The Role of Sex in the Risk of Mortality From COVID-19 Amongst Adult Patients: A Systematic Review.

A worldwide outbreak of coronavirus disease 2019 (COVID-19), identified as being caused by the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), was classified as a Public Health Emergency of International Concern by the World Health Organisation (WHO) on January 30, 2020. Initial sex-disaggregated mortality data emerging from the Wuhan province of China identified male sex as a risk factor for increased COVID-19 mortality.   In this systematic review, we aimed to assess the role of sex in the risk of mortality from COVID-19 in adult patients through comparison of clinical markers and inflammatory indexes.   A systematic search was conducted on the following databases: PubMed, WHO COVID-19 database, Ovid MEDLINE, and Web of Science between the dates of June 15, 2020, and June 30, 2020. Key search terms used included: "sex", "gender", "SARS-COV-2", "COVID" and "mortality". We accepted the following types of studies concerning adult COVID-19 patients: retrospective cohort, observational cohort, case series, and applied research. Further studies were extracted from reference searching. The risk of bias was determined using the National Institutes of Health Quality Assessment Tool for Observational Cohort, Cross-Sectional Studies, and Case Series.  We identified a total of 16 studies published between January 2020 and June 2020 for analysis in this systematic review. Our study population consisted of 11 cohort studies, four case series, and one genetic study, including a total of 76,555 participants. Ten of the studies included in this review observed a higher risk of mortality among males compared to females, and eight of these studies found this risk to be statistically significant.    Sex-disaggregated COVID-19 mortality data identifies male patients with comorbidities as being at an increased risk of mortality worldwide. Further investigation revealed differences in immune response regulated by sex hormones, angiotensin-converting enzyme 2 (ACE2) expression, and health behaviours as contributing factors to increased risk of mortality from COVID-19 among males.    Nine out of the 16 studies included were conducted in China. In order to comprehensively assess sex-differences in the risk of mortality from COVID-19, more studies will need to be conducted worldwide. Sex-disaggregated COVID-19 data published in the medical literature is limited, however it has become evident that male sex is an important risk factor for mortality. Further exploration into the impact of sex on this pandemic is required in order to develop targeted therapies, as well as public health policies, and to prevent sex bias in treatment.

Clinical Efficacy of Ketamine for Treatment-resistant Depression.

Depression is a common psychiatric disorder affecting more than 300 million people worldwide. According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), the diagnosis of depression requires at least two weeks of either low mood or anhedonia as well as four or more other symptoms such as appetite or weight changes, insomnia or hypersomnia, psychomotor agitation or retardation, loss of energy, inability to concentrate, feelings of worthlessness or excessive guilt, and suicidality. Selective serotonin reuptake inhibitors (SSRIs) target the monoaminergic system and are the commonest drugs used for treating depression, but have certain limitations, such as their delayed onset of action. Ketamine, a non-competitive NMDA receptor antagonist, has shown in several randomized controlled trials (RCTs) promising results with rapid antidepressant effects, especially in patients with severe treatment-resistant depression (TRD), which is depression that has not responded to more than two antidepressants. In this review, the clinical efficacy of ketamine in TRD has been discussed, with emphasis placed on the evidence from RCTs.

Efficacy and Safety of Deep Brain Stimulation in the Treatment of Parkinson's Disease: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

Deep brain stimulation (DBS) is a neurosurgical procedure indicated for patients with advanced Parkinson's disease (PD). Whether similar benefits may be realized by patients with early PD, however, is currently unclear, especially given the potential risks of the procedure. This systematic review and meta-analysis aimed to investigate the relative efficacy and safety of DBS in comparison to best medical therapy (BMT) in the treatment of PD. It also aimed to compare the efficacy of DBS between patients with early and advanced PD. A systematic search was performed in Medline, Embase, and Cochrane Central Register of Controlled Trials (CENTRAL). Randomized controlled trials (RCTs) comparing DBS to BMT in PD patients were included. Outcome measures were impairment/disability using the Unified Parkinson's Disease Rating Scale (UPDRS), quality of life (QoL) using the Parkinson's Disease Questionnaire (PDQ-39), levodopa equivalent dose (LED) reduction, and rates of serious adverse events (SAE). Eight eligible RCTs (n = 1,189) were included in the meta-analysis, two of which recruited early PD patients. Regarding efficacy outcomes, there were significant improvements in UPDRS, PDQ-39, and LED scores in favour of DBS (P < 0.00001). There was a significantly greater reduction of LED in patients with early PD (P < 0.00001), but no other differences between early and advanced PD patients were found. The risk of a patient experiencing an SAE was significantly higher in the DBS group (P = 0.005), as was the total number of SAEs (P < 0.00188). Overall, DBS was superior to BMT at improving impairment/disability, QoL, and reducing medication doses, but these benefits need to be weighed against the higher risk of SAEs. There was insufficient evidence to determine the impact of the PD stage on the efficacy of DBS.

