Aristolochic acid, a plant extract used in the treatment of pain and linked to Balkan endemic nephropathy, is a regulator of K2P channels

Background and Purpose: Aristolochic acid (AristA) is found in plants used in traditional medicines to treat pain. We investigated the action of AristA on TREK and TRESK, potassium (K2P) channels, which are potential therapeutic targets in pain. Balkan endemic nephropathy (BEN) is a renal disease associated with AristA consumption. A mutation of TASK-2 (K2P 5.1) channels (T108P) is seen in some patients susceptible to BEN, so we investigated how both this mutation and AristA affected TASK-2 channels. Experimental Approach: Currents through wild-type and mutated human K2P channels expressed in tsA201 cells were measured using whole-cell patch-clamp recordings in the presence and absence of AristA. Key Results: TREK-1- and TREK-2-mediated currents were enhanced by AristA (100 ?M), whereas TRESK was inhibited. Inhibition of TRESK did not depend on the phosphorylation of key intracellular serines but was completely blocked by mutation of bulky residues in the inner pore (F145A_F352A). The TASK-2_T108P mutation markedly reduced both current density and ion selectivity. A related mutation (T108C) had similar but less marked effects. External alkalization and application of flufenamic acid enhanced TASK-2 and TASK-2_T108C current but did not affect TASK-2_T108P current. AristA (300 ?M) produced a modest enhancement of TASK-2 current. Conclusions and Implications: Enhancement of TREK-1 and TREK-2 and inhibition of TRESK by AristA may contribute to therapeutically useful effects of this compound in pain. Whilst AristA is unlikely to interact directly with TASK-2 channels in BEN, loss of functional TASK-2 channels may indirectly increase susceptibility to AristA toxicity

Effects of the ventilatory stimulant, doxapram on human TASK‐3 (KCNK9, K2P9.1) channels and TASK‐1 (KCNK3, K2P3.1) channels

Aims The mode of action by which doxapram acts as a respiratory stimulant in humans is controversial. Studies in rodent models, have shown that doxapram is a more potent and selective inhibitor of TASK-1 and TASK-1/TASK-3 heterodimer channels, than TASK-3. Here we investigate the direct effect of doxapram and chirally separated, individual positive and negative enantiomers of the compound, on both human and mouse, homodimeric and heterodimeric variants of TASK-1 and TASK-3. Methods Whole-cell patch clamp electrophysiology on tsA201 cells was used to assess the potency of doxapram on cloned human or mouse TASK-1, TASK-3 and TASK-2 channels. Mutations of amino acids in the pore-lining region of TASK-3 channels were introduced using site-directed mutagenesis. Results Doxapram was an equipotent inhibitor of human TASK-1 and TASK-3 channels, compared with mouse channel variants, where it was more selective for TASK-1 and heterodimers of TASK-1 and TASK-3. The effect of doxapram could be attenuated by either the removal of the C-terminus of human TASK-3 channels or mutations of particular hydrophobic residues in the pore-lining region. These mutations, however, did not alter the effect of a known extracellular inhibitor of TASK-3, zinc. The positive enantiomer of doxapram, GAL-054, was a more potent antagonist of TASK channels, than doxapram, whereas the negative enantiomer, GAL-053, had little inhibitory effect. Conclusion These data show that in contrast to rodent channels, doxapram is a potent inhibitor of both TASK-1 and TASK-3 human channels, providing further understanding of the pharmacological profile of doxapram in humans and informing the development of new therapeutic agents

Pharmacists detecting atrial fibrillation in general practice: a qualitative focus group study

Background: Atrial fibrillation (AF) affects up to 10% of people aged ≥65 years, yet a third of all cases remain undetected. Practice-based pharmacists are in an ideal position to facilitate opportunistic AF screening, while increasing general practice capacity at a time of workforce crisis. Aim: To explore the perspectives of three stakeholder groups involved in the ‘Pharmacists Detecting Atrial Fibrillation’ (PDAF) study to elucidate the facilitators and barriers to pharmacist-led AF screening in general practice. Design & setting: A qualitative study took place, comprising homogeneous focus groups with stakeholders in Kent, UK. Method: The stakeholder groups — patients, general practice staff (GPS), and clinical pharmacists (CPs) — were recruited using convenience sampling. Audio-recordings were transcribed verbatim and analysed using a deductive Theoretical Domains Framework (TDF) approach. Results: Twenty-five patients, four pharmacists, and nine practice staff participated in six focus groups. Three main themes were identified: knowledge and awareness; prioritisation of resources; and environmental considerations. The public’s lack of awareness of AF-related risks and pharmacist-led screening services was highlighted. Practice-based pharmacists were perceived as an underutilised educational resource which, together with novel electrocardiogram devices, enabled convenient access to screening while reducing GPs’ workload. Participants agreed that AF screening should be incorporated into personalised health checks and at-risk groups should be prioritised, such as care home residents. Patients favoured the general practice environment over the community pharmacy where concerns of privacy, staffing, and commercialisation were raised. Conclusion: The findings of this study support the introduction of pharmacist-led AF screening programmes in general practice surgeries. Commissioners should consider the added value of utilising CPs and focus on the delivery of AF screening within an integrated service

