Personalizing rehabilitation for individuals with musculoskeletal impairments: Feasibility of implementation of the Measures Associated to Prognostic (MAPS) tool.

INTRODUCTION: The Measures Associated to PrognoStic (MAPS) tool is a standardized questionnaire that integrates validated prognostic tools to detect the presence of biopsychosocial prognostic factors in patients consulting for musculoskeletal disorders. PURPOSE: The objectives were to assess the: 1) feasibility of implementation of the MAPS tool, 2) clinicians' acceptability of the dashboard, and 3) patients' acceptability of the MAPS tool. METHODS: Twenty physiotherapists and two occupational therapists from seven outpatient musculoskeletal clinics were recruited to implement the MAPS tool during a 3-month timeframe, where new patients completed the questionnaire upon initial assessment. The results were presented to the clinicians via a dashboard. Surveys and semi-structured interviews were conducted to measure feasibility and acceptability. RESULTS: Six out of 11 feasibility criteria (55%) and 21 out of 24 acceptability criteria (88%) reached the a priori threshold for success. The interviews allowed us to identify three main themes to facilitate implementation: 1) limiting the burden, 2) ensuring patients' understanding of the tool's purpose, and 3) integrating the dashboard as a clinical information tool. CONCLUSION: Our quantitative and qualitative results support the feasibility of implementation and acceptability of the MAPS tool pending minor adjustments. Depicting the patients' prognostic profile has the potential to help clinicians optimize their interventions for patients presenting with musculoskeletal disorders.

Establishing the prognostic profile of patients with work-related musculoskeletal disorders: Development and acceptability of the MAPS questionnaire.

PURPOSE: Work-related musculoskeletal disorders (WRMD) are the most common causes of disability worldwide and are associated with significant use of healthcare. One way to optimize the clinical outcomes of injured workers receiving rehabilitation is to identify and address individual prognostic factors (PF), which can facilitate the personalization of the treatment plan. As there is no pragmatic and systematic method to collect prognostic-related data, the purpose of the study was to develop and assess the acceptability of a set of questionnaires to establish the "prognostic profile" of workers with WRMD. METHODS: We utilized a multistep process to inform the acceptability of the Measures Associated to PrognoStic (MAPS) questionnaire. During STEP-1, a preliminary version of the was developed through a literature search followed by an expert consensus including a patient-advisor. During STEP-2, future users (rehabilitation professionals, healthcare administrators and compensation officers) were consulted through an online survey and were asked to rate the relevance of each content item; items that obtained ≥80% of "totally agree" answers were included. They were also asked to prioritize PF according to their usefulness for clinical decision-making, as well as perceived efficacy to enhance the treatment plan. RESULTS: The questionnaire was developed with three categories: the outcome predicted, the unique PF, and prognostic tools. Personal PF (i.e.: coping strategies, fear-avoidance beliefs), pain related PF (i.e.: pain intensity/severity, duration of pain), and work-related PF (i.e.: work physical demands, work accommodations) were identified to be totally relevant and included in the questionnaire. 84% of the respondents agreed that their patients could complete the MAPS questionnaire in their clinical setting, while 75% totally agreed that the questionnaire is useful to personalize rehabilitation interventions. CONCLUSION: The MAPS questionnaire was deemed acceptable to establish the "prognostic profile" of injured workers and help the clinicians in the treatment decision-making process.

Experiences and Perceptions of Medical Cannabis among People Living with Chronic Pain and Community Pharmacists: A Qualitative Study in Canada.

Background: The use of cannabis to treat chronic pain is under debate despite high expectations from patients. Qualitative data obtained by exploring both patients' and health professionals' perspectives are scarce. Aims: This study aimed to understand the experiences and perceptions of people living with chronic pain and community pharmacists regarding the role of cannabis in chronic pain treatment in the Canadian context where both medical and recreational cannabis are legal. Methods: We conducted 12 online focus groups (July 2020-February 2021) with 26 patients and 19 community pharmacists using semistructured discussion guides. All discussions were audio recorded and transcribed verbatim were analyzed using a reflexive thematic approach. Results: We developed three themes related to patients' perspectives and three themes related to pharmacists' perspectives. Patients' perspectives included (1) cannabis as an alternative to other pain medications, (2) a new treatment with potential health-related risks, and (3) a therapy rather than a recreational drug. Pharmacists' perspectives included (1) challenges in monitoring drug interactions with cannabis in the context of scarce research data, (2) informing and treating patients self-medicating with cannabis amid its growing popularity, and (3) financial costs and legal constraints for patients. Conclusions: This study highlights patients' and pharmacists' urgent need for reliable information regarding the benefits and risks of cannabis. Training tailored to pharmacists' needs and evidence-based information for patients should be developed to support pharmacists' practice, improve patients' experiences, and promote safe cannabis use.

