Inhibition of SP signaling pathway or activity of p38 MAPK significantly improved the EA analgesia In EA poor-responders with SCD, which provides a promising way to treat the chronic pain in patients with SCD.

Mast Cells Induce Blood Brain Barrier Damage in SCD by Causing Endoplasmic Reticulum Stress in the Endothelium.

Endothelial dysfunction underlies the pathobiology of cerebrovascular disease. Mast cells are located in close proximity to the vasculature, and vasoactive mediators released upon their activation can promote endothelial activation leading to blood brain barrier (BBB) dysfunction. We examined the mechanism of mast cell-induced endothelial activation via endoplasmic reticulum (ER) stress mediated P-selectin expression in a transgenic mouse model of sickle cell disease (SCD), which shows BBB dysfunction. We used mouse brain endothelial cells (mBECs) and mast cells-derived from skin of control and sickle mice to examine the mechanisms involved. Compared to control mouse mast cell conditioned medium (MCCM), mBECs incubated with sickle mouse MCCM showed increased, structural disorganization and swelling of the ER and Golgi, aggregation of ribosomes, ER stress marker proteins, accumulation of galactose-1-phosphate uridyl transferase, mitochondrial dysfunction, reactive oxygen species (ROS) production, P-selectin expression and mBEC permeability. These effects of sickle-MCCM on mBEC were inhibited by Salubrinal, a reducer of ER stress. Histamine levels in the plasma, skin releasate and in mast cells of sickle mice were higher compared to control mice. Compared to control BBB permeability was increased in sickle mice. Treatment of mice with imatinib, Salubrinal, or P-selectin blocking antibody reduced BBB permeability in sickle mice. Mast cells induce endothelial dysfunction via ER stress-mediated P-selectin expression. Mast cell activation contributes to ER stress mediated endothelial P-selectin expression leading to increased endothelial permeability and impairment of BBB. Targeting mast cells and/or ER stress has the potential to ameliorate endothelial dysfunction in SCD and other pathobiologies.

Electroacupuncture in conscious free-moving mice reduces pain by ameliorating peripheral and central nociceptive mechanisms.

Integrative approaches such as electroacupuncture, devoid of drug effects are gaining prominence for treating pain. Understanding the mechanisms of electroacupuncture induced analgesia would benefit chronic pain conditions such as sickle cell disease (SCD), for which patients may require opioid analgesics throughout life. Mouse models are instructive in developing a mechanistic understanding of pain, but the anesthesia/restraint required to administer electroacupuncture may alter the underlying mechanisms. To overcome these limitations, we developed a method to perform electroacupuncture in conscious, freely moving, unrestrained mice. Using this technique we demonstrate a significant analgesic effect in transgenic mouse models of SCD and cancer as well as complete Freund's adjuvant-induced pain. We demonstrate a comprehensive antinociceptive effect on mechanical, cold and deep tissue hyperalagesia in both genders. Interestingly, individual mice showed a variable response to electroacupuncture, categorized into high-, moderate-, and non-responders. Mechanistically, electroacupuncture significantly ameliorated inflammatory and nociceptive mediators both peripherally and centrally in sickle mice correlative to the antinociceptive response. Application of sub-optimal doses of morphine in electroacupuncture-treated moderate-responders produced equivalent antinociception as obtained in high-responders. Electroacupuncture in conscious freely moving mice offers an effective approach to develop a mechanism-based understanding of analgesia devoid of the influence of anesthetics or restraints.

Comparative Analysis of Pain Behaviours in Humanized Mouse Models of Sickle Cell Anemia.

