The Journal of Pharmacology and Experimental Therapeutics (JPET) is one of the leading journals serving the membership of the American Society for Pharmacology and Experimental Therapeutics (ASPET). JPET focuses on the publication of important and ground-breaking innovative science. We publish

ID 26669 Poster Board 395 The Pharmacology Knowledge Objectives (KOs) are a compilation of concepts and content originally developed to guide faculty members in the education of medical students in the mid-1980s by the Association of Medical School Pharmacology Chairs (AMSPC), and to guide the development of core pharmacology content and suggested contact hours for undergraduate medical student education. They provided guidance towards a standardized knowledge base for content decisions. The KOs were maintained by AMSPC, with the last iteration occurring in 2012. The need for such guidance seems apparent when one considers the variety of curricula in medical education today. To provide some consistency in content and concepts for undergraduate medical students, and perhaps other healthcare students, seems an important goal. To bring the KOs up to date, AMSPC and ASPET’s Division for Pharmacology Education (DPE) combined to update them, with the DPE providing oversight of updates and future maintenance. AMSPC and DPE formed working groups chaired by content experts with committee members from numerous institutions. Peer review was minimal since each committee was comprised of content experts who currently teach in each area. Co-editors reviewed the documents for formatting, grammar, and spelling, plus any clarification needed. Important drugs in each class were listed along with content recommendations for faculty consideration with suggestions of relevant physiology and pathophysiology, pharmacodynamics, pharmacokinetics, and pharmacogenomics. Faculty can modify suggestions to meet the needs of their individual students and/or the culture of their institution. The final document contains over 170 pages of guidance in fifteen major areas of pharmacology for undergraduate medical education. It has been compiled and reviewed by content experts who are pharmacology educators in universities and medical schools around the country, providing their expertise to help all faculty who request assistance. The current edition of the Knowledge Objectives in Pharmacology, 2022, is maintained on the ASPET/DPE and the AMSPC websites and is available to all. The KOs may also benefit international medical education faculty and curriculum development. In conclusion, this document is a concept and content document that contains prototypical and representative drugs in each drug class. The Knowledge Objectives have served the discipline of pharmacology well for almost 4 decades. They provide a guide for consistency in pharmacology education of undergraduate medical students. The update, and the relationship between AMSPC and ASPET/DPE will allow a timely maintenance of the KOs to the benefit of medical students, faculty, and the public, enhancing the consistency and quality of undergraduate medical education, and hopefully, clinical practice. They may find use in the international educational community.

We have developed a conceptual framework for promoting antiracism in pharmacology education by categorizing topics based on those where cultural competencies are clearly established and those where a more nuanced discussion is required with learners, as well as identifying and reducing bias in assessment. These are generalizable across health professions in which pharmacology education is a discipline in the curriculum.

ID 13913 Poster Board 393 Introduction: In medicine, pharmacology content introduced during pre-clerkship years must align with best practices and guidelines taught in the clinical years of training. Due to the large volume of content students must master during pre-clerkship training, learners often rely on board preparation resources, which may contain errors. Furthermore, the rapidly changing landscape of new drug approvals and practice guidelines requires frequent updates to pharmacology curricula. These issues prompted an update to the Pharmacology Knowledge Objectives (KO) last published in 2012, which have traditionally served as a standard content guide for educators. The purpose of this abstract is to present our approach in reviewing and updating the KO for the Cardiovascular System. Methods: During the ASPET annual meeting in Philadelphia in 2022, the Division of Pharmacology Education sponsored a session detailing plans to update the KO, which in the past had been updated by the American Association of Pharmacology Chairs (AMSPC). A call for ASPET members was made to participate in the process and committees were formed. The Cardiovascular System Committee discussed the intent of the KO and best practices for the update. A shared Google document was used for timely updates and commentaries, and weekly meetings via Zoom were used to facilitate dialogue. A 3rd year medical student who completed the required medical licensing examination and clerkship rotations was invited to provide input from a medical student perspective. Results: A numbering system with subheadings was proposed with a process of referencing KO from other organ systems. Objectives were revised to be more concise, with a focus on using terminology that is more specific and accurate. Action verbs were updated to target higher order cognitive processes of learning. Where appropriate in the clinical pharmacology section, a reference on most recent recommendations by the American College of Cardiology guidelines were included. Conclusions: Since last published in 2012, drug discontinuation and/or removal from the market and new drug approval with novel mechanisms of action led to updated practice guideline for the management of hypertension (2017), hyperlipidemias (2018), and heart failure (2022). New classifications were also proposed for antiarrhythmic drugs (2018). Pharmacology textbooks lag in describing new therapies and treatments. Leading electronic resource tools often do not provide specific information related to basic science content. The process for updating the KO was a learning experience for all involved. Pharmacology educators are encouraged to use the KO when developing new curricula and even provide the KO to learners to guide their self-directed study. Likely in 2023, additional updates to the Cardiovascular System KO will be needed as the field is constantly evolving.