Contexte: L'utilisation du cannabis pour traiter la douleur chronique fait l'objet d'un débat, malgré les fortes attentes des patients. Les données qualitatives issues de l'exploration des perspectives à la fois des patients et des professionnels de la santé, demeurent rares.Objectifs: Cette étude visait à comprendre les expériences et les perceptions des personnes vivant avec une douleur chronique et des pharmaciens communautaires concernant le rôle du cannabis dans le traitement de la douleur chronique dans le contexte canadien, où le cannabis médical et récréatif est légal.Méthodes: Nous avons mené 12 groupes de discussion en ligne entre juillet 2020 et février 2021, réunissant 26 patients et 19 pharmaciens communautaires, à l'aide de guides de discussion semi-structurés. Toutes les discussions ont été enregistrées et transcrites mot à mot, puis analysées à l'aide d'une approche thématique réflexive.Résultats: Nous avons développé trois thèmes liés aux perspectives des patients et trois thèmes liés aux perspectives des pharmaciens. Le point de vue des patients incluait (1) le cannabis comme option de rechange à d'autres médicaments contre la douleur, (2) un nouveau traitement avec des risques potentiels pour la santé, et (3) un traitement plutôt qu'une drogue récréative. Le point de vue des pharmaciens portait sur (1) les défis liés à la surveillance des interactions médicamenteuses avec le cannabis dans le contexte de la rareté des données de recherche, (2) l'information et le traitement des patients qui s'auto-médicamentent avec du cannabis dans un contexte de popularité croissante, et (3) les coûts financiers et les contraintes légales pour les patients.Conclusions: Cette étude met en évidence le besoin urgent des patients et des pharmaciens de disposer d'informations fiables sur les avantages et les risques du cannabis. Une formation adaptée aux besoins des pharmaciens et des informations fondées sur des données probantes pour les patients devraient être développées pour soutenir la pratique des pharmaciens, améliorer l'expérience des patients et promouvoir une utilisation sûre du cannabis.

Prognostic factors specific to work-related musculoskeletal disorders: An overview of recent systematic reviews.

PURPOSE: Work-related injuries affect a considerable number of people each year and represent a significant burden for society. To reduce this burden, optimizing rehabilitation care by integrating prognostic factors (PF) into the clinical decision-making process is a promising way to improve clinical outcomes. The aim of this study was to identify PF specific to work-related musculoskeletal disorders. METHODS: We performed an overview of systematic reviews reporting on PF that had the following outcomes of interest: Return to work, pain, disability, functional status, or poor outcomes. Each extracted PF was categorized according to its level of evidence (grade A or B) and whether it was modifiable or not. The risk of bias of each study was assessed with the ROBIS tool. RESULTS: We retrieved 757 citations from 3 databases. After removing 307 duplicates, 450 records were screened, and 20 studies were retained. We extracted a total of 20 PF with a Grade A recommendation, where 7 were deemed modifiable, 11 non-modifiable and 2 were index test. For example, return to work expectations, previous sick leave, delay in referral and pain intensity were found to be predictors of return-to-work outcomes. We also identified 17 PF with a Grade B recommendation, where 11 were deemed modifiable. For example, poor general health, negative recovery expectations, coping and fear-avoidance beliefs, pain severity, and particularly physical work were found to predict return to work outcomes. CONCLUSION: We found numerous modifiable PFs that can help clinicians personalize their treatment plan beyond diagnostic-related information for work-related musculoskeletal disorders.

Characteristics of Persons Seeking Care for Moderate to Severe Pain Due to Chronic Low Back Pain and Osteoarthritis: A Cross-Sectional Study.