Checkpoint inhibitors for malignant melanoma: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: Rates of malignant melanoma are continuing to increase, and until recently effective treatments were lacking. However, since 2011 three immunotherapeutic agents, known as checkpoint inhibitors, have been approved. This review aims to establish whether these three drugs - ipilimumab, nivolumab, and pembrolizumab - offer greater efficacy and tolerability compared to control interventions (placebo, immunotherapy, or chemotherapy) in patients with stage III or IV unresectable cutaneous melanoma. MATERIALS AND METHODS: A search on four major medical and scientific databases yielded 7,553 records, of which seven met the inclusion criteria, with a total study population of 3,628. Only prospective Phase II or III randomized controlled trials on checkpoint inhibitors for patients with unresectable cutaneous melanoma that reported data on survival (overall or progression-free), tumor response, or adverse events were included. Three meta-analyses were carried out. RESULTS: The hazard ratio for progression or death was 0.54 (95% confidence interval [CI]: 0.44-0.67), and the odds ratio for best overall response rate was 4.48 (95% CI: 2.77-7.24), both in favor of checkpoint inhibitors. However, control treatments were associated with an insignificantly lower rate of discontinuation of treatment due to adverse effects or treatment-related adverse events (odds ratio =1.63 [95% CI: 0.55-4.88]). CONCLUSION: This study finds that checkpoint inhibitors are more effective than control interventions, both in terms of survival and tumor response, and yet no less tolerable. PD1 therapies (nivolumab and pembrolizumab) appear to offer greater efficacy than CTLA4 therapy (ipilimumab). The combination of nivolumab and ipilimumab was, however, the most effective, but significantly less tolerable than monotherapy. The lack of published clinical data does, however, limit this study. Further research is needed in two areas in particular: 1) to determine the optimal use of checkpoint inhibitors, specifically in terms of combination therapy, and 2) to identify reliable biomarkers to predictive responders and guide treatment assignment.

Effects of sleep deprivation on cognitive and physical performance in university students.

Sleep deprivation is common among university students, and has been associated with poor academic performance and physical dysfunction. However, current literature has a narrow focus in regard to domains tested, this study aimed to investigate the effects of a night of sleep deprivation on cognitive and physical performance in students. A randomized controlled crossover study was carried out with 64 participants [58% male (n = 37); 22 ± 4 years old (mean ± SD)]. Participants were randomized into two conditions: normal sleep or one night sleep deprivation. Sleep deprivation was monitored using an online time-stamped questionnaire at 45 min intervals, completed in the participants' homes. The outcomes were cognitive: working memory (Simon game© derivative), executive function (Stroop test); and physical: reaction time (ruler drop testing), lung function (spirometry), rate of perceived exertion, heart rate, and blood pressure during submaximal cardiopulmonary exercise testing. Data were analysed using paired two-tailed T tests and MANOVA. Reaction time and systolic blood pressure post-exercise were significantly increased following sleep deprivation (mean ± SD change: reaction time: 0.15 ± 0.04 s, p = 0.003; systolic BP: 6 ± 17 mmHg, p = 0.012). No significant differences were found in other variables. Reaction time and vascular response to exercise were significantly affected by sleep deprivation in university students, whilst other cognitive and cardiopulmonary measures showed no significant changes. These findings indicate that acute sleep deprivation can have an impact on physical but not cognitive ability in young healthy university students. Further research is needed to identify mechanisms of change and the impact of longer term sleep deprivation in this population.

Blended learning: how can we optimise undergraduate student engagement?