Bone marrow skeletal stem cells (SSCs) secrete many cytokines including stromal derived factor-1 or CXCL12, which influences cell proliferation, migration, and differentiation. All CXCL12 splice variants are rapidly truncated on their N-terminus by dipeptidyl peptidase 4 (DPP4). This includes the common variant CXCL12 alpha (1-68) releasing a much less studied metabolite CXCL12(3-68). Here, we found that CXCL12(3-68) significantly inhibited SSC osteogenic differentiation and RAW-264.7 cell osteoclastogenic differentiation and induced a senescent phenotype in SSCs. Importantly, pre-incubation of SSCs with CXCL12(3-68) significantly diminished their ability to migrate toward CXCL12(1-68) in transwell migration assays. Using a high-throughput G-protein-coupled receptor (GPCR) screen (GPCRome) and bioluminescent resonance energy transfer molecular interaction assays, we revealed that CXCL12(3-68) acts via the atypical cytokine receptor 3-mediated β-arrestin recruitment and as a competitive antagonist to CXCR4-mediated signaling. Finally, a reverse phase protein array assay revealed that DPP4-cleaved CXCL12 possesses a different downstream signaling profile from that of intact CXCL12 or controls. The data presented herein provides insights into regulation of CXCL12 signaling. Importantly, it demonstrates that DPP4 proteolysis of CXCL12 generates a metabolite with significantly different and previously overlooked bioactivity that helps explain discrepancies in the literature. This also contributes to an understanding of the molecular mechanisms of osteoporosis and bone fracture repair and could potentially significantly affect the interpretation of experimental outcomes with clinical consequences in other fields where CXCL12 is vital, including cancer biology, immunology, cardiovascular biology, neurobiology, and associated pathologies.

Using fourth-year medical students as academic coaches 1 | WHAT PROBLEMS WERE ADDRESSED?Coaching helps learners visualise their future, promotes a trusting environment, facilitates self-directed learning, assists with goal setting and fosters accountability.Some of the biggest barriers to coaching students effectively are learner engagement, scheduling, coach training and resources to support a coaching programme.We piloted a coaching programme that would address these challenges and also benefit the coaches in a meaningful way.

The American Society for Pharmacology and Experimental Therapeutics (ASPET) held its annual meeting at the Experimental Biology 2022 conference in Philadelphia, PA on April 2-5, 2022. The authors provide a synopsis and discussion of each of the four sessions presented at the meeting under the ASPET Division for Pharmacology Education (DPE).

A grounded knowledge of pharmacology is essential for healthcare providers to improve the quality of patients' lives, avoid medical errors, and circumvent potentially dangerous drug-drug interactions. One of the greatest tools to achieve this foundational knowledge of pharmacology is the dedicated pharmacology educators who teach in health sciences programs. Too often, the pharmacology educators responsible for teaching this material are left siloed at their own institutions with little room for dialog and collaboration. As scientists, we know that it is through dialog and collaboration that ideas grow, are refined, and improve. More collaborative work is needed to identify and describe best practices for pharmacology education in health sciences programs. While evidence-based, outcomes-focused studies are the optimum standard for this work, there is also a place for descriptive studies and innovative reports.

