Opioids are currently the primary treatment method used to manage both acute and chronic pain. In the past two to three decades, there has been a surge in the use, abuse and misuse of opioids. The mechanism by which opioids relieve pain and induce euphoria is dependent on the drug crossing the blood–brain barrier and accessing the central nervous system. This suggests the blood brain barrier plays a central role in both the benefits and risks of opioid use. The complex physiological responses to opioids that provide the benefits and drive the abuse also needs to be considered in the resolution of the opioid epidemic.
P-glycoprotein (PgP), a drug efflux pump in blood-brain barrier endothelial cells, is a major clinical obstacle for effective central nervous system drug delivery. Identifying PgP regulatory pathways that can be exploited clinically is critical for improving central nervous system drug delivery. We previously found that PgP activity increases in rat brain microvessels concomitant with decreased central nervous system drug delivery in response to acute peripheral inflammatory pain. In the current study, we tested the hypothesis that PgP traffics to the luminal plasma membrane of the microvessel endothelial cells from intracellular stores during peripheral inflammatory pain. Using immunofluorescence microscopy, we detected PgP in endothelial cell nuclei and in the luminal plasma membrane in control animals. Following peripheral inflammatory pain, luminal PgP staining increased while staining in the nucleus decreased. Biochemical analysis of nuclear PgP content confirmed our visual observations. Peripheral inflammatory pain also increased endothelial cell luminal staining of polymerase 1 and transcript release factor/cavin1 and serum deprivation response protein/cavin2, two caveolar scaffold proteins, without changing caveolin1 or protein kinase C delta binding protein/cavin3 location. Our data (a) indicate that PgP traffics from stores in the nucleus to the endothelial cell luminal membrane in response to peripheral inflammatory pain; (b) provide an explanation for our previous observation that peripheral inflammatory pain inhibits central nervous system drug uptake; and (c) suggest a novel regulatory mechanism for PgP activity in rat brain. KeywordsCaveolin1, P-glycoprotein, protein kinase C delta binding protein/cavin3, polymerase 1 and transcript release factor/ cavin1, serum deprivation response protein/cavin2, trafficking
Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1 and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.
The rates of opioid prescription and use have continued to increase over the last few decades resulting in a greater number of opioid tolerant patients. Treatment of acute pain from surgery and injury is a clinical challenge for these patients. Several pain management strategies including prescribing increased opioids are used clinically with limited success; all currently available strategies have significant limitations. Many opioids are a substrate for p-glycoprotein (p-gp), an efflux transporter at the blood-brain barrier (BBB). Increased p-gp is associated with a decreased central nervous system uptake and analgesic efficacy of morphine. Our laboratory previously found that acute peripheral inflammatory pain (PIP) induces p-gp trafficking from the nucleus to the luminal surface of endothelial cells making up the BBB concomitant with increased p-gp activity and decreased morphine analgesic efficacy. In the current study, we tested whether PIP-induced p-gp trafficking could contribute to decreased opioid efficacy in morphine tolerant rats. A 6-day continuous dosing of morphine from osmotic minipumps was used to establish morphine tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase in morphine tolerant rats. This observation suggests that p-gp trafficking contributes to the decreased morphine analgesic effects in morphine tolerant rats experiencing an acute pain stimulus. Attenuating p-gp trafficking during an acute pain stimulus could improve pain management by increasing the amount of opioid that could reach CNS analgesic targets and decrease the need for the dose escalation that is a serious challenge in pain management.
