Propranolol Promotes Bone Formation and Limits Resorption Through Novel Mechanisms During Anabolic Parathyroid Hormone Treatment in Female C57BL/6J Mice

Although the nonselective β-blocker, propranolol, improves bone density with parathyroid hormone (PTH) treatment in mice, the mechanism of this effect is unclear. To address this, we used a combination of in vitro and in vivo approaches to address how propranolol influences bone remodeling in the context of PTH treatment. In female C57BL/6J mice, intermittent PTH and propranolol administration had complementary effects in the trabecular bone of the distal femur and fifth lumbar vertebra (L₅), with combination treatment achieving microarchitectural parameters beyond that of PTH alone. Combined treatment improved the serum bone formation marker, procollagen type 1 N propeptide (P1NP), but did not impact other histomorphometric parameters relating to osteoblast function at the L₅. In vitro, propranolol amplified the acute, PTH-induced, intracellular calcium signal in osteoblast-like cells. The most striking finding, however, was suppression of PTH-induced bone resorption. Despite this, PTH-induced receptor activator of nuclear factor κ-B ligand (RANKL) mRNA and protein levels were unaltered by propranolol, which led us to hypothesize that propranolol could act directly on osteoclasts. Using in situ methods, we found Adrb2 expression in osteoclasts in vivo, suggesting β-blockers may directly impact osteoclasts. Consistent with this, we found propranolol directly suppresses osteoclast differentiation in vitro. Taken together, this work suggests a strong anti-osteoclastic effect of nonselective β-blockers in vivo, indicating that combining propranolol with PTH could be beneficial to patients with extremely low bone density. © 2022 American Society for Bone and Mineral Research (ASBMR).     

A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.     

Mitochondrial function and energy metabolism in neuronal HT22 cells resistant to oxidative stress

Background and Purpose

The hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Extracellular glutamate depletes cellular glutathione by blocking the glutamate/cystine antiporter system xc−. Glutathione depletion induces a well-defined programme of cell death characterized by an increase in reactive oxygen species and mitochondrial dysfunction.

Experimental Approach

We compared the mitochondrial shape, the abundance of mitochondrial complexes and the mitochondrial respiration of HT22 cells, selected based on their resistance to glutamate, with those of the glutamate-sensitive parental cell line.

Key Results

Glutamate-resistant mitochondria were less fragmented and displayed seemingly contradictory features: mitochondrial calcium and superoxide were increased while high-resolution respirometry suggested a reduction in mitochondrial respiration. This was interpreted as a reverse activity of the ATP synthase under oxidative stress, leading to hydrolysis of ATP to maintain or even elevate the mitochondrial membrane potential, suggesting these cells endure ineffective energy metabolism to protect their membrane potential. Glutamate-resistant cells were also resistant to oligomycin, an inhibitor of the ATP synthase, but sensitive to deoxyglucose, an inhibitor of hexokinases. Exchanging glucose with galactose rendered resistant cells 1000-fold more sensitive to oligomycin. These results, together with a strong increase in cytosolic hexokinase 1 and 2, a reduced lactate production and an increased activity of glucose-6-phosphate dehydrogenase, suggest that glutamate-resistant HT22 cells shuttle most available glucose towards the hexose monophosphate shunt to increase glutathione recovery.

Conclusions and Implications

These results indicate that mitochondrial and metabolic adaptations play an important role in the resistance of cells to oxidative stress.

Linked Articles

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

QT correction using Bazett’s formula remains preferable in long QT syndrome type 1 and 2

BackgroundThe heart rate (HR) corrected QT interval (QTc) is crucial for diagnosis and risk stratification in the long QT syndrome (LQTS). Although its use has been questioned in some contexts, Bazett''s formula has been applied in most diagnostic and prognostic studies in LQTS patients. However, studies on which formula eliminates the inverse relation between QT and HR are lacking in LQTS patients.We therefore determined which QT correction formula is most appropriate in LQTS patients including the effect of beta blocker therapy and an evaluation of the agreement of the formulae when applying specific QTc limits for diagnostic and prognostic purposes.MethodsAutomated measurements from routine 12‐lead ECGs from 200 genetically confirmed LQTS patients from two Swedish regions were included (167 LQT1, 33 LQT2). QT correction was performed using the Bazett, Framingham, Fridericia, and Hodges formulae. Linear regression was used to compare the formulae in all patients, and before and after the initiation of beta blocking therapy in a subgroup (n = 44). Concordance analysis was performed for QTc ≥ 480 ms (diagnosis) and ≥500 ms (prognosis).ResultsThe median age was 32 years (range 0.1–78), 123 (62%) were female and 52 (26%) were children ≤16 years. Bazett''s formula was the only method resulting in a QTc without relation with HR. Initiation of beta blocking therapy did not alter the result. Concordance analyses showed clinically significant differences (Cohen''s kappa 0.629–0.469) for diagnosis and prognosis in individual patients.ConclusionBazett''s formula remains preferable for diagnosis and prognosis in LQT1 and 2 patients.     