Expectations for physicians are rapidly changing, as is the environment in which they will practice. In response, preclerkship medical education curricula are adapting to meet these demands, often by reducing the time for foundational sciences. This descriptive study compares preclerkship pharmacology education curricular practices from seven allopathic medical schools across the United States. We compare factors and practices that affect how pharmacology is integrated into the undergraduate medical education curriculum, including teaching techniques, resources, time allocated to pharmacology teaching, and assessment strategies. We use data from seven medical schools in the United States, along with results from a literature survey, to inform the strengths and weaknesses of various approaches and to raise important questions that can guide future research regarding integration of foundational sciences in medical school and health professions’ curricula. In this comparative study, we found that there is significant heterogeneity in the number of hours dedicated to pharmacology in the preclerkship curriculum, whereas there was concordance in the use of active learning pedagogies for content delivery. Applying the ICAP (Interactive, Constructive, Active, Passive) Framework for cognitive engagement, our data showed that pharmacology was presented using more highly engaging pedagogies during sessions that are integrated with other foundational sciences. These findings can serve as a model that can be applied beyond pharmacology to other foundational sciences such as genetics, pathology, microbiology, biochemistry, etc.

Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the US. AGS3, a receptor-independent activator of G-protein signaling has been shown to affect different cellular processes and cell cycle activity. The aim of this study is to assess the role of AGS3 on the development and progression of prostate cancer as well as to understand the molecular dynamics involved. Preliminary results indicate a differential in the level of expression of AGS3 in PCa cells with the African American cell line (MDA PCa 2b) expressing high amount of AGS3 while the Caucasian cell lines (PC3 and LNCAP) do not. Overexpression of AGS3 in PC3 augmented tumor development in nude mice xenografts whereas depletion of AGS3 in MDA PCa 2b decreased tumor size. Interestingly, a xenograft model of Lewis Lung Carcinoma (LLC) cells in AGS3 conditional knockout (AGS3−/−) mice resulted in a significant increase in tumor progression when compared with the wild-type (AGS3+/+) mice. Taken together, these results indicate that AGS3 expression modulates tumor development and progression and may play a role in PCa health disparity.

Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein-coupled receptors are essential in transducing signals from the microenvironment, and heterotrimeric G alpha signaling links these receptors to downstream effectors. Several G alpha(i)-coupled G-protein-coupled receptors have been implicated in macrophage polarization. In this study, we use genetically modified mice to investigate the role of G alpha(i2) on inflammasome activity and macrophage polarization. We report that G alpha(i2) in murine bone marrow-derived macrophages (BMDMs) regulates IL1 beta release after activation of the NLRP3, AIM2, and NLRC4 inflammasomes. We show this regulation stems from the biased polarity of G alpha(i2) deficient (Gnai2(-/-)) and RGS-insensitive G alpha(i2) (Gnai2(G184S/G184S)) BMDMs. We determined that although Gnai2(G184S/G184S) BMDMs (excess G alpha(i2) signaling) have a tendency toward classically activated proinflammatory (M1) phenotype, Gnai2(-/-) BMDMs (G alpha(i2) deficient) are biased toward alternatively activated anti-inflammatory (M2) phenotype. Finally, we find that G alpha(i2)-deficient macrophages have increased Akt activation and IFN-beta production but defects in ERK1/2 and STAT3 activation after LPS stimulation. G alpha(i2)-deficient macrophages also exhibit increased STAT6 activation after IL-4 stimulation. In summary, our data indicates that excess G alpha(i2) signaling promotes an M1 macrophage phenotype, whereas G alpha(i2) signaling deficiency promotes an M2 phenotype. Understanding G alpha(i2)-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.

