The calcium-sensing receptor (CaS) is the principal controller of extracellular calcium (Ca 21 o) homeostasis and is inhibited in vitro and in vivo by protein kinase C (PKC)-mediated phosphorylation at CaS T888. However, PKC inhibition enhances signaling even in CaSs lacking Thr-888, suggesting that an additional inhibitory site exists. An apparently equivalent PKC regulatory site in metabotropic glutamate receptor 5 (Ser-839) aligns not with CaS T888 but instead with CaS S875 , which was not previously considered to be a PKC site. CaS S875A (nonphosphorylatable) exhibited significantly enhanced Ca 21 o sensitivity of both intracellular Ca 21 mobilization and extracellular signal-regulated kinase 1/2 activation, whereas the phosphomimetic CaS S875D mutant exhibited a loss of function. The CaS S875A/T888A double mutant exhibited even greater Ca 21 o sensitivity than CaS T888A alone, a response no longer enhanced by PKC inhibition. Finally, when expressed in CaS lacking its extracellular domain, the CaS S875A/T888A double mutation elicited maximal activation even under control conditions, but remained sensitive to negative allosteric modulation [N-(2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl)-1,1-dimethyl-2-(2-nephthyl)ethylamine] or Ca 21 o removal. Therefore, we have now identified CaS S875 as the missing PKC phosphorylation site that, together with CaS T888 , shapes the CaS signaling that underpins Ca 21 o homeostasis. Together with the inactive form of the CaS extracellular domain, these sites attenuate Ca 21 o sensitivity to attain appropriate physiologic Ca 21 o sensing. SIGNIFICANCE STATEMENT Serine-875 represents the missing inhibitory PKC phosphorlyation site in CaS that in tandem with Thr-888 controls receptor activity. s This article has supplemental material available at molpharm.aspetjournals.org.

The role of cannabinoid receptors in nephropathy is gaining much attention. This study investigated the effects of two neutral CB1 receptor antagonists, AM6545 and AM4113, on nephropathy associated with metabolic syndrome (MetS). MetS was induced in rats by high-fructose high-salt feeding for 12 weeks. AM6545, the peripheral silent antagonist and AM4113, the central neutral antagonist were administered in the last 4 weeks. At the end of study, blood and urine samples were collected for biochemical analyses while the kidneys were excised for histopathological investigation and transforming growth factor beta 1 (TGFβ1) measurement. MetS was associated with deteriorated kidney function as indicated by the elevated proteinuria and albumin excretion rate. Both compounds equally inhibited the elevated proteinuria and albumin excretion rate while having no effect on creatinine clearance and blood pressure. In addition, AM6545 and AM4113 alleviated the observed swelling and inflammatory cells infiltration in different kidney structures. Moreover, AM6545 and AM4113 alleviated the observed histopathological alterations in kidney structure of MetS rats. MetS was associated with a ten-fold increase in urine uric acid while both compounds blocked this increase. Furthermore, AM6545 and AM4113 completely prevented the collagen deposition and the elevated expression of the TGFβ1 seen in MetS animals. In conclusion, AM6545 and AM4113, possess reno-protective effects by interfering with TGFβ1-mediated renal inflammation and fibrosis, via peripheral action.

Coronavirus disease (COVID-19) has spread from China to the world since December 2019. In addition to being a pandemic with mortality risk, COVID-19 affected global mental health. This study is the first to address the mental health aspects among Saudi pharmacy students during this pandemic and to explore the potential effect on their academic development. An online cross-sectional survey was distributed among 677 students from the Faculty of Pharmacy at King Abdulaziz University, Jeddah, Saudi Arabia. Participants were assessed with demographic information, the validated Generalized Anxiety Disorder 7-item (GAD-7) scale, the validated Patient Health Questionnaire-2 (PHQ-2), and multiple stress factors and motivators that might influence their academic development. Collected data were statistically analyzed. Results demonstrated that 32% of the participants were experiencing mild anxiety, 22% moderate anxiety, and 19% severe anxiety. Statistical significance in anxiety level was seen in students living alone, previously diagnosed with mental illness, and within their 4th pharmacy academic year. 49.5% of the participants were PHQ-2 positive and maybe showing symptoms of depressions. Major academic stressor for students with anxiety was their grades while major academic motivator was the coordination of each course studied. Over 70% of pharmacy students had anxiety, and half of them were PHQ-2 positive. We recommend universities to address all students’ psychological needs more often in the future, especially during a crisis like this pandemic and look into the possibility of having a Pass/Fail outcome rather than a grade-based GPA.

25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyses the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca 2+ (Ca 2+o) depending on the tissue. However, the Ca 2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca 2+o-dependent control of 1α-hydroxylase using HEK-293 cells stably expressing the CaSR (HEK-CaSR cells). In HEK-CaSR cells, but not control HEK-293 cells, co-transfected with reporter genes for CYP27B1-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca 2+o from 0.5 to 3.0 mM induced a 2-3 fold increase in firefly-luciferase activity as well as mRNA and protein levels. Surprisingly, firefly-luciferase was specifically suppressed at Ca 2+o ≥ 5.0 mM, demonstrating biphasic Ca 2+o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca 2+o induced simple monotonic increases in firefly-luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca 2+o was post-transcriptional. Studies with inhibitors and the CaSR C-terminal mutant T888A identified roles for PKC, phosphorylation of T888, and ERK1/2 in high Ca 2+o-dependent suppression of firefly-luciferase. Blockade of both PKC and ERK1/2 abolished Ca 2+o-stimulated firefly-luciferase, demonstrating that either PKC or ERK1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (–74 bp to –68 bp), which is regulated downstream of PKC and ERK1/2 was required for both basal transcription and Ca 2+o-mediated transcriptional upregulation. The CaSR mediates Ca 2+o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca 2+o can promote luciferase and possibly 1α-hydroxylase breakdown.