Purpose: To assess the associations between pain severity or physical (pQoL) and mental (mQoL) health-related quality of life and disability status or health-care utilization among persons living with moderate/severe pain due to chronic low back pain (CLBP) or osteoarthritis (OA), who received treatments in Quebec's tertiary care pain centers. Materials and Methods: This retrospective study was carried out using the Quebec Pain Registry (Canada) from 2008 to 2014 and contains data on persons referred to tertiary pain management clinics. Participants were selected if they were diagnosed with CLBP (N = 2663) or OA (N = 139) of more than 3 months duration and of pain intensity ≥5 on the Numeric Rating Scale (0-10) and completed baseline questionnaires. Results: Less than 5% of persons were hospitalized in the 6 months before their first visit at the pain clinic, and 11.9% and 18.9% of persons with OA and CLBP, respectively, had a pain-related emergency room (ER) visit. Less than 1/5 and more than 1/4 of persons with OA and CLBP were receiving disability benefits, respectively. Persons with CLBP who had visited the ER, those on disability and those receiving disability benefits, reported higher levels of pain severity, interference, and lower levels of mQoL (and pQoL for those on disability or receiving benefits) compared to those who did not consult the ER, those not on disability or not receiving disability benefits, respectively (all p < 0.05). For OA, disability status was the only variable associated with pain interference and QoL (all p < 0.05). Conclusion: Pain severity, pain interference and mQoL were associated with health-care utilization and disability status in persons with CLBP. These results were globally not found among persons with OA, which might be due to smaller sample size or unique characteristics of this population.

Swallowing the pill of adverse effects: A qualitative study of patients' and pharmacists' experiences and decision-making regarding the adverse effects of chronic pain medications.

INTRODUCTION: Pharmacological treatments of chronic pain can lead to numerous and sometimes serious adverse effects. Drawing on a social science approach to chronic illness, this study aimed to understand the experiences of people living with chronic pain and community pharmacists regarding the definition, prevention and management of analgesic adverse effects. METHODS: This qualitative study proceeded through 12 online focus groups (FGs) with people living with chronic pain (n = 26) and community pharmacists (n = 19), conducted between July 2020 and February 2021 in the province of Quebec, Canada. The semistructured discussion guides covered participants' definitions of adverse effects and decision-making regarding their prevention and management. Discussions were audio-recorded, transcribed verbatim and analysed using grounded theory. RESULTS: Both people with chronic pain and pharmacists provided varying definitions of analgesic adverse effects depending on patients' social and clinical characteristics. Present quality of life and serious long-term risks related to treatment were described as key dimensions influencing adverse effect appraisal. Dilemmas and discrepancies occurred between patients and pharmacists when choosing to prioritize pain relief or adverse effect prevention. Some patients lacked information about their medications and wanted to be more involved in decisions, while many pharmacists were concerned by patients' self-management of adverse effects. Preventing opioid-related overdoses often led pharmacists to policing practices. Despite most pharmacists wishing they could have a key role in the management of pain and adverse effects face organizational and financial barriers. CONCLUSION: Defining, preventing and managing adverse effects in the treatment of chronic pain requires a person-centred approach and shared decision-making. Clinical training improvements and healthcare organization changes are needed to support pharmacists in providing patients with community-based follow-up and reliable information about the adverse effects of chronic pain treatments. PATIENT OR PUBLIC CONTRIBUTION: A person with lived experience of chronic pain was involved as a coinvestigator in the study. He contributed to shaping the study design and objectives, including major methodological decisions such as the choice of pharmacists as the most appropriate professionals to investigate. In addition, 26 individuals with chronic pain shared their experiences extensively during the FGs.

Distinct behavioral responses evoked by selective optogenetic stimulation of the major TRPV1+ and MrgD+ subsets of C-fibers.

