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Summary. The study examined the effect of pramipexole (2-amino-4,5,6,7-tetrahydro-6-propyl-aminobenzthiazole dihydrochloride; PRA), a new potent dopamine receptor agonist with the high preference for D3 receptors, as compared to D2 or D4, on the central dopamine D3 system. Experiments were conducted on male Wistar rats. PRA was injected subcutaneously. PRA given repeatedly (14 days, twice a day, in doses of 0.3 and 1 mg/kg), but not acutely, potentiated the locomotor hyperactivity induced by (±)-7-OH-DPAT (3 mg/kg s.c.), when given 24 h after the single or the last dose of PRA. Administration of PRA, 1 mg/kg, for 3 or 7 days produced an effect similar to that described above, whereas a dose of 0.3 mg/kg produced such an effect only after 7, but not 3, days. Repeated treatment with PRA (0.3 and 1 mg/kg, 14 days, twice daily) also enhanced the D3 receptor binding in the islands of Calleja and nucleus accumbens (shell) – the brain region known to be rich in D3 receptors – when [3H]7-OH-DPAT was used as a ligand. Repeated PRA administration did not change the concentration of mRNA coding for D3 receptors in the islands of Calleja. The obtained results indicate that – like the previously studied typical antidepressants given repeatedly – PRA increases the functional responsiveness and the binding to the brain dopamine D3 receptors. Hence PRA may be considered as a potential antidepressant drug. [ABSTRACT FROM AUTHOR]

Venlafaxine (VEN), a representative of a new class of antidepressants (serotonin and noradrenaline reuptake inhibitors, SNRI), administered repeatedly affects—as was demonstrated by us previously—the behavioural responsiveness of α[sub 1] -adrenergic, dopaminergic (D[sub 2] and D[sub 3] ) and serotonergic systems to their agonists. In the present study we aimed to find out whether parallel changes in the binding to the respective receptors also occurred. The experiment was carried out on male Wistar rats. VEN was administered in a dose of 10 mg/kg once or repeatedly (14 days, twice daily). The obtained results showed that VEN did not change the binding (B [sub max] and K [sub D] ) of α[sub 1] -adrenergic receptors to [[sup 3] H]-prazosin in the cerebral cortex, having increased only its displacement by phenylephrine. The binding (B [sub max] and K [sub D] ) to D[sub 1] and D[sub 2] receptors in the limbic forebrain and the striatum was not affected by repeated venlafaxine when [[sup 3] H];-SCH 23390 and [[sup 3] H]-spiperone, respectively, were used as ligands. When [[sup 3] H]-quinpirole was used as a ligand, the binding was enhanced in the striatum, the nucleus accumbens (shell and core) and islands of Calleja. VEN also increased the binding of [[sup 3] H]-7-OH-DPAT to D[sub 3] receptors in islands of Calleja and the nucleus accumbens (shell). In the serotonergic system, a decrease in the density of 5-HT[sub 1A] receptors was observed in the hippocampus, whereas no changes occurred in the binding of 5-HT[sub 2] receptors in the cortex. Thus VEN given repeatedly enhanced the binding (of the ligands that are agonists) to dopamine D[sub 2] and D[sub 3] receptors. Weaker effects were observed in the α[sub 1] -adrenergic and the serotonergic systems. Copyright © 1999 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Trimipramine (TRI), which shows a clinical antidepressant activity, is chemically related to imipramine but does not inhibit the reuptake of noradrenaline and 5-hydroxytryptamine, nor does it induce β-adrenergic down-regulation. The mechanism of its antidepressant activity is still unknown. The aim of the present study was to find out whether TRI given repeatedly was able to induce adaptive changes in the dopaminergic and α1-adrenergic systems, demonstrated by us previously for various antidepressants. TRI was given to male Wistar rats and male Albino Swiss mice perorally twice daily for 14 days. In the acute experiment TRI (given i.p.) does not antagonize the reserpine hypothermia in mice and does not potentiate the 5-hydroxytryptophan