Pain is a hallmark feature of sickle cell anemia (SCA) but management of chronic as well as acute pain remains a major challenge. Mouse models of SCA are essential to examine the mechanisms of pain and develop novel therapeutics. To facilitate this effort, we compared humanized homozygous BERK and Townes sickle mice for the effect of gender and age on pain behaviors. Similar to previously characterized BERK sickle mice, Townes sickle mice show more mechanical, thermal, and deep tissue hyperalgesia with increasing age. Female Townes sickle mice demonstrate more hyperalgesia compared to males similar to that reported for BERK mice and patients with SCA. Mechanical, thermal and deep tissue hyperalgesia increased further after hypoxia/reoxygenation (H/R) treatment in Townes sickle mice. Together, these data show BERK sickle mice exhibit a significantly greater degree of hyperalgesia for all behavioral measures as compared to gender- and age-matched Townes sickle mice. However, the genetically distinct "knock-in" strategy of human α and ß transgene insertion in Townes mice as compared to BERK mice, may provide relative advantage for further genetic manipulations to examine specific mechanisms of pain.

Cannabinoid receptor-specific mechanisms to alleviate pain in sickle cell anemia via inhibition of mast cell activation and neurogenic inflammation.

Sickle cell anemia is a manifestation of a single point mutation in hemoglobin, but inflammation and pain are the insignia of this disease which can start in infancy and continue throughout life. Earlier studies showed that mast cell activation contributes to neurogenic inflammation and pain in sickle mice. Morphine is the common analgesic treatment but also remains a major challenge due to its side effects and ability to activate mast cells. We, therefore, examined cannabinoid receptor-specific mechanisms to mitigate mast cell activation, neurogenic inflammation and hyperalgesia, using HbSS-BERK sickle and cannabinoid receptor-2-deleted sickle mice. We show that cannabinoids mitigate mast cell activation, inflammation and neurogenic inflammation in sickle mice via both cannabinoid receptors 1 and 2. Thus, cannabinoids influence systemic and neural mechanisms, ameliorating the disease pathobiology and hyperalgesia in sickle mice. This study provides 'proof of principle' for the potential of cannabinoid/cannabinoid receptor-based therapeutics to treat several manifestations of sickle cell anemia.

3D Cine Magnetic Resonance Imaging of Rat Lung ARDS using Gradient-modulated SWIFT with Retrospective Respiratory Gating.

SWeep Imaging with Fourier Transformation (SWIFT) with gradient modulation and DC navigator retrospective gating is introduced as a 3D cine magnetic resonance imaging (MRI) method for the lung. The quasi-simultaneous excitation and acquisition in SWIFT enabled extremely high sensitivity to the fast-decaying parenchymal signals (TE=~4 µs), which are invisible with conventional MRI techniques. Based on respiratory motion information extracted from DC navigator signals, the SWIFT data were reconstructed to 3D cine images with 16 respiratory phases. To test the capability of the proposed technique, rats exposed to > 95% O2 for 60 hours for induction of acute respiratory distress syndrome (ARDS), were imaged and compared with normal rat lungs (N=7 and 5 for ARDS and normal group, respectively). SWIFT images showed lung tissue density difference along the gravity direction. In the cine SWIFT images, parenchymal signal drop at the inhalation phase was consistently observed for both normal and ARDS rats due to inflation of the lung (i.e. decrease of the proton density), but the drop was less for ARDS rats. Depending on the respiration phase and lung region, the lungs from the ARDS rats showed 1-24% higher parenchymal signal intensities relative to the normal rat lungs, which would be mainly from accumulation of extravascular water (EVLW). Those results demonstrate that SWIFT has high enough sensitivity for detecting the lung proton density changes due to gravity, different respiration phases and accumulation of EVLW in the rat ARDS lungs.

Src kinase integrates PI3K/Akt and MAPK/ERK1/2 pathways in T3-induced Na-K-ATPase activity in adult rat alveolar cells.

