Novel regulatory properties of adenylate cyclase type 6

Several studies have examined the regulation of recombinant adenylate cyclase type 6 (AC6) in vitro. Both PKC phosphorylation and calcium inhibit AC6 in vitro. In cultured cells or in tissues, neither PKC activation nor calcium release has been demonstrated to inhibit AC6. Furthermore, downstream effectors of PKC and calcium, Raf1 and calmodulin, respectively, have been implicated in the heterologous sensitization of AC6, suggesting that both PKC and calcium signaling may differentially regulate AC6 in vitro vs. in intact cells. To specifically investigate the regulation of AC6 by PKC, we constructed HEK293 cells that stably express recombinant AC6. PKC activation in intact cells enhances AC6 activation and the positive effects of PKC on AC6 activity are Raf1-dependent. Activation of Raf1 via receptor tyrosine kinases also enhances AC6 activation and AC6 and Raf1 may interact physically in intact cells. Subsequent studies investigated the ability of Gαqcoupled receptors upstream of PKC to potentiate AC6. Activation of endogenous Gαqcoupled muscarinic receptors in HEK-AC6 cells potentiates drug-stimulated cyclic AMP accumulation, however this potentiation was independent of PKC. Chelation of intracellular calcium or antagonism of calmodulin attenuated the effects of Gαq-coupled muscarinic receptors on AC6 activation. These studies suggest that although calcium directly inhibits AC6, Gαq-mediated release of intracellular calcium potentiates AC6 activation. To investigate the role of these novel signaling mechanisms in heterologous sensitization of AC6, we constructed HEK-AC6–D2L cells. AC6 is robustly sensitized after pretreatment (2 hr) with the D2L agonist, quinpirole. Neither inhibition of PKC nor Raf1 effectively attenuated D2L-mediated sensitization of AC6. However, activation of PKC robustly potentiated D2L-receptor mediated sensitization without altering acute D2L-mediated inhibition of AC6. The potentiation of sensitization of AC6 by PKC was blocked by inhibition of novel PKC isoforms and Raf1. These data demonstrate that although PKC and Raf1 are not required for heterologous sensitization of AC6, they robustly potentiate the magnitude of sensitization. Due to the broad expression pattern of AC6 in human tissue, these studies may aid in understanding the pathology and treatment of neuronal disorders and cardiac disease

Neuroprotective effects of direct activation and transactivation of PDGFβ receptors

Platelet-derived growth factor (PDGF) receptors are expressed throughout the body, including the central nervous system (CNS). Although the physiological role of PDGF receptors in the developed CNS is not fully characterized, PDGF signaling appears to provide neuroprotective effects against several neuronal insults. One of the best-characterized neuroprotective effects of PDGF type-β receptors is against human immunodeficiency virus (HIV) protein-induced neurotoxicity, with potential physiological relevance to HAD. PDGFβ receptors are also neuroprotective against glutamate excitotoxicity, which is associated with both stroke and neurodegenerative diseases, including Alzheimer’s disease. The neuroprotective effects of PDGFβ receptors occur both via direct activation by ligand (PDGF-BB), as well as by PDGFβ receptors activated downstream of G protein-coupled receptor signaling. In addition to the involvement of PDGF signaling in various pathologies and potential therapies, there is also an emerging body of evidence that PDGF may serve as a biomarker for neurological or psychiatric diseases

Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.</p

Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons

Estimating implicit and explicit racial and ethnic bias among community pharmacists in Canada

