Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is an antimicrobial agent whose actions as a zinc or copper ionophore and an iron chelator revived the interest in similar compounds for the treatment of fungal and bacterial infections, neurodegeneration and cancer. Recently, we reported zinc ionophores, including clioquinol, cause vasorelaxation in isolated arteries through mechanisms that involve sensory nerves, endothelium and vascular smooth muscle. Here, we report that clioquinol also uniquely acts as a competitive alpha-1 (α1) adrenoceptor antagonist. We employed ex vivo functional vascular contraction and pharmacological techniques in rat isolated mesenteric arteries, receptor binding assays using stabilized solubilized α1 receptor variants, or wild-type human α1-adrenoceptors transfected in COS-7 cells (African green monkey kidney fibroblast-like cells), and molecular dynamics homology modelling based on the recently published α1A adrenoceptor cryo-EM and α1B crystal structures. At higher concentrations, all ionophores including clioquinol cause a non-competitive antagonism of agonist-mediated contraction due to intracellular zinc delivery, as reported previously. However, at lower concentration ranges, clioquinol has an additional mechanism of competitively inhibiting α1-adrenoceptors that contributes to decreasing vascular contractility. Molecular dynamic simulation showed that clioquinol binds stably to the orthosteric binding site (Asp106) of the receptor, confirming the structural basis for competitive α1-adrenoceptor antagonism by clioquinol.

Cannabidiol is increasingly considered for treatment of a wide range of medical conditions. Binding studies suggest that cannabidiol binds to CB1 receptors. In the rat isolated vas deferens bioassay, a single electrical pulse causes a biphasic contraction from nerve-released ATP and noradrenaline. WIN 55,212-2 acts on prejunctional CB1 receptors to inhibit release of these transmitters. In this bioassay, we tested whether cannabidiol and SR141716 were acting as competitive antagonists of this receptor. Monophasic contractions mediated by ATP or noradrenaline in the presence of prazosin or NF449 (P2X1 inhibitor), respectively, were measured to a single electrical pulse delivered every 30 min. Following treatment with cannabidiol (10-100 mu M) or SR141716 (0.003-10 mu M), cumulative concentrations of WIN 55,212-2 (0.001-30 mu M) were applied followed by a single electrical pulse. The WIN 55,212-2 concentration-contraction curve EC50 values were applied to global regression analysis to determine the pK(B). The antagonist potency of cannabidiol at the CB1 receptor in the rat vas deferens bioassay matched the reported receptor binding affinity. Cannabidiol was a competitive antagonist of WIN 55,212-2 with pK(B) values of 5.90 when ATP was the effector transmitter and 5.29 when it was noradrenaline. Similarly, SR141716 was a competitive antagonist with pK(B) values of 8.39 for ATP and 7.67 for noradrenaline as the active transmitter. Cannabidiol's low micromolar CB1 antagonist pK(B) values suggest that at clinical blood levels (1-3 mu M) it may act as a CB1 antagonist at prejunctional neuronal sites with more potency when ATP is the effector than for noradrenaline.

The pharmacology of cannabidiol, the non-psychoactive major component of Cannabis saliva, is of growing interest as it becomes more widely prescribed. This study aimed to examine the effects of cannabidiol on a wide range of contractile agents in rat small resistance arteries, in comparison with large arteries, and to explore its mechanism of action. The vascular actions of cannabidiol were also contrasted with effects on the contractions of bronchial, urogenital, cardiac and skeletal muscles. Isolated small or large arteries were incubated with cannabidiol (0.3-3 mu M) or vehicle and concentration-contraction response curves were completed to various agents, including endothelin-1, arginine vasopressin, methoxamine, 5-HT, alpha-methyl 5-HT and U46619. In small arteries, the effects of cannabidiol were tested in the presence of antagonists of CB1 or CB2 receptors, calcitonin generelated peptide (CGRP), nitric oxide synthase, cyclooxygenase, PPAR gamma or a combination. The role of L-type voltage-operated calcium channels was also assessed. Cannabidiol 1-3 mu M significantly inhibited the contraction of small resistance arteries to all tested agents through a combination of mechanisms that include CGRP and L-type calcium channels. However, large arteries were insensitive to cannabidiol. Cannabidiol (10-100 mu M) was largely without effect in bronchi, atria and hemidiaphragm, but 100 mu M attenuated maximum contractions in vasa deferentia. Cannabidiol's effects in the clinical range (1-3 mu M) appear to be specific to small resistance arteries. This high sensitivity of the resistance arterial circulation to cannabidiol may offer a therapeutic opportunity in peripheral vascular disease that excludes off-target sites such as the heart and non-vascular smooth muscle.