Developing a Competency‐based Curriculum in Basic and Clinical Pharmacology – A Delphi Study among Physicians

A new curriculum is planned for the medical school at Lund University, Sweden. Pharmacology, in a broad sense, has been identified as a subject that needs to be strengthened based on needs in the healthcare system. The aim was to identify the competencies in basic and clinical pharmacology that a newly qualified physician needs. Using a modified three‐round Delphi technique, 31 physicians were invited to list necessary competencies (round 1). After content analysis, these panel members classified the list by importance on two occasions (rounds 2 and 3) using a 4‐point scale (4 = necessary, 3 = desirable, 2 = useful, 1 = not necessary). Competencies with the highest ranks based on necessity were retained. Thirty physicians accepted the invitation and 25 (83%) of them completed all three rounds. Round 1 resulted in 258 suggestions, which were subsequently reduced to 95 competencies. Of these 95 competencies, 40 were considered necessary by at least 75% of the panel members. The degree of consensus increased between round 2 and round 3. Using a modified Delphi technique, we identified 40 competencies that could be transferred to learning outcomes for a new curriculum in basic and clinical pharmacology at medical school.     

Activated platelets promote increased monocyte expression of CXCR5 through prostaglandin E2-related mechanisms and enhance the anti-inflammatory effects of CXCL13

Background

We have previously shown that the homeostatic chemokine CXCL13 is up-regulated in monocytes in atherosclerosis, mediating anti-apoptotic and anti-inflammatory effects.

Objective

To investigate the regulation of CXCL13s receptor, CXCR5.

Methods/patients

In vitro studies in THP-1 and primary monocytes and studies of CXCR5 expression in thrombus material obtained at the site of plaque rupture during myocardial infarction (MI).

Results

Our major findings were: (i) toll-like receptor agonists and particularly β-adrenergic receptor activation and releasate from thrombin-activated platelets increased CXCR5 mRNA levels in monocytes. (ii) The platelet-mediated induction of CXCR5 involved prostaglandin E₂/cAMP/protein kinase A-dependent as well as RANTES-dependent pathways with NFκB activation as a potential common down-stream mediator. (iii) Releasate from thrombin-activated platelets augmented the anti-inflammatory effects of CXCL13 in monocytes at least partly by enhancing the effects of CXCL13 on CXCR5 expression. (iv) We found strong immunostaining of CXCR5 in thrombus material obtained at the site of plaque rupture in patients with ST elevation MI (STEMI) and in unstable carotid lesions, co-localized with platelets.

Conclusion

Our findings suggest that platelet-mediated signaling through CXCR5 may be active in vivo during plaque destabilization, potentially representing a counteracting mechanism to inflammation.     

Handheld ECG in analysis of arrhythmia and heart rate variability in children with Fontan circulation

Background

Our aim was to evaluate the intermittent use of a handheld ECG system for detecting silent arrhythmias and cardiac autonomic dysfunction in children with univentricular hearts.

Methods

Twenty-seven patients performed intermittent ECG recordings with handheld devices during a 14-day period. A manual arrhythmia analysis was performed. We analyzed heart rate variability (HRV) using scatter plots of all interbeat intervals (Poincaré plots) from the total observation period. Reference values of HRV indices were determined from Holter-ECGs in 41 healthy children.

Results

One asymptomatic patient had frequent ventricular extra systoles. Another patient had episodes with supraventricular tachycardia (with concomitant palpitations). Seven patients showed reduced HRV.

Conclusions

Asymptomatic arrhythmia was detected in one patient. The proposed method for pooling of intermittent recordings from handheld or similar devices may be used for detection of arrhythmias as well as for cardiac autonomic dysfunction.     

Engaging a state medical society in oral health: a community action report

Increasingly, the medical community is recognizing how oral health relates to overall health and engaging in oral health promotion. In 2012, the Massachusetts Medical Society set out to explore how to prioritize oral health as a part of its public health agenda. It began with an Oral Health Forum, which evolved into a Committee on Oral Health. This committee is the first of its kind in a state medical society and is comprised of a diverse group of medical and dental professionals. In this article, we review the process by which the committee was established, report on projects, and reflect on lessons learned.