BACKGROUND: Blended learning is a combination of online and face-to-face learning and is increasingly of interest for use in undergraduate medical education. It has been used to teach clinical post-graduate students pharmacology but needs evaluation for its use in teaching pharmacology to undergraduate medical students, which represent a different group of students with different learning needs. METHODS: An existing BSc-level module on neuropharmacology was redesigned using the Blended Learning Design Tool (BLEnDT), a tool which uses learning domains (psychomotor, cognitive and affective) to classify learning outcomes into those taught best by self-directed learning (online) or by collaborative learning (face-to-face). Two online courses were developed, one on Neurotransmitters and the other on Neurodegenerative Conditions. These were supported with face-to-face tutorials. Undergraduate students' engagement with blended learning was explored by the means of three focus groups, the data from which were analysed thematically. RESULTS: Five major themes emerged from the data 1) Purpose and Acceptability 2) Structure, Focus and Consolidation 3) Preparation and workload 4) Engagement with e-learning component 5) Future Medical Education. CONCLUSION: Blended learning was acceptable and of interest to undergraduate students learning this subject. They expressed a desire for more blended learning in their courses, but only if it was highly structured, of high quality and supported by tutorials. Students identified that the 'blend' was beneficial rather than purely online learning.

Ins(1,4,5)P3 interacts with PIP2 to regulate activation of TRPC6/C7 channels by diacylglycerol in native vascular myocytes.

We investigated synergism between inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG) on TRPC6-like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P(3) at 10 microm increased 3-fold TRPC6-like activity induced by 10 microm 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue. Ins(1,4,5)P(3) had no effect on OAG-induced TRPC6 activity in mesenteric artery myocytes. Anti-TRPC6 and anti-TRPC7 antibodies blocked channel activity in portal vein but only anti-TRPC6 inhibited activity in mesenteric artery. TRPC6 and TRPC7 proteins strongly associated in portal vein but only weakly associated in mesenteric artery tissue lysates. Therefore in portal vein the conductance consists of TRPC6/C7 subunits, while OAG activates a homomeric TRPC6 channel in mesenteric artery myocytes. Wortmannin at 20 microm reduced phosphatidylinositol 4,5-bisphosphate (PIP(2)) association with TRPC6 and TRPC7, and produced a 40-fold increase in OAG-induced TRPC6/C7 activity. Anti-PIP(2) antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8-PIP(2), a water-soluble PIP(2) analogue, inhibited OAG-induced TRPC6/C7 activity with an IC(50) of 0.74 microm. Ins(1,4,5)P(3) rescued OAG-induced TRPC6/C7 activity from inhibition by diC8-PIP(2) in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P(3) receptor antagonist heparin. In contrast, Ins(1,4,5)P(3) did not overcome diC8-PIP(2)-induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6-Tri-O-butyryl-Ins(1,4,5)P(3)/AM (6-Ins(1,4,5)P(3)), a cell-permeant analogue of Ins(1,4,5)P(3), at 10 microm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP(2), but not TRPC6 and PIP(2). In contrast, 10 microm OAG reduced association between TRPC6 and PIP(2), but not between TRPC7 and PIP(2). The present work provides the first evidence that Ins(1,4,5)P(3) modulates native TRPC channel activity through removal of the inhibitory action of PIP(2) from TRPC7 subunits.

Activation of native TRPC1/C5/C6 channels by endothelin-1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes.

We investigate activation mechanisms of native TRPC1/C5/C6 channels (termed TRPC1 channels) by stimulation of endothelin-1 (ET-1) receptor subtypes in freshly dispersed rabbit coronary artery myocytes using single channel recording and immunoprecipitation techniques. ET-1 evoked non-selective cation channel currents with a unitary conductance of 2.6 pS which were not inhibited by either ET(A) or ET(B) receptor antagonists, respectively BQ-123 and BQ788, when administered separately. However, in the presence of both antagonists, ET-1-evoked channel activity was abolished indicating that both ET(A) and ET(B) receptor stimulation activate this conductance. Stimulation of both ET(A) and ET(B) receptors evoked channel activity which was inhibited by the protein kinase C (PKC) inhibitor chelerythrine and by anti-TRPC1 antibodies indicating that activation of both receptor subtypes causes TRPC1 channel activation by a PKC-dependent mechanism. ET(A) receptor-mediated TRPC1 channel activity was selectively inhibited by phosphoinositol-3-kinase (PI-3-kinase) inhibitors wortmannin (50 nM) and PI-828 and by antibodies raised against phosphoinositol-3,4,5-trisphosphate (PIP(3)), the product of PI-3-kinase-mediated phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Moreover, exogenous application of diC8-PIP(3) stimulated PKC-dependent TRPC1 channel activity. These results indicate that stimulation of ET(A) receptors evokes PKC-dependent TRPC1 channel activity through activation of PI-3-kinase and generation of PIP(3). In contrast, ET(B) receptor-mediated TRPC1 channel activity was inhibited by the PI-phospholipase C (PI-PLC) inhibitor U73122. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), an analogue of diacylglycerol (DAG), which is a product of PI-PLC, also activated PKC-dependent TRPC1 channel activity. OAG-induced TRPC1 channel activity was inhibited by anti-phosphoinositol-4,5-bisphosphate (PIP(2)) antibodies and high concentrations of wortmannin (20 microM) which depleted tissue PIP(2) levels. In addition exogenous application of diC8-PIP(2) activated PKC-dependent TRPC1 channel activity. These data indicate that stimulation of ET(B) receptors evokes PKC-dependent TRPC1 activity through PI-PLC-mediated generation of DAG and requires a permissive role of PIP(2). In conclusion, we provide the first evidence that stimulation of ET(A) and ET(B) receptors activate native PKC-dependent TRPC1 channels through two distinct phospholipids pathways involving a novel action of PIP(3), in addition to PIP(2), in rabbit coronary artery myocytes.