Zinc, an abundant transition metal, serves as a signalling molecule in several biological systems. Zinc transporters are genetically associated with cardiovascular diseases but the function of zinc in vascular tone regulation is unknown. We found that elevating cytoplasmic zinc using ionophores relaxed rat and human isolated blood vessels and caused hyperpolarization of smooth muscle membrane. Furthermore, zinc ionophores lowered blood pressure in anaesthetized rats and increased blood flow without affecting heart rate. Conversely, intracellular zinc chelation induced contraction of selected vessels from rats and humans and depolarized vascular smooth muscle membrane potential. We demonstrate three mechanisms for zinc-induced vasorelaxation: (1) activation of transient receptor potential ankyrin 1 to increase calcitonin gene-related peptide signalling from perivascular sensory nerves; (2) enhancement of cyclooxygenase-sensitive vasodilatory prostanoid signalling in the endothelium; and (3) inhibition of voltage-gated calcium channels in the smooth muscle. These data introduce zinc as a new target for vascular therapeutics.

Human envenoming by Australian brown snakes (Pseudonaja spp.) may result in potentially life-threatening hypotension and subsequent cardiovascular collapse. There have been relatively few studies of the cardiovascular and sympathetic effects of Pseudonaja spp. venoms. In this study, we have examined the effects of venom from five brown snake species—P. affinis, aspidorhyncha, inframacula, nuchalis, and textilis—on cardiac inotropic and chronotropic responses, vascular tone, and sympathetic nerve-induced vascular contractions in rat isolated tissues. The role of phospholipases A2 (PLA2s) in venom-induced effects was assessed with the sPLA2 inhibitor varespladib. In rat isolated left and right atria, there were no physiologically relevant effects of Pseudonaja venoms (0.1–30 µg/ml) on left atrial force of contraction (inotropy) or right atrial rate (chronotropy). In contrast, in isolated small mesenteric arteries precontracted with a thromboxane mimetic, each of the five brown snake venoms (at 30 µg/ml) caused marked vasorelaxation (−60 to –90% of contractile tone). Pretreatment with varespladib (1 µM) significantly inhibited the vasorelaxation caused by P. aspidorhyncha, P. nuchalis, and P. textilis venoms. Electrically induced sympathetic nerve-mediated contractions of mesenteric arteries were significantly attenuated by only P. textilis, and P. affinis venoms (30 µg/ml) and these sympatholytic effects were inhibited by varespladib (1 µM). Based on their inhibition with the sPLA2 inhibitor varespladib, we conclude that PLA2 toxins in P. aspidorhyncha, P. nuchalis, and P. textilis venoms are involved in brown snake venom-induced vasorelaxation and the sympatholytic effects of P. affinis, and P. textilis venoms. Our study supports the promising potential role of varespladib as an initial (pre-referral) and/or adjunct (in combination with antivenom) therapeutic agent for brown snake envenoming.

Objectives: The vascular amplifier in hypertension is a result of structural changes in resistance arteries. We estimated the vascular amplifier hypertensive:normotensive (H:N) ratio in the renal bed compared with the total peripheral bed in conscious rabbits during infusion of vasoconstrictor and vasodilator stimuli. Methods: Rabbits were subjected to bilateral renal cellophane wrap or sham operation. A perivascular ultrasonic flow probe was implanted on the left renal artery to measure renal blood flow. A catheter was inserted into the thoracic aorta for agonist administration. Blood pressure, heart rate and renal blood flow were measured on three separate days in conscious rabbits with intact effectors, ganglionic block or neurohumoral block. Dose-response curves were constructed to intra-arterial infusion of noradrenaline, angiotensin II, adenosine and acetylcholine. Results: Resting renal vascular resistance in hypertensive rabbits was markedly decreased by ganglionic block and further by neurohumoral block. With effectors intact, ganglionic block or neurohumoral block, the H:N ratio for renal vascular resistance was 2.32, 1.72 or 1.72, respectively. The ratio was generally maintained during the infusion of constrictor and dilator drugs although distortions occurred at higher concentrations of constrictor or dilator drugs. Conclusions: Estimation of the renal resistance amplifier in renal wrap hypertension with neurohumoral block accords with our earlier estimates of the total peripheral resistance amplifier (1.79). This vascular resistance amplifier is consistent with a decrease in internal radius through structural remodelling in the renal vascular bed as is reflected in the total arterial circulation in hypertension.