Primary C-fiber nociceptors are broadly divided into peptidergic and nonpeptidergic afferents. TRPV1 is a thermosensitive cation channel mainly localized in peptidergic nociceptors, whereas MrgD is a sensory G protein-coupled receptor expressed in most nonpeptidergic nociceptive afferents. TRPV1 and MrgD fibers have been reported to be primarily involved in thermal and mechanical nociception, respectively. Yet, their functional assessment in somatosensory transmission relied on ablation strategies that do not account for compensatory mechanisms. To achieve selective activation of these 2 major subsets of C-fibers in vivo in adult mice, we used optogenetics to specifically deliver the excitatory opsin channelrhodopsin-2 (ChR2) to TRPV1 or MrgD primary sensory neurons, as confirmed by histology and electrophysiology. This approach allowed, for the first time, the characterization of behavioral responses triggered by direct noninvasive activation of peptidergic TRPV1 or nonpeptidergic MrgD fibers in freely moving mice. Transdermal blue light stimulation of the hind paws of transgenic mice expressing ChR2 in TRPV1 neurons generated nocifensive behaviors consisting mainly of paw withdrawal and paw licking, whereas paw lifting occurrence was limited. Conversely, optical activation of cutaneous MrgD afferents produced mostly withdrawal and lifting. Of interest, in a conditioned place avoidance assay, blue light induced aversion in TRPV1-ChR2 mice, but not in MrgD-ChR2 mice. In short, we present novel somatosensory transgenic models in which control of specific subsets of peripheral unmyelinated nociceptors with distinct functions can be achieved with high spatiotemporal precision. These new tools will be instrumental in further clarifying the contribution of genetically identified C-fiber subtypes to chronic pain.

Will optogenetics be used to treat chronic pain patients?

Chronic pain affects a third of the population and current treatments produce limited relief and severe side effects. An alternative strategy to decrease pain would be to directly modulate somatosensory pathways using optogenetics. Optogenetics involves the use of genetically encoded and optically active proteins, namely opsins, to control neuronal circuits. In preclinical animal models, optical silencing of peripheral nociceptors has been shown to alleviate both inflammatory and neuropathic pain. An opsin-based gene therapy to treat chronic pain patients is not ready yet, but encouraging advances have been made in optical and viral technology. In view of the increasing burden of chronic pain in our aging society, innovative analgesic approaches based on optogenetics are definitely worth exploring.

Optogenetic Silencing of Nav1.8-Positive Afferents Alleviates Inflammatory and Neuropathic Pain.

We report a novel transgenic mouse model in which the terminals of peripheral nociceptors can be silenced optogenetically with high spatiotemporal precision, leading to the alleviation of inflammatory and neuropathic pain. Inhibitory archaerhodopsin-3 (Arch) proton pumps were delivered to Nav1.8(+) primary afferents using the Nav1.8-Cre driver line. Arch expression covered both peptidergic and nonpeptidergic nociceptors and yellow light stimulation reliably blocked electrically induced action potentials in DRG neurons. Acute transdermal illumination of the hindpaws of Nav1.8-Arch(+) mice significantly reduced mechanical allodynia under inflammatory conditions, while basal mechanical sensitivity was not affected by the optical stimulation. Arch-driven hyperpolarization of nociceptive terminals was sufficient to prevent channelrhodopsin-2 (ChR2)-mediated mechanical and thermal hypersensitivity in double-transgenic Nav1.8-ChR2(+)-Arch(+) mice. Furthermore, prolonged optical silencing of peripheral afferents in anesthetized Nav1.8-Arch(+) mice led to poststimulation analgesia with a significant decrease in mechanical and thermal hypersensitivity under inflammatory and neuropathic conditions. These findings highlight the role of peripheral neuronal inputs in the onset and maintenance of pain hypersensitivity, demonstrate the plasticity of pain pathways even after sensitization has occurred, and support the involvement of Nav1.8(+) afferents in both inflammatory and neuropathic pain. Together, we present a selective analgesic approach in which genetically identified subsets of peripheral sensory fibers can be remotely and optically inhibited with high temporal resolution, overcoming the compensatory limitations of genetic ablations.

Recent advances on the δ opioid receptor: from trafficking to function.