Deep breaths are one of three breathing patterns in rodents characterized by an increased tidal volume. While humans incorporate deep breaths into vocal behavior, it was unknown whether nonhuman mammals use deep breaths for vocal production. We have utilized subglottal pressure recordings in awake, spontaneously behaving male Sprague-Dawley rats in five contexts: sleep, rest, noxious stimulation, exposure to a female in estrus, and exposure to an unknown male. Deep breaths were produced at rates ranging between 17.5 and 90.3 deep breaths per hour. While overall breathing and vocal rates were higher in social and noxious contexts, the rate of deep breaths was only increased during the male’s interaction with a female. Results also inform our understanding of vocal-respiratory integration in rats. The rate of deep breaths that were associated with a vocalization during the exhalation phase increased with vocal activity. The proportion of deep breaths that were associated with a vocalization (on average 22%) was similar to the proportion of sniffing or eupnea breaths that contain a vocalization. Therefore, vocal motor patterns appear to be entrained to the prevailing breathing rhythm, i.e., vocalization uses the available breathing pattern rather than recruiting a specific breathing pattern. Furthermore, the pattern of a deep breath was different when it was associated with a vocalization, suggesting that motor planning occurs. Finally, deep breaths are a source for acoustic variation; for example, call duration and fundamental frequency modulation were both larger in 22-kHz calls produced following a deep inhalation. NEW & NOTEWORTHY The emission of a long, deep, audible breath can express various emotions. The investigation of deep breaths, also known as sighing, in a nonhuman mammal demonstrated the occasional use of deep breaths for vocal production. Similar to the human equivalent, acoustic features of a deep breath vocalization are characteristic.
P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.
Rocky Mountain spotted fever (RMSF) is a rapidly‐progressing, tick‐borne, zoonotic disease caused by the bacterium Rickettsia rickettsii. Most of the symptoms of RMSF are flu‐like in nature; including fever, nausea, fatigue, and muscle weakness. The one non‐specific symptom is the presence of petechiae, but by the time they appear it is often too late. Even with treatment, RMSF can have a fatality rate of up to 35% and if left untreated it can rise to as high as 80%. In 2003, a new vector for RMSF was identified; Rhipicephalus sanguineus, or the brown dog tick. Unlike its Dermacentor counterparts, R. sanguineus is found nationally, yet is only known to act as a vector in the Arizona region. The number of cases in Arizona has been on the rise year over year and, perhaps more troubling, just south of the Arizona border, the number of cases in Mexico has surpassed 4000 in the last three years, with an annual fatality rate as high as 47%. Recently, the brown dog tick was indicated as the vector for this outbreak in Mexico. Therefore, understanding why R. sanguineus can act as a vector for RMSF in the Arizona region, but not elsewhere has become more urgent. We propose three hypotheses for the recent emergence of R. sanguineus as a vector for RMSF: (1) there exists a distinct sub‐population of R. sanguineus which better transmits R. rickettsii, (2) a distinct strain of R. rickettsii maintains more favorably in the R. sanguineus populations in this region, and (3) changing environmental factors and dog‐keeping practices in the Arizona region have contributed to the spread of RMSF. To test these hypotheses, we have partnered with the Midwestern Veterinary Mobile Clinic as well as the Arizona Animal Welfare League to collect brown dog tick and serum samples from 16 locations spanning Arizona, New Mexico, and Mexico. We isolated and sequenced 12S, 16S, and COX‐1 tick mitochondrial genes and used a maximum likelihood phylogenetic method on the concatenated sequences. To determine rickettsial prevalence, we tested for the rickettsial‐specific, rOmpA gene and sequenced it. Furthermore, immunofluorescence assay (IFA) was conducted on canine serum samples to evaluate canine anti‐rickettsial antibody seroprevalence in these locations. Finally, all these data were mapped on a geographic climate model. Results indicate variation in tick phylogeographic distribution, rickettsial prevalence rates ranging from 18% to 64% among sub‐populations of R. sanguineus, and seroprevalence rates as high as 33%, indicating areas with endemic levels of RMSF (>15%). These data indicate that no single factor, but rather a confluence of factors drive the spread of RMSF in the region. With this data we can begin to predict and prevent future outbreaks of RMSF and improve canine and human health. Support or Funding Information MWU One Health Award to JVB, MWU REAP Award to JVB, MWU CGS Funding to AO, Kenneth A. Suarez Award to SM
The Leadership and Management Technical Area of the US Amy Research Institute is primarily involved in applying the principles of organizational effectiveness (OE) to the Army, thereby developing new technology for use by Organizational Effectiveness Staff and Non-Commissioned Officers (OESOs and OENCOs). The development of a coherent and uniform case study methodology will enhance the retrieval of OE information by OESOs, key managers, and commanders. This manual presents OESOs with a step-by-step approach on how to document organizational interventions using a case study methodology. It was developed by the System Development Corporation under Contract DAHC19-78-C-0008 with guidance from ARI personnel, as part of Army Project 2Q163731A781. The Deputy Chief of Staff for Personnel is the sponsor.
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