Recently, the beta(2)-adrenoceptor agonist terbutaline was shown to have alpha(1)-adrenolytic activity in mouse isolated pulmonary arteries in vitro and to lower pulmonary artery pressure in anaesthetised mice. The aim of our study was to determine the alpha(1)-adrenoceptor antagonist activity of terbutaline and its structurally close resorcinol, orciprenaline, in rat isolated small mesenteric arteries set up for myography. Their alpha(1)-adrenoceptor antagonist potency was then compared with their potency as beta(2)-adrenoceptor agonists. Concentration-response curves to methoxamine were competitively antagonised by terbutaline (30-300 mu M) or orciprenaline (30-300 mu M) with a pK(B) of 4.70 +/- 0.09 or 4.79 +/- 0.17, respectively. Both terbutaline and orciprenaline fulfilled the criteria for simple, silent competitive antagonism. Terbutaline (30-300 mu M) had no effect on endothelin-1 concentration-contraction curves. Our findings suggest that after oral dosing of terbutaline, the maximum plasma levels would NOT reach levels to show alpha(1)-adrenoceptor antagonist activity. In conclusion, our work has provided additional quantitative evidence that terbutaline and orciprenaline are weak competitive alpha(1)-adrenoceptor antagonists, but this additional property is probably not therapeutically important in the clinical treatment of asthma or pulmonary artery hypertension with these more potent beta(2)-adrenoceptor agonists.

In the pulmonary vasculature there is clearance of endothelin-1 from the circulation mediated by endothelin ETB receptors. This study explored the haemodynamic effects of endothelin-1 and its clearance in the pulmonary and hindquarter vasculature in anaesthetised rats. Carotid and pulmonary artery pressures and pulmonary and hindquarter blood flows were measured. In each rat, a single endothelin-1 or sarafotoxin S6C cumulative dose-response curve was generated with or without antagonist pretreatment (i.v.). Endothelin-1 caused an acute fall in MAP and rise in hindquarter vascular conductance (HVC) followed by a marked increase in MAP at 5 min with falls in HVC and pulmonary vascular conductance (PVC). Bosentan (10, 20 & 30 mg/kg) pretreatment caused dose-dependent inhibition of the MAP increase as well as PVC and HVC decreases to endothelin-1. Similarly, macitentan (30 mg/kg) or ambrisentan (10 mg/kg) caused significant block of responses to endothelin-1. Sarafotoxin S6C caused acute falls in MAP and increases in HVC and then small falls in PVC and HVC, all prevented by pretreatment with ET B antagonist BQ788 (1 mg/kg). Pretreatment with BQ788 enhanced endothelin-1 potency by 2.5-fold in PVC and 2.4-fold in HVC. With BQ788 and bosentan, the fall in HVC response was completely blocked, but there were residual MAP rises and PVC falls at the highest endothelin-1 dose. Our work confirms the role of ETB receptors in the pulmonary vasculature that decrease the circulating levels of endothelin-1. This has important consequences in selecting an appropriate ETA and ETB dual receptor antagonist to effectively block endothelin-1-mediated pulmonary vasoconstriction.

Valproic acid (VPA) is a medication that is widely used in the treatment of epilepsy and bipolar disorder, despite a known potential for liver damage in some patients and a specific risk of teratogenic effects if taken by pregnant women. Here the metabolism of VPA has been investigated in male adult Sprague-Dawley rats using the twin-ion method and HiTIME (High-resolution Twin-Ion Metabolite Extraction) analysis under acute dosing. VPA and 13C4-VPA were administered to rats via intra venous infusion and then blood extracts were analysed by liquid chromatography mass spectrometry (LC-MS). Following HiTIME analysis, 13 high-scoring data regions were identified that were not present in control samples and corresponded to VPA together with 12 known VPA metabolites. Tandem mass spectrometry was performed on selected VPA metabolites. Increasing the dose of VPA resulted in an increase in the proportion of phase II metabolites formed at the expense of oxidative phase I products. A twin ion corresponding to a putative VPA-ascorbate adduct was observed and confirmed via independent synthesis. The origin of twin-ion artefacts detected in LC-MS is also discussed as well as proposed methods to reduce their formation.