We previously reported that the 3,5,3'-triiodo-L-thyronine (T3)-induced increase of Na-K-ATPase activity in rat alveolar epithelial cells (AECs) required activation of Src kinase, PI3K, and MAPK/ERK1/2. In the present study, we assessed the role of Akt in Na-K-ATPase activity and the interaction between the PI3K and MAPK in response to T3 by using MP48 cells, inhibitors, and constitutively active mutants in the MP48 (alveolar type II-like) cell line. The Akt inhibitor VIII blocked T3-induced increases in Na-K-ATPase activity and amount of plasma membrane Na-K-ATPase protein. The Akt inhibitor VIII also abolished the increase in Na-K-ATPase activity induced by constitutively active mutants of either Src kinase or PI3K. Moreover, constitutively active mutants of Akt increased Na-K-ATPase activity in the absence of T3. Thus activation of Akt was required for T3-induced Na-K-ATPase activity in AECs and is sufficient in the absence of T3. Inhibitors of Src kinase (PP1), PI3K (wortmannin), and ERK1/2 (U0126) all blocked the T3-induced Na-K-ATPase activity. PP1 blocked the activation of PI3K and also ERK1/2 by T3, whereas U0126 did not prevent T3 activation of Src kinase or PI3K activity. Wortmannin did not significantly alter T3-increased MAPK/ERK1/2 activity, suggesting that T3-activated PI3K/Akt and MAPK/ERK1/2 pathways acted downstream of the Src kinase. Furthermore, in the absence of T3, a constitutively active mutant of Src kinase increased activities of Na-K-ATPase, PI3K, and MAPK/ERK1/2. A constitutively active mutant of PI3K enhanced Na-K-ATPase activity but did not alter the MAPK/ERK1/2 activity significantly. In summary, in adult rat AECs T3-stimulated Src kinase activity can activate both PI3K/Akt and MAPK/ERK1/2, and activation of Akt is necessary for T3-induced Na-K-ATPase activity.

Triiodo-L-thyronine rapidly stimulates alveolar fluid clearance in normal and hyperoxia-injured lungs.

RATIONALE: Edema fluid resorption is critical for gas exchange and requires active epithelial ion transport by Na, K-ATPase and other ion transport proteins. OBJECTIVES: In this study, we sought to determine if alveolar fluid clearance (AFC) is stimulated by 3,3',5 triiodo-L-thyronine (T(3)). METHODS: AFC was measured in in situ ventilated lungs and ex vivo isolated lungs by instilling isosmolar 5% bovine serum albumin solution with fluorescein-labeled albumin tracer and measuring the change in fluorescein isothiocyanate-albumin concentration over time. MEASUREMENTS AND MAIN RESULTS: Systemic treatment with intraperitoneal injections of T(3) for 3 consecutive days increased AFC by 52.7% compared with phosphate-buffered saline-injected control rats. Membranes prepared from alveolar epithelial cells from T(3)-treated rats had higher Na, K-ATPase hydrolytic activity. T(3) (10(-6) M), but not reverse T(3) (3,3',5' triiodo-L-thyronine), applied to the alveolar space increased AFC by 31.8% within 1.5 hours. A 61.5% increase in AFC also occurred by airspace instillation of T(3) in ex vivo isolated lungs, suggesting a direct effect of T(3) on the alveolar epithelium. Exposure of rats to an oxygen concentration of greater than 95% for 60 hours increased wet-to-dry lung weights and decreased AFC, whereas the expression of thyroid receptor was not markedly changed. Airspace T(3) rapidly restored the AFC in rat lungs with hyperoxia-induced lung injury. CONCLUSIONS: Airspace T(3) rapidly stimulates AFC by direct effects on the alveolar epithelium in rat lungs with and without lung injury.

T3 increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells.

Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and beta-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase alpha(1)-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.

Thyroid hormone rapidly stimulates alveolar Na,K-ATPase by activation of phosphatidylinositol 3-kinase.

PURPOSE OF REVIEW: Nongenomic actions of 3,3',5-triiodo-L-thyronine (T3) occur quite rapidly usually via activation of signaling cascades. In this review, we focus on recent advances made in the understanding of activation of the phosphatidylinositol 3-kinase pathway by T3 in alveolar epithelial cells, resulting in upregulation of Na,K-ATPase hydrolytic activity and potential physiological significance of this finding. RECENT FINDINGS: T3 stimulates the Src family of kinases. Activation of Src-kinase and phosphatidylinositol 3-kinase/protein kinase B is required for the T3-induced stimulation of alveolar epithelial Na,K-ATPase activity in rat alveolar epithelial cells. The stimulation does not require transcription. This T3-sensitive Na,K-ATPase stimulation in rat alveolar epithelial cells is switched on late in gestation. In skin fibroblasts phosphatidylinositol 3-kinase is also involved in the nongenomic T3 stimulation of ZAK1-4alpha protein expression, an endogenous calcineurin inhibitor. SUMMARY: T3 plays an important role in cell survival and differentiation. Nongenomic regulation of phosphatidylinositol 3-kinase and downstream molecules by T3 is being recognized in different tissues. Upregulation of alveolar Na,K-ATPase is one such molecule, which plays an important role in removal of edema fluid from the alveolar space. These effects are rapid and do not require direct nuclear gene transcription.