head twitches in rats. TRI given repeatedly to rats increases the locomotor hyperactivity induced by d-amphetamine, quinpirole and (±)-7-hydroxy-dipropylo-aminotetralin (dopamine D2 and D3 effects). The stereotypies induced by d-amphetamine or apomorphine are not potentiated by TRI. It increases the behaviour stimulation evoked by phenylephrine (given intraventricularly) in rats, evaluated in the open field test as well as the aggressiveness evoked by clonidine in mice, both these effects being mediated by an α1-adrenergic receptor. It may be concluded that, like other tricyclic antidepressants studied previously, TRI given repeatedly increases the responsiveness of brain dopamine D2 and D3 (locomotor activity but not stereotypy) as well as α1-adrenergic receptors to their agonists. A question arises whether the re-uptake inhibition is of any importance to the adaptive changes induced by repeated antidepressants, suggested to be responsible for the antidepressant activity. [ABSTRACT FROM AUTHOR]

Introduction: The phase 3 XTEND-1 trial (NCT04161495) demonstrated that efanesoctocog alfa prophylaxis provided superior bleed protection compared with pre-trial factor VIII (FVIII) prophylaxis in patients with severe haemophilia A. The aim of this study was to indirectly compare the efficacy of efanesoctocog alfa with non-factor replacement therapy emicizumab in adolescent and adult patients with severe haemophilia A without inhibitors., Methods: A systematic literature review was conducted to identify phase 3 trials of emicizumab. Matching-adjusted indirect comparisons were used to compare annualised bleeding rates (ABRs) for any, treated, joint, and spontaneous bleeds, and joint health (measured using Hemophilia Joint Health Score [HJHS]), between efanesoctocog alfa and emicizumab. Estimated effects for different emicizumab regimens were pooled using random-effect meta-analysis to evaluate the overall difference in bleed outcomes between efanesoctocog alfa and emicizumab., Results: One emicizumab trial was included (HAVEN 3), which investigated three dosing regimens. In meta-analyses, efanesoctocog alfa once-weekly (Q1W) was associated with significantly lower ABRs for any (incidence rate ratio [95% CI] 0.33 [0.20; 0.53]), any treated (0.49 [0.30; 0.80]) and treated joint (0.51 [0.28; 0.91]) bleeds compared with emicizumab Q1W in non-inhibitor patients with prior prophylaxis or on-demand treatment. Efanesoctocog alfa Q1W was also associated with a significantly better improvement from baseline in HJHS Joint Score (mean difference [95% CI] -2.06 [-3.97; -0.14]) and Total Score (-2.37 [-4.36; -0.39]) versus emicizumab Q1W or every 2 weeks., Conclusion: Efanesoctocog alfa prophylaxis was associated with significantly lower rates of any, treated, and joint bleeds and improved joint health compared with emicizumab in patients with severe haemophilia A., Competing Interests: Declarations. Conflict of Interest: Alix Arnaud, Amanda Wilson, and Patricia Guyot are Sanofi employees and may hold stock/stock options in Sanofi. Nana Kragh and Elena Santagostino are employees of Sobi and may hold stock/stock options in Sobi. Marlena Wdowiak and Wojciech Margas are employees of Putnam PHMR. Piotr Wojciechowski is a co-founder of Assignity and an external consultant at Putnam PHMR. María Teresa Álvarez Román has received reimbursement for attending symposia/congresses and/or honoraria for speaking and/or honoraria for consulting, and/or funds for research from Takeda, Bayer, CSL-Behring, Novo Nordisk, Sobi, Roche, Biomarin, Novartis, Amgen, and Pfizer. Piotr Wojciechowski’s and Wojciech Margas’ affiliation Assignity is now called Clever-Access. Ethical Approval: This is a post hoc analysis and modelling of data already collected and/or published data. Original studies were all approved by the relevant institutional review boards at each study site and were carried out in accordance with the International Conference on Harmonisation good clinical practice guidelines and the Declaration of Helsinki., (© 2024. The Author(s).)