Effects of the ventilatory stimulant, doxapram on human TASK-3 (KCNK9, K2P9.1) channels and TASK-1 (KCNK3, K2P3.1) channels

Aims: The mode of action by which doxapram acts as a respiratory stimulant in humans is controversial. Studies in rodent models, have shown that doxapram is a more potent and selective inhibitor of TASK-1 and TASK-1/TASK-3 heterodimer channels, than TASK-3. Here we investigate the direct effect of doxapram and chirally separated, individual positive and negative enantiomers of the compound, on both human and mouse, homodimeric and heterodimeric variants of TASK-1 and TASK-3. Methods: Whole-cell patch-clamp electrophysiology on tSA201 cells was used to assess the potency of doxapram on cloned human or mouse TASK-1, TASK-3 and TASK-2 channels. Mutations of amino acids in the pore-lining region of TASK-3 channels were introduced using site-directed mutagenesis. Results: Doxapram was an equipotent inhibitor of human TASK-1 and TASK-3 channels, compared with mouse channel variants, where it was more selective for TASK-1 and heterodimers of TASK-1 and TASK-3. The effect of doxapram could be attenuated by either the removal of the C-terminus of human TASK-3 channels or mutations of particular hydrophobic residues in the pore-lining region. These mutations, however, did not alter the effect of a known extracellular inhibitor of TASK-3, zinc. The positive enantiomer of doxapram, GAL-054, was a more potent antagonist of TASK channels, than doxapram, whereas the negative enantiomer, GAL-053, had little inhibitory effect. Conclusion: These data show that in contrast to rodent channels, doxapram is a potent inhibitor of both TASK-1 and TASK-3 human channels, providing further understanding of the pharmacological profile of doxapram in humans and informing the development of new therapeutic agents. This article is protected by copyright. All rights reserved

TRESK is a key regulator of nocturnal suprachiasmatic nucleus dynamics and light adaptive responses

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours

Opportunistic screening for atrial fibrillation by clinical pharmacists in UK general practice during the influenza vaccination season: a cross-sectional feasibility study

Background: Growing prevalence of atrial fibrillation (AF) in the ageing population, and its associated life-changing health and resource implications, have led to a need to improve its early detection. Primary care is an ideal place to screen for AF, however this is limited by shortages in general practitioner (GP) resources. Recent increases in the number of clinical pharmacists within primary care, makes them ideally placed to conduct AF screening. This study aimed to determine the feasibility of GP practice-based clinical pharmacists to screen the over 65s for AF, using digital technology and pulse palpation during the influenza vaccination season. Methods and Findings: Screening was conducted over two influenza vaccination seasons, 2017-2018 and 2018-2019 in four GP practices in Kent, UK. Pharmacists were trained by a cardiologist to pulse palpate, record and interpret a single-lead ECG (SLECG). Eligible persons aged ≥65 years, attending an influenza vaccination clinic were offered a free heart rhythm check. 604 participants were screened (median age 73 years, 42.7% male). Total prevalence of AF was 4.3%. All participants with AF qualified for anticoagulation and were more likely to be male (57.7%); older; have an increased BMI and have a CHA2DS2-VASc (Congestive heart failure, Hypertension, Age ≥ 75, Diabetes, previous Stroke, Age 65-74 years, Sex category) score ≥ 3. The sensitivity and specificity of clinical pharmacists diagnosing AF using pulse palpation was 76.9% [95% CI: 56.4-91.0] and 92.2% [89.7-94.3], respectively. This rose to 88.5% (69.9-97.6) and 97.2% [95.5-98.4] with a SLECG. At follow-up, four participants (0.7%) were diagnosed with new AF and 3 (0.5%) were initiated on anticoagulation. Screening with SLECG also helped identify new non-AF cardiovascular diagnoses, such as left ventricular hypertrophy, in 28 participants (4.6%). The screening strategy was cost-effective in 71.8% and 64.3% of the estimates for SLECG or pulse palpation, respectively. Feedback from participants (422/604) was generally positive. Key limitations of the study were that the intervention did not reach individuals who did not attend the practice for an influenza vaccination and there was a limited representation of UK ethnic minority groups in the study cohort. Conclusions: This study demonstrates that AF screening performed by GP practice-based pharmacists was feasible, economically viable and positively endorsed by participants. Furthermore, diagnosis of AF by the clinical pharmacist using a SLECG was more sensitive and more specific than the use of pulse palpation alone. Future research should explore the key barriers preventing the adoption of national screening programmes

Screening for atrial fibrillation within a South Asian community setting using single-lead ECG

Timely detection and treatment of AF is amongst the Government’s cardiovascular priorities. Several initiatives have demonstrated the potential of AF detection using single-lead electrocardiogram (SLECG) technology in primary care settings, such as general practitioners’ (GP) surgeries. Unfortunately, some AF patients, including those from ethnic minorities, may not routinely engage with main-stream healthcare and may go undetected. Compared to White British, South Asians have a higher prevalence of cardiovascular risk factors, however fewer are diagnosed with AF. This study aimed to ascertain the feasibility of opportunistic AF screening within a South Asian community delivered by supervised pharmacy undergraduates using SL-ECG devices