Activator of G-protein signaling 4 (AGS4)/G-protein signaling modulator 3 (Gpsm3) contains three G-protein regulatory (GPR) motifs, each of which can bind Gαi-GDP free of Gβγ. We previously demonstrated that the AGS4-Gαi interaction is regulated by seven transmembrane-spanning receptors (7-TMR), which may reflect direct coupling of the GPR-Gαi module to the receptor analogous to canonical Gαβγ heterotrimer. We have demonstrated that the AGS4-Gαi complex is regulated by chemokine receptors in an agonist-dependent manner that is receptor-proximal. As an initial approach to investigate the functional role(s) of this regulated interaction in vivo, we analyzed leukocytes, in which AGS4/Gpsm3 is predominantly expressed, from AGS4/Gpsm3-null mice. Loss of AGS4/Gpsm3 resulted in mild but significant neutropenia and leukocytosis. Dendritic cells, T lymphocytes, and neutrophils from AGS4/Gpsm3-null mice also exhibited significant defects in chemoattractant-directed chemotaxis and extracellular signal-regulated kinase activation. An in vivo peritonitis model revealed a dramatic reduction in the ability of AGS4/Gpsm3-null neutrophils to migrate to primary sites of inflammation. Taken together, these data suggest that AGS4/Gpsm3 is required for proper chemokine signal processing in leukocytes and provide further evidence for the importance of the GPR-Gαi module in the regulation of leukocyte function.

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

Bioluminescence resonance energy transfer (BRET) is a valuable tool to detect protein-protein interactions. BRET utilizes bioluminescent and fluorescent protein tags with compatible emission and excitation properties, making it possible to examine resonance energy transfer when the tags are in close proximity (<10 nm) as a typical result of protein-protein interactions. Here we describe a protocol for detecting BRET from two known protein binding partners (G alpha i1 and RGS14) in HEK 293 cells using Rend la luciferase and yellow fluorescent protein tags. We discuss the calculation of the acceptor/donor ratio as well as net BRET and demonstrate that BRET can be used as a platform to investigate the regulation of protein-protein interactions in live cells in real time.

Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gβγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαβγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oβγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gβγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oβγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαβγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.

Activator of G-protein signaling 3 (AGS3) is an accessory protein that functions to regulate the activation status of heterotrimeric G-protein subunits. To date, however, the downstream signaling pathways regulated by AGS3 remain to be fully elucidated, particularly in renal epithelial cells. In the present study, normal rat kidney (NRK-52E) proximal tubular epithelial cells were genetically modified to regulate the expression of AGS3 to investigate its role on MAPK and mTOR signaling to control epithelial cell number. Knockdown of endogenous AGS3 protein was associated with a reduced phosphorylated form of ERK5 and increased apoptosis as determined by elevated cleaved caspase-3. In the presence of the ERK5 inhibitor, BIX02189, a significant 2-fold change (P < 0.05) in G2/M transition state was detected compared to control conditions. Neither of the other MAPK, ERK1/2 or p38 MAPK, nor another pro-survival pathway, mTOR, was significantly altered by the changes in AGS3 protein levels in the renal epithelial cells. The selective ERK5 inhibitor, BIX02189, was found to dose-dependently reduce NRK cell number by up to 41% (P < 0.05) compared to control cells. In summary, these findings demonstrated that cell viability was regulated by AGS3 and was associated with ERK5 activation in renal epithelial cells.

Activators of G protein signaling (AGS), initially discovered in the search for receptor-independent activators of G protein signaling, define a broad panel of biologic regulators that influence signal transfer from receptor to G-protein, guanine nucleotide binding and hydrolysis, G protein subunit interactions, and/or serve as alternative binding partners for Gα and Gβγ independently of the classic heterotrimeric Gαβγ. AGS proteins generally fall into three groups based upon their interaction with and regulation of G protein subunits: group I, guanine nucleotide exchange factors (GEF); group II, guanine nucleotide dissociation inhibitors; and group III, entities that bind to Gβγ. Group I AGS proteins can engage all subclasses of G proteins, whereas group II AGS proteins primarily engage the Gi/Go/transducin family of G proteins. A fourth group of AGS proteins with selectivity for Gα16 may be defined by the Mitf-Tfe family of transcription factors. Groups I-III may act in concert, generating a core signaling triad analogous to the core triad for heterotrimeric G proteins (GEF + G proteins + effector). These two core triads may function independently of each other or actually cross-integrate for additional signal processing. AGS proteins have broad functional roles, and their discovery has advanced new concepts in signal processing, cell and tissue biology, receptor pharmacology, and system adaptation, providing unexpected platforms for therapeutic and diagnostic development.