The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ± 7% and raising Mg2+ to 3 mM decreased it to 39 ± 3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ± 3%) but was decreased to 63 ± 4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.

BackgroundThis chapter describes analyses of data from the GP Patient Survey investigating which patients have a preference for seeing a particular GP and how successful they are in seeing that doctor. We report these trends over a 4-year period. In addition, we undertook analyses to examine whether or not patients’ expectations of who they wished to see and who they did see (a doctor or a nurse) influenced their assessment of the consultation.Methods

Patients who experience a cardiac event are at higher risk of developing depression than the general population. Despite this, cardiac rehabilitation (CR) programmes do not provide a systematic approach to psychological care for depression. The CADENCE study aimed to develop and pilot an enhanced psychological care (EPC) intervention consisting of behavioural activation (BA) and mental health care coordination. Following original research commissioning guidance, the intervention was planned to be embedded in routine care and delivered by CR nurses to patients with depression attending CR. This paper describes how qualitative methods were used to develop, embed and refine the intervention.

BackgroundDespite widespread adoption of patient feedback surveys in the NHS, evidence of a demonstrable impact of surveys on service improvement is sparse. The objective of this study was to explore the views of primary care practice staff regarding the utility of patient experience surveys.MethodsWe conducted focus groups with staff from 14 practices following the receipt of feedback from a recent patient experience survey.ResultsAlthough participants engaged with feedback from patient experience surveys, they routinely questioned its validity and reliability. Participants identified surveys as having a number of useful functions: for patients, as a potentially therapeutic way of getting their voice heard; for practice staff, as a way of identifying areas of improvement; and for GPs, as a source of evidence for professional development and appraisal. Areas of potential change stimulated by survey feedback included …

BackgroundTo date, little research has focused on doctors’ attitudes to patient experience surveys that give them personalised feedback. Although national surveys, such as the GP Patient Survey, report results at a practice level, GPs are additionally required to reflect on individual-level patient feedback for the purposes of appraisal and revalidation. This chapter examines doctors’ perceptions of patient experience surveys, and the receipt of personal feedback from these, in primary care settings.MethodsWe analysed data from 21 interviews conducted with GPs across 14 practices. Participants were sampled from doctors who had participated in our patient experience survey (reported in Chapter 9) and had recently received individual-level survey feedback.ResultsGeneral practitioners expressed commitment to incorporating patient feedback in quality improvement efforts. However, they also expressed negative …