UNLABELLED: Within the opioid family of receptors, δ (DOPrs) and µ opioid receptors (MOPrs) are typical GPCRs that activate canonical second-messenger signalling cascades to influence diverse cellular functions in neuronal and non-neuronal cell types. These receptors activate well-known pathways to influence ion channel function and pathways such as the map kinase cascade, AC and PI3K. In addition new information regarding opioid receptor-interacting proteins, downstream signalling pathways and resultant functional effects has recently come to light. In this review, we will examine these novel findings focusing on the DOPr and, in doing so, will contrast and compare DOPrs with MOPrs in terms of differences and similarities in function, signalling pathways, distribution and interactions. We will also discuss and clarify issues that have recently surfaced regarding the expression and function of DOPrs in different cell types and analgesia. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Pain after discontinuation of morphine treatment is associated with synaptic increase of GluA4-containing AMPAR in the dorsal horn of the spinal cord.

Withdrawal from prescribed opioids results in increased pain sensitivity, which prolongs the treatment. This pain sensitivity is attributed to neuroplastic changes that converge at the spinal cord dorsal horn. We have recently reported that repeated morphine administration triggers an insertion of GluA2-lacking (Ca(2+)-permeable) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) in the hippocampus. This finding together with the reported involvement of AMPAR in the mechanisms underlying inflammatory pain led us to hypothesize a role for spinal AMPAR in opioid-induced pain behavior. Mice treated with escalating doses of morphine showed hypersensitivity to mechanical stimulation. Intrathecal administration of a Ca(2+)-permeable AMPAR selective blocker disrupted morphine-induced mechanical sensitivity. Analysis of the expression and phosphorylation levels of AMPAR subunits (GluA1/2/3/4) in homogenates and in postsynaptic density fractions from spinal cord dorsal horns showed an increase in GluA4 expression and phosphorylation in the postsynaptic density after morphine. Co-immunoprecipitation analyses suggested an increase in GluA4 homomers (Ca(2+)-permeable AMPAR) and immunohistochemical staining localized the increase in GluA4 levels in laminae III-V. The excitatory postsynaptic currents (EPSCs) recorded in laminae III-V showed enhanced sensitivity to Ca(2+)-permeable AMPAR blockers in morphine-treated mice. Furthermore, current-voltage relationships of AMPAR-mediated EPSCs showed that rectification index (an indicator of Ca(2+)-permeable AMPAR contribution) is increased in morphine-treated but not in saline-treated mice. These effects could be reversed by infusion of GluA4 antibody through patch pipette. This is the first direct evidence for a role of GluA4-containing AMPAR in morphine-induced pain and highlights spinal GluA4-containing AMPAR as targets to prevent the morphine-induced pain sensitivity.

Increased anxiety-like behaviors in rats experiencing chronic inflammatory pain.

For many patients, chronic pain is often accompanied, and sometimes amplified, by co-morbidities such as anxiety and depression. Although it represents important challenges, the establishment of appropriate preclinical behavioral models contributes to drug development for treating chronic inflammatory pain and associated psychopathologies. In this study, we investigated whether rats experiencing persistent inflammatory pain induced by intraplantar injection of complete Freund's adjuvant (CFA) developed anxiety-like behaviors, and whether clinically used analgesic and anxiolytic drugs were able to reverse CFA-induced anxiety-related phenotypes. These behaviors were evaluated over 28 days in both CFA- and saline-treated groups with a variety of behavioral tests. CFA-induced mechanical allodynia resulted in increased anxiety-like behaviors as evidenced by: (1) a significant decrease in percentage of time spent and number of entries in open arms of the elevated-plus maze (EPM), (2) a decrease in number of central squares visited in the open field (OF), and (3) a reduction in active social interactions in the social interaction test (SI). The number of entries in closed arms in the EPM and the distance traveled in the OF used as indicators of locomotor performance did not differ between treatments. Our results also reveal that in CFA-treated rats, acute administration of morphine (3mg/kg, s.c.) abolished tactile allodynia and anxiety-like behaviors, whereas acute administration of diazepam (1mg/kg, s.c) solely reversed anxiety-like behaviors. Therefore, pharmacological treatment of anxiety-like behaviors induced by chronic inflammatory pain can be objectively evaluated using multiple behavioral tests. Such a model could help identify/validate alternative potential targets that influence pain and cognitive dimensions of anxiety.

Activation of spinal mu- and delta-opioid receptors potently inhibits substance P release induced by peripheral noxious stimuli.