Characterisation and regulation of wild type and mutant TASK-1 two pore domain potassium channels indicated in pulmonary arterial hypertension

KEY POINTS SUMMARY The TASK-1 channel gene (KCNK3) has been identified as a possible disease-causing gene in heritable pulmonary arterial hypertension (PAH). In this study we show that novel mutated TASK-1 channels, seen in PAH patients, have a substantially reduced current compared to wild type TASK-1 channels. These mutated TASK-1 channels are located at the plasma membrane to the same degree as wild type TASK-1 channels. ONO-RS-082 and alkaline pH 8.4 both activate TASK-1 channels but do not recover current through mutant TASK-1 channels. We show that the guanylate cyclase activator, riociguat, a novel treatment for PAH, enhances current through TASK-1 channels but does not recover current through mutant TASK-1 channels. ABSTRACT Pulmonary arterial hypertension (PAH) affects approximately 15-50 people per million. KCNK3, the gene that encodes the two pore domain potassium channel TASK-1 (K2P3.1), has been identified as a possible disease-causing gene in heritable PAH. Recently two new mutations have been identified in KCNK3, G106R and L214R, in PAH patients. The aim of this study is to characterise the functional properties and regulation of wildtype (WT) and mutated TASK-1 channels and understand how these might contribute to PAH and its treatment. Currents through WT and mutated human TASK-1 channels transiently expressed in tsA201 cells were measured using whole-cell patch-clamp electrophysiology. Localisation of fluorescently-tagged channels was visualised using confocal microscopy and quantified with in-cell and on-cell Westerns. G106R or L214R mutated channels were located at the plasma membrane to the same degree as WT channels, however their current was markedly reduced compared to WT TASK-1 channels. Functional current through these mutated channels could not be restored using activators of WT TASK-1 channels (pH 8.4, ONO-RS-082). The guanylate cyclase activator, riociguat, enhanced current through WT TASK-1 channels, however, like the other activators investigated, riociguat did not have any effect on current through mutated TASK-1 channels. Thus, novel mutations in TASK-1 seen in PAH, substantially alter the functional properties of these channels. Current through these channels could not be restored by activators of TASK-1 channels. Riociguat enhancement of current through TASK-1 channels could contribute to its therapeutic benefit in the treatment of PAH

Enhanced inflammatory cell profiles in schistosomiasis-induced pulmonary vascular remodeling.

Schistosomiasis (bilharzia) is a neglected parasitic disease caused by trematode flatworms of the genus Schistosoma which affects over 240 million people worldwide. It is characterized by the formation of inflammatory granulomas around deposited parasite eggs. Recent studies have revealed that immune and inflammatory responses play a crucial role in pathogenesis of schistosomiasis. The aim of this paper is to systematically evaluate the number and distribution of inflammatory cells in S. mansoni-infected mice at different doses and time points. Immunohistochemistry was performed on lung and liver tissue sections from Schistosoma-infected mice and uninfected healthy controls. Positively stained cells in whole-lung/liver tissue sections, surrounding the eggs, and in the different compartments of the tissues, were counted. We found a significant increase in the number of mast cells (toluidine blue(+)), CD3(+) cells, CD14(+) cells, CD68(+) cells, and CD15(+) cells in Schistosoma-infected tissues compared with untreated healthy controls (P???0.05 for all). Our findings revealed altered and enhanced immune cell infiltration in schistosomiasis. We suggest that these cells may contribute to the pathophysiology of Schistosoma resulting in pulmonary vascular remodeling

Screening for atrial fibrillation in care homes using pulse palpation and the AliveCor Kardia Mobile® device: a comparative cross-sectional pilot study.

Atrial fibrillation (AF) is a major cause of stroke in older people. Exacerbated by age and co-morbidities, residents of care homes are more likely to develop AF and less likely to receive oral anticoagulants. To determine the prevalence of AF using the design and methodology of the Pharmacists Detecting Atrial Fibrillation (PDAF) study in a care home setting. A cross-sectional AF screening pilot study within four UK care homes, three residential and one residential/nursing. Screening followed the original PDAF protocol: a manual pulse check, followed by a single-Lead ECG ( ECG, AliveCor Kardia Mobile (KMD)) delivered by a pharmacist. All recorded ECG were reviewed by a cardiologist and any residents requiring follow-up investigations were referred to their general practitioner. Fifty-three of 112 care home residents participated. From 52 ECGs recorded, the cardiologist interpreted 13.5% (7/52) as having possible AF of which 9.6% (5/52) were previously unknown. One resident with previously unknown AF received anticoagulation. This study has shown a need for AF screening in care homes and that elements of the PDAF screening protocol are transferable in this setting. Early diagnosis and treatment of AF are essential to reduce the risk of stroke in this population. [Abstract copyright: © 2023. The Author(s).