Background: There has been an increased focus towards improving quality of care within the NHS in the last 15 years; as part of this, there has been an emphasis on the importance of patient feedback within policy, through National Service Frameworks and the Quality and Outcomes Framework. The development and administration of large-scale national patient surveys to gather representative data on patient experience, such as the national GP Patient Survey in primary care, has been one such initiative. However, it remains unclear how the survey is used by patients and what impact the data may have on practice. Objectives: Our research aimed to gain insight into how different patients use surveys to record experiences of general practice; how primary care staff respond to feedback; and how to engage primary care staff in responding to feedback. Methods: We used methods including quantitative survey analyses, focus groups, interviews, an exploratory trial and an experimental vignette study. Results: (1) Understanding patient experience data. Patients readily criticised their care when reviewing consultations on video, although they were reluctant to be critical when completing questionnaires. When trained raters judged communication during a consultation to be poor, a substantial proportion of patients rated the doctor as ‘good’ or ‘very good’. Absolute scores on questionnaire surveys should be treated with caution; they may present an overoptimistic view of general practitioner (GP) care. However, relative rankings to identify GPs who are better or poorer at communicating may be acceptable, as long as statistically reliable figures are obtained. Most patients have a particular GP whom they prefer to see; however, up to 40% of people who have such a preference are unable regularly to see the doctor of their choice. Users of out-of-hours care reported worse experiences when the service was run by a commercial provider than when it was run by a not-for profit or NHS provider. (2) Understanding patient experience in minority ethnic groups. Asian respondents to the GP Patient Survey tend to be registered with practices with generally low scores, explaining about half of the difference in the poorer reported experiences of South Asian patients than white British patients. We found no evidence that South Asian patients used response scales differently. When viewing the same consultation in an experimental vignette study, South Asian respondents gave higher scores than white British respondents. This suggests that the low scores given by South Asian respondents in patient experience surveys reflect care that is genuinely worse than that experienced by their white British counterparts. We also found that service users of mixed or Asian ethnicity reported lower scores than white respondents when rating out-of-hours services. (3) Using patient experience data. We found that measuring GP–patient communication at practice level masks variation between how good individual doctors are within a practice. In general practices and in out-of-hours centres, staff were sceptical about the value of patient surveys and their ability to support service reconfiguration and quality improvement. In both settings, surveys were deemed necessary but not sufficient. Staff expressed a preference for free-text comments, as these provided more tangible, actionable data. An exploratory trial of real-time feedback (RTF) found that only 2.5% of consulting patients left feedback using touch screens in the waiting room, although more did so when reminded by staff. The representativeness of responding patients remains to be evaluated. Staff were broadly positive about using RTF, and practices valued the ability to include their own questions. Staff benefited from having a facilitated session and protected time to discuss patient feedback. Conclusions: Our findings demonstrate the importance of patient experience feedback as a means of informing NHS care, and confirm that surveys are a valuable resource for monitoring national trends in quality of care. However, surveys may be insufficient in themselves to fully capture patient feedback, and in practice GPs rarely used the results of surveys for quality improvement. The impact of patient surveys appears to be limited and effort should be invested in making the results of surveys more meaningful to practice staff. There were several limitations of this programme of research. Practice recruitment for our in-hours studies took place in two broad geographical areas, which may not be fully representative of practices nationally. Our focus was on patient experience in primary care; secondary care settings may face different challenges in implementing quality improvement initiatives driven by patient feedback. Recommendations for future research include consideration of alternative feedback methods to better support patients to identify poor care; investigation into the factors driving poorer experiences of communication in South Asian patient groups; further investigation of how best to deliver patient feedback to clinicians to engage them and to foster quality improvement; and further research to support the development and implementation of interventions aiming to improve care when deficiencies in patient experience of care are identified. Funding: The National Institute for Health Research Programme Grants for Applied Research programme.

BackgroundPatient feedback instruments used in national survey programmes are robustly tested and evaluated, yet there remains a paucity of evidence on the drivers of a patient’s choice of response option. The objective of this study was to understand how patients’ responses to a questionnaire relate to their actual experience of a consultation with a GP, focusing on both implicit and explicit processes that respondents use to answer survey items.MethodsWe video recorded GP–patient consultations at 13 practices. Immediately following the consultation, patients were asked to complete a questionnaire about the GPs’ communication skills. We purposively approached a sample of these patients to take part in a video elicitation interview (n= 52), in which they were shown the video of their consultation and asked to reflect on their completion of the questionnaire.ResultsAlthough participants were able to raise …

Aggregation of tau protein into intracellular deposits is a pathognomonic feature of tauopathies such as Alzheimer's disease (AD) and lowering tau is a prominent therapeutic strategy under development. However, the physiological function of tau protein is not well known, particularly in the periphery. Lowering tau protein risks disrupting its physiological role leading to unwanted effects. In this study, the presence of tau protein in cardiac tissue is confirmed and the functional role in the cardiovascular system and the consequences of its loss were explored. Isolated right and left atria and small mesenteric arteries from wild type and tau deficient (KO) mice of two age groups (13 and 23 months old) were used to assess cardiovascular phenotypes. Tau KO mice showed an increased systolic blood pressure and cardiac hypertrophy at 13 months, whichwas accompanied by a significantly lower right atrial rate and a subtle decrease in the maximum contractility to calcium, isoprenaline, and electrical sympathetic nerve stimulation. Aging tau KO mice to 23 months resulted in cardiac hypertrophy with significantly attenuated left atrial contractility, increased blood pressure, and sensitivity of isolated mesenteric arteries to angiotensin II contraction and isoprenaline relaxation compared to their younger counterparts. This study supports a functional role of tau in the heart and loss of this protein leads to a deterioration in cardiovascular performance which worsens with age. Taken together, these results provide insight into the peripheral function of tau protein, and give caution to the therapeutic strategy of lowering tau protein.