Over the past few years, δ-opioid receptors (DOPRs) and µ-opioid receptors (MOPRs) have been shown to interact with each other. We have previously seen that expression of MOPR is essential for morphine and inflammation to potentiate the analgesic properties of selective DOPR agonists. In vivo, it is not clear whether MOPRs and DOPRs are expressed in the same neurons. Indeed, it was recently proposed that these receptors are segregated in different populations of nociceptors, with MOPRs and DOPRs expressed by peptidergic and nonpeptidergic fibers, respectively. In the present study, the role and the effects of DOPR- and MOPR-selective agonists in two different pain models were compared. Using preprotachykinin A knock-out mice, we first confirmed that substance P partly mediates intraplantar formalin- and capsaicin-induced pain behaviors. These mice had a significant reduction in pain behavior compared with wild-type mice. We then measured the effects of intrathecal deltorphin II (DOPR agonist) and DAMGO (MOPR agonist) on pain-like behavior, neuronal activation, and substance P release following formalin and capsaicin injection. We found that both agonists were able to decrease formalin- and capsaicin-induced pain, an effect that was correlated with a reduction in the number of c-fos-positive neurons in the superficial laminae of the lumbar spinal cord. Finally, visualization of NK(1) (neurokinin 1) receptor internalization revealed that DOPR and MOPR activation strongly reduced formalin- and capsaicin-induced substance P release via direct action on primary afferent fibers. Together, our results indicate that functional MOPRs and DOPRs are both expressed by peptidergic nociceptors.

Selective angiotensin II AT2 receptor agonists: Benzamide structure-activity relationships.

In the investigation of the structure-activity relationship of nonpeptide AT(2) receptor agonists, a series of substituted benzamide analogues of the selective nonpeptide AT(2) receptor agonist M024 have been synthesised. In a second series, the biphenyl scaffold was compared to the thienylphenyl scaffold and the impact of the isobutyl substituent and its position on AT(1)/AT(2) receptor selectivity was also investigated. Both series included several compounds with high affinity and selectivity for the AT(2) receptor. Three of the compounds were also proven to function as agonists at the AT(2) receptor, as deduced from a neurite outgrowth assay, conducted in NG108-15 cells.

Role of tyrosine kinase receptors in angiotensin II AT2 receptor signaling: involvement in neurite outgrowth and in p42/p44mapk activation in NG108-15 cells.

NG108-15 cells, which have a rounding-up morphology when cultured in serum-supplemented medium, extend neurites when stimulated for 3 d with angiotensin II (Ang II). The aim of the present study was to investigate whether growth factor receptors are necessary for mediating the effects of Ang II. A 3-d treatment with AG879, an inhibitor of nerve growth factor receptor TrkA, strongly affected neurite outgrowth and phosphorylation of p42/p44(mapk) induced by Ang II. PD168393, an inhibitor of epidermal growth factor (EGF) receptor slightly decreased Ang II-induced neurite outgrowth, whereas AG213, an inhibitor of both platelet-derived growth factor receptor and EGF receptor, stimulated neurite outgrowth and p42/p44(mapk) phosphorylation on its own, without affecting further stimulation with Ang II. Moreover, Ang II induced the phosphorylation of TrkA (maximum at 5 min of incubation in the presence of serum or at 20 min in cells depleted in serum for 2 h) and a rapid increase in Rap1 activity, both effects abolished in cells preincubated with 10 microm AG879. In summary, the present results demonstrate that AT(2) receptor-induced sustained activation of p42/p44(mapk) and corresponding neurite outgrowth are mediated by phosphorylation of the nerve growth factor TrkA receptor. However, the results also point out that the presence of other growth factors, such as EGF or PDFG, may interfere with the effect of Ang II. Altogether, the current findings clearly indicate that the effects of the AT(2) receptor on neurite outgrowth dynamics are modulated by the presence of growth factors in the culture medium.

Short pseudopeptides containing turn scaffolds with high AT2 receptor affinity.