Developmental acquisition of T3-sensitive Na-K-ATPase stimulation by rat alveolar epithelial cells.

Late in gestation, the developing air space epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na-K-ATPase acting in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption, and thyroid hormone [triiodothyronine (T3)] stimulates adult alveolar epithelial cell (AEC) Na-K-ATPase. This study explored the developmental regulation of Na-K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) cells. T3 increased Na-K-ATPase activity in primary FDLE cells from gestational day 19 [both primary FDLE cells at embryonic day 19 (E19) and the cell line FD19 derived from FDLE cells at E19]. However, T3 did not increase the Na-K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the cell line FD18 (derived from FDLE cells at E18). Subsequent experiments assessed the T3 signal pathway to define whether it was similar in the late FDLE and adult AEC and to determine the site of the switch in responsiveness to T3. As in adult AEC, in the FD19 cell line, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked the T3-induced increase in Na-K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active mutant of the PI3K catalytic p110 subunit significantly augmented the Na-K-ATPase activity and the cell surface expression of Na-K-ATPase alpha(1) protein. In conclusion, FDLE cells from E17 and E18 lacked T3-sensitive Na-K-ATPase activity but acquired this response at E19. The developmental stimulation of Na-K-ATPase by T3 in rat FDLE cells requires activation of PI3K, and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway.

3,3',5-Triiodo-L-thyronine up-regulation of Na,K-ATPase activity and cell surface expression in alveolar epithelial cells is Src kinase- and phosphoinositide 3-kinase-dependent.

We previously reported that thyroid hormone, 3,3',5-triiodo-l-thyronine (T3), increased Na,K-ATPase activity of adult rat alveolar epithelial cells in a transcription-independent manner via increased cell surface expression of the alpha(1) and beta(1) subunits of Na,K-ATPase. Now we sought to identify signaling molecules necessary for T3 stimulation of Na,K-ATPase activity in alveolar epithelial cells. Whereas protein kinase A inhibitor H-8 and protein kinase C inhibitor bisindolymaleimide did not block the T3-induced increase in Na,K-ATPase activity, two inhibitors of phosphoinositide 3-kinase (PI3K), wortmannin and Ly294002, and two Src kinase inhibitors, PP1 and PP2, blocked the T3-induced Na,K-ATPase activity. T3 stimulated the activity of PI3K as measured by phosphatidylinositol 3-phosphate. T3 also stimulated the serine 473 phosphorylation of the PI3K downstream molecule PKB/Akt in a dose-dependent manner. Transient expression of a constitutively active mutant of the PI3K catalytic subunit p110 augmented Na,K-ATPase activity and increased the amount of cell surface Na,K-ATPase alpha(1) subunit protein. T3 also stimulated Src family kinase activity. Transient expression of a constitutively active Src kinase increased Na,K-ATPase activity, PI3K activity, and phosphorylation of PKB/Akt at serine 473. PP1 or PP2 blocked T3-stimulated PKB/Akt phosphorylation at serine 473 and PI3K activity that was activated by an active mutant of Src; however, wortmannin did not inhibit the T3-stimulated Src kinase activity. Although PP1 and wortmannin abolished the increase in Na,K-ATPase activity induced by the active mutant of Src, PP1 did not inhibit the active mutant of PI3K-up-regulated Na,K-ATPase activity. In summary, T3 stimulates the PI3K/PKB pathway via the Src family of tyrosine kinases, and activation of both the Src family kinases and PI3K is required for the T3-induced stimulation of Na,K-ATPase activity and its cell surface expression in adult rat alveolar epithelial cells.