Identification of canonical transient receptor potential (TRPC) channel proteins in native vascular smooth muscle cells.

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable non-selective cation channels, which on stimulation allow influx of Na(+) and Ca(2+) ions into cells. It is proposed that stimulation of TRPC conductances by neurotransmitters and hormones such as noradrenaline, angiotensin II and endothelin-1 have important functions in vascular smooth muscle cells including vasoconstriction, cell growth and proliferation. Moreover constitutive TRPC activity contributes to setting the resting membrane potential of vascular myocytes. Activation of TRPC channels is thought to provide a direct Ca(2+) influx pathway and evoke indirect Ca(2+) entry by inducing depolarisation and opening of voltage-gated Ca(2+) channels and by stimulating the reverse mode of the Na(+)/Ca(2+) exchanger. Therefore identification of native TRPC channel proteins, which underlie these mechanisms, will provide important information on physiological functioning of vascular tissue and these conductances are pharmacological targets for the prevention of cardiovascular diseases such as hypertension. This review focuses on different experimental approaches that have been used to elucidate the molecular identity of TRPCs in native vascular myocytes. It will discuss the advantages and problems associated with using siRNA and anti-sense technologies in primary cell cultures, cell lines and transgenic mice models. In addition we describe recent work, which combines studies on the effect of anti-TRPC antibodies and pharmacological agents on biophysically characterised single cation channel currents to identify TRPC channel proteins in freshly dispersed vascular myocytes. These data provide strong evidence that native vascular myocytes contain diverse TRPC-mediated channels which are usually composed of complex heterotetrameric structures possessing marked pharmacological differences.

Role of phosphoinositol 4,5-bisphosphate and diacylglycerol in regulating native TRPC channel proteins in vascular smooth muscle.

Stimulation of receptor-operated (ROCs) and store-operated (SOCs) Ca(2+)-permeable cation channels by vasoconstrictors has many important physiological functions in vascular smooth muscle. The present review indicates that ROCs and SOCs with diverse properties in different blood vessels are likely to be explained by composition of different subunits from the canonical transient receptor potential (TRPC) family of cation channel proteins. In addition we illustrate that activation of native TRPC ROCs and SOCs involves different phospholipase-mediated transduction pathways linked to generation of diacylglycerol (DAG). Moreover we describe recent novel data showing that the endogenous phospholipid phosphoinositol 4,5-bisphosphate (PIP(2)) has profound and contrasting actions on TRPC ROCs and SOCs. Optimal activation of a native TRPC6 ROC by angiotensin II (Ang II) requires both depletion of PIP(2) and generation of DAG which leads to stimulation of TRPC6 via a PKC-independent mechanism. The data also indicate that PIP(2) has a marked constitutive inhibitory action of TRPC6 and DAG and PIP(2) are physiological antagonists on TRPC6 ROCs. In contrast PIP(2) stimulates TRPC1 SOCs and has an obligatory role in activation of these channels by store-depletion which requires PKC-dependent phosphorylation of TRPC1 proteins. Finally, we conclude that interactions between PIP(2) bound to TRPC proteins at rest, generation of DAG and PKC-dependent phosphorylation of TRPC proteins have a fundamental role in activation mechanisms of ROCs and SOCs in vascular smooth muscle.

Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytes.

In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP(2), a water soluble form of PIP(2), to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP(2)-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP(2) antibodies and by depletion of tissue PIP(2) levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP(2) to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP(2) at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP(2) whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP(2). Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP(2) can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP(2) in SOC activation requires PKC-dependent phosphorylation of TRPC1.

Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes.