Two pentapeptides, Ac-Tyr-Ile-His-Pro-Phe/Ile, were synthesized and shown to have angiotensin II AT2 receptor affinity and agonistic activity. Based on these peptides, a new series of 13 pseudopeptides was synthesized via introduction of five different turn scaffolds replacing the Tyr-Ile amino acid residues. Pharmacological evaluation disclosed subnanomolar affinities for some of these compounds at the AT2 receptor. Substitution of Phe by Ile in this series of ligands enhanced the AT2 receptor affinity of all compounds. These results suggest that the C-terminal amino acid residues can be elaborated on to enhance the AT2 receptor affinity in truncated Ang II analogues.

Involvement of protein kinase C alpha (PKC alpha) in the early action of angiotensin II type 2 (AT2) effects on neurite outgrowth in NG108-15 cells: AT2-receptor inhibits PKC alpha and p21ras activity.

The aim of the present study was to investigate whether protein kinase C (PKC) isoforms may be among the putative candidates implicated in the primary effects of the Ang II type 2 (AT2) receptor. Western blot analyses revealed the presence of PKC alpha,epsilon, iota, and zeta in NG108-15 cells. After a 3-d treatment with 3 nm Gö6976, a specific inhibitor of classical PKC isoforms, cells were characterized by the presence of one elongated process similar to that observed after treatment with Ang II or with CGP42112, a selective AT2 receptor agonist. Similar findings were observed in cells expressing a dominant-negative mutant of PKC alpha (K368A). Inhibition of PKC alpha in NG108-15 cells also decreased cell number and proliferation. In conditions of acute stimulation, Ang II induced a time-dependent and transient inhibition of PKC alpha activity, as well as a decrease in PKC alpha levels associated with the membrane. Treatment of cells with Gö6976 was also found to inhibit p21(ras) (between 1-10 min) but stimulated Rap1 activity (1-5 min) in a time-course similar to that of Ang II. Incubation of NG108-15 cells with Gö6976 (3 nm) inhibited basal p42/p44(mapk) phosphorylation, but failed to interfere with its activation by the AT(2) receptor, indicating that inhibition of PKC alpha is not directly involved in the Rap1-MEK-p42/p44(mapk) cascade. Taken together, these results indicate that PKC alpha is a primary target of the AT2 receptor. Inhibition of PKC alpha leads to a decrease in both p21(ras) activity and cell proliferation, which may facilitate AT2 receptor signaling through p42/p44(mapk), thereby leading to neurite outgrowth.

New selective AT2 receptor ligands encompassing a gamma-turn mimetic replacing the amino acid residues 4-5 of angiotensin II act as agonists.

New benzodiazepine-based gamma-turn mimetics with one or two amino acid side chains were synthesized. The gamma-turn mimetics were incorporated into angiotensin II (Ang II) replacing the Val(3)-Tyr(4)-Ile(5) or Tyr(4)-Ile(5) peptide segments. All of the resulting pseudopeptides displayed high AT(2)/AT(1) receptor selectivity and exhibited AT(2) receptor affinity in the low nanomolar range. Molecular modeling was used to investigate whether the compounds binding to the AT(2) receptor could position important structural elements in common areas. A previously described benzodiazepine-based gamma-turn mimetic with high affinity for the AT(2) receptor was also included in the modeling. It was found that the molecules, although being structurally quite different, could adopt the same binding mode/interaction pattern in agreement with the model hypothesis. The pseudopeptides selected for agonist studies were shown to act as AT(2) receptor agonists being able to induce outgrowth of neurite cells, stimulate p42/p44(mapk), and suppress proliferation of PC12 cells.

Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist.

The first druglike selective angiotensin II AT(2) receptor agonist (21) with a K(i) value of 0.4 nM for the AT(2) receptor and a K(i) > 10 microM for the AT(1) receptor is reported. Compound 21, with a bioavailability of 20-30% after oral administration and a half-life estimated to 4 h in rat, induces outgrowth of neurite cells, stimulates p42/p44(mapk), enhances in vivo duodenal alkaline secretion in Sprague-Dawley rats, and lowers the mean arterial blood pressure in anesthetized, spontaneously hypertensive rats. Thus, the peptidomimetic 21 exerts a similar biological response as the endogenous peptide angiotensin II after selective activation of the AT(2) receptor. Compound 21, derived from the prototype nonselective AT(1)/AT(2) receptor agonist L-162,313 will serve as a valuable research tool, enabling studies of the function of the AT(2) receptor in more detail.