Background: Bias, whether implicit (unconscious) or explicit (conscious), can lead to preferential treatment of specific social groups and antipathy towards others. When healthcare professionals (HCPs), including pharmacists, act on these biases, patient care and health outcomes can be adversely affected. This study aims to estimate implicit and explicit racial/ethnic bias towards Black and Arab people among community pharmacists in Ontario, Canada. Methods: Community pharmacists participated in a secure, web-based survey using a cross-sectional design that included Harvard’s Race and Arab Implicit Association Tests (IATs) to examine bias towards Black and Arab people. Explicit (stated) preferences were measured by self-report. Data were analyzed using descriptive and inferential statistics. Results: The study surveyed 407 community pharmacists, 56.1 % of whom were women with an average age of 46.9. Implicit Association Test (IAT) results showed a statistically significant moderate preference for white people over both Black (mean IAT = 0.41) and Arab people (mean IAT = 0.35). However, most pharmacists explicitly stated that they had no racial/ethnic preference, with 75.7 % expressing a neutral preference between Black and white and 66.6 % neutral between Arab and white. However, a slight preference for white individuals was observed. Demographic factors such as age, place of birth, race/ethnicity, and experience significantly impacted IAT scores. For example, older, Canadian-born, white pharmacists with more experience displayed higher implicit bias scores. A mild correlation was found between implicit and explicit bias, indicating as implicit bias increases, explicit bias tends to become more negative. Conclusions: This study is the first to explore the issue of pharmacist bias in Canada and concentrate on anti-Arab bias. Our findings reveal that Ontario community pharmacists tend to have an unconscious inclination towards white people, which calls for further understanding of this matter. Additionally, we discovered a moderate degree of anti-Arab bias, indicating that studies on other HCPs should consider bias against this social group. Educational interventions are needed to address the implicit biases among community pharmacists in Ontario, Canada. These findings should aim to raise self-awareness of biases, educate about the potential implications of these biases on patient care, and provide strategies to reduce bias

Abelson Tyrosine Kinase Links PDGFbeta Receptor Activation to Cytoskeletal Regulation of NMDA Receptors in CA1 Hippocampal Neurons

Background: We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results: Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDAevoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion: This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons

Differential Effects of Endocannabinoids on Amyloid-Beta Aggregation and Toxicity

The regulation and metabolism of the endocannabinoid system has received extensive attention for their potential neuroprotective effect in neurodegenerative diseases such as Alzheimer’s disease (AD), which is characterized by amyloid β (Aβ) -induced cell toxicity, inflammation, and oxidative stress. Using in vitro techniques and two cell lines, the mouse hippocampus-derived HT22 cells and Chinese hamster ovary (CHO) cells expressing human cannabinoid receptor type 1 (CB1), we investigated the ability of endocannabinoids to inhibit Aβ aggregation and protect cells against Aβ toxicity. The present study provides evidence that endocannabinoids N-arachidonoyl ethanol amide (AEA), noladin and O-arachidonoyl ethanolamine (OAE) inhibit Aβ42 aggregation. They were able to provide protection against Aβ42 induced cytotoxicity via receptor-mediated and non-receptor-mediated mechanisms in CB1-CHO and HT22 cells, respectively. The aggregation kinetic experiments demonstrate the anti-Aβ aggregation activity of some endocannabinoids (AEA, noladin). These data demonstrate the potential role and application of endocannabinoids in AD pathology and treatment

Investigating Community Pharmacy Take Home Naloxone Dispensing during COVID-19: The Impact of One Public Health Crisis on Another

A recent report found that the number of opioid-related deaths in Ontario in the first 15 weeks of the COVID-19 pandemic was 38.2% higher than in the 15 weeks before the pandemic. Our study sought to determine if pharmacy professionals self-reported an increase or decrease in naloxone provision due to the pandemic and to identify adjustments made by pharmacy professionals to dispense naloxone during the pandemic. A total of 231 Ontario community pharmacy professionals completed an online survey. Pharmacy professionals’ barriers, facilitators, and comfort level with dispensing naloxone before and during the pandemic were identified. The sample consisted of mostly pharmacists (99.1%). Over half (51.1%) reported no change in naloxone dispensing, while 22.9% of respondents reported an increase and 24.7% a decrease. The most common adjustments made during the pandemic were training patients how to administer naloxone over video or phone, delivering naloxone kits, and pharmacy technicians offering naloxone at prescription intake. Over half (55%) of participants said the top barrier for dispensing was that patients did not request naloxone. Naloxone distribution through pharmacies could be further optimized to address the increased incidence of overdose deaths during the pandemic. Future research should investigate the reasons for changes in naloxone dispensing