The present work investigates the effect of phosphatidylinositol-4,5-bisphosphate (PIP(2)) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co-immunoprecipitation methods. Inclusion of 100 microM diC8-PIP(2) in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)-evoked whole-cell cation currents and TRPC6 channel activity by over 90%. In inside-out patches diC8-PIP(2) also inhibited TRPC6 activity induced by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) with an IC(50) of 7.6 microM. Anti-PIP(2) antibodies potentiated Ang II- and OAG-evoked TRPC6 activity by about 2-fold. Depleters of tissue PIP(2) wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP(2) scavenger poly-L-lysine. Wortmannin reduced Ang II-evoked TRPC6 activity by over 75% but increased OAG-induced TRPC6 activity by over 50-fold. Co-immunoprecipitation studies demonstrated association between PIP(2) and TRPC6 proteins in tissue lysates. Pre-treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP(2). These results provide for the first time compelling evidence that constitutively produced PIP(2) exerts a powerful inhibitory action on native TRPC6 channels.

Diverse properties of store-operated TRPC channels activated by protein kinase C in vascular myocytes.

In vascular smooth muscle, store-operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)-induced whole-cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin-1, CPA and the cell-permeant Ca(2+) chelator BAPTA-AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mM [Ca(2+)](o) the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mM [Ca(2+)](o) the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13-dibutyrate (PDBu), the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti-TRPC1 and -TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti-TRPC6 blocked SOCs only in coronary artery and anti-TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

Stimulation of Ca2+-gated Cl- currents by the calcium-dependent K+ channel modulators NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one] and isopimaric acid.

Because chloride (Cl(-)) channel blockers such as niflumic acid enhance large-conductance Ca(2+)-activated potassium channels (BK(Ca)), the aim of this study was to determine whether there is a reciprocal modification of Ca(2+)-activated chloride Cl(-) currents (I(ClCa)) by two selective activators of BK(Ca). Single smooth muscle cells were isolated by enzymatic digestion from murine portal vein and rabbit pulmonary artery. The BK(Ca) activators NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl-)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one] and isopimaric acid (IpA) augmented macroscopic I(ClCa) elicited by pipette solutions containing [Ca(2+)](i) > 100 nM without any alteration in current kinetics. Enhanced currents recorded in the presence of NS1619 or IpA reversed at the theoretical Cl(-) equilibrium potential, which was shifted by approximately -40 mV upon replacement of the external anion with the more permeable thiocyanate anion. NS1619 increased the sensitivity of calcium-activated chloride channel (Cl(Ca)) to Ca(2+) (approximately 100 nM at +60 mV) and induced a leftward shift in their voltage dependence (approximately 80 mV with 1 micro Ca(2+)). Single-channel experiments revealed that NS1619 increased the number of open channels times the open probability of small-conductance (1.8-3.1 pS) Cl(Ca) without any alteration in their unitary amplitude or number of observable unitary levels of activity. These data, in addition to the established stimulatory effects of niflumic acid on BK(Ca), show that there is similarity in the pharmacology of calcium-activated chloride and potassium channels. Although nonspecific interactions are possible, one alternative hypothesis is that the channel underlying vascular I(ClCa) shares some structural similarity to the BK(Ca) or that the latter K(+) channel physically interacts with Cl(Ca).

Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release.

The present study describes the first characterization of Ca(2+)-activated Cl(-) currents (I(ClCa)) in single smooth muscle cells from a murine vascular preparation (portal veins). I(ClCa) was recorded using the perforated patch version of the whole cell voltage-clamp technique and was evoked using membrane depolarization. Generation of I(ClCa) relied on Ca(2+) entry through dihydropyridine-sensitive Ca(2+) channels because I(ClCa) was abolished by 1 microM nicardipine and enhanced by raising external Ca(2+) concentration or by application of BAY K 8644. I(ClCa) was characterized by the sensitivity to Cl(-) channel blockers and the effect of altering the external anion on reversal potential. Activation of I(ClCa) after membrane depolarization was dependent on Ca(2+) release from intracellular stores. Thus the amplitude of I(ClCa) was diminished by the SR-ATPase inhibitor cyclopiazonic acid, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the ryanodine receptor blocker tetracaine. The degree of inhibition produced by the application of 2-APB and tetracaine together was significantly greater than the effect of each agent applied alone. In current-clamp mode, injection of depolarizing current elicited a biphasic action potential, with the later depolarization being sensitive to niflumic acid (NFA; 10 microM). In isometric tension recordings, NFA inhibited spontaneous contractions. These data support a role for this conductance in portal vein excitability.