Introduction: In the Phase 3 XTEND-1 trial, (NCT04161495) efanesoctocog alfa prophylaxis provided superior bleed protection versus pre-study factor VIII (FVIII) replacement therapy in patients with severe haemophilia A. The aim of this study was to indirectly compare bleed outcomes between efanesoctocog alfa and standard/extended half-life (SHL and EHL) FVIII replacement therapies in adolescent and adult patients with severe haemophilia A without inhibitors., Methods: A systematic literature review was conducted to identify Phase 3 trials of EHL and SHL FVIII replacement therapies for comparison with efanesoctocog alfa data from XTEND-1. Matching-adjusted indirect comparisons were used to compare annualised bleeding rates (ABRs) for any, treated, joint, and spontaneous bleeds between efanesoctocog alfa and comparators. The estimates from respective comparisons were pooled using random-effect meta-analyses to evaluate the overall difference between efanesoctocog alfa and comparator therapies., Results: Four EHL therapies (rurioctocog alfa pegol, efmoroctocog alfa, turoctocog alfa pegol, damoctocog alfa pegol) and two octocog alfa SHL therapies were included. In meta-analyses, efanesoctocog alfa was associated with significantly lower ABRs for any [mean difference (95% CI) - 2.24 ( - 3.24; - 1.25)], spontaneous [ - 1.52 ( - 2.33; - 0.72)], and joint bleeds [ - 1.60 ( - 2.32; - 0.88)] versus EHL therapies, and with significantly lower ABRs for any [ - 3.61 ( - 4.43; - 2.79)], treated [ - 1.55 ( - 1.89; - 1.20)], spontaneous [ - 2.52 ( - 3.31; - 1.72)], and joint bleeds [ - 3.42 ( - 4.77; - 2.08)] versus SHL therapies., Conclusion: Efanesoctocog alfa was associated with significantly lower ABRs (any, spontaneous and joint) compared with EHL or SHL prophylaxis therapies. Patients had, on average, 2.2 and 3.6 fewer bleeds per year versus EHL and SHL therapies, respectively., Competing Interests: Declarations. Conflict of Interest: Alix Arnaud, Amanda Wilson, and Patricia Guyot are Sanofi employees and may hold stock/stock options in Sanofi. Nana Kragh and Linda Bystrická are employees of Sobi and may hold stock/stock options in Sobi. Piotr Wojciechowski is co-founder at Assignity and an external consultant at Putnam PHMR. Marlena Wdowiak and Wojciech Margas are employees of Putnam PHMR. Robert Klamroth has received honoraria and/or been a member of advisory committees for Bayer, Biomarin, Biotest, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche/Chugai, Sanofi, Sobi, and Takeda. Alberto Tosetto has no relevant conflicts of interest. Piotr Wojciechowski’s and Wojciech Margas’ affiliation Assignity is now called Clever-Access. Ethical Approval: This is a post hoc analysis and modelling of data already collected and/or published data. Original studies were all approved by the relevant institutional review boards at each study site and were carried out in accordance with the International Conference on Harmonisation good clinical practice guidelines and the Declaration of Helsinki., (© 2024. The Author(s).)

Background: Globally, healthcare has shouldered much of the socioeconomic brunt of the COVID-19 pandemic leading to numerous clinical trials suspended or discontinued., Objective: To estimate the COVID-19 impact on the number of clinical trials worldwide., Methods: Data deposited by 219 countries in the ClinicalTrials.gov database (2007-2020) were interrogated using targeted queries. A time series model was fitted to the data for studies ongoing, initiated, or ended between 2007 Quarter (Q) 1 and 2019 Q4 to predict the expected trials number in 2020 in the COVID-19 absence. The predicted values were compared with the actual 2020 data to quantify the pandemic impact., Results: The ongoing registered trials number grew from 2007 Q1 (33,739) to 2019 Q4 (80,319). By contrast, there were markedly fewer ongoing trials in all four quarters of 2020 compared with forecasted values (1.6%-2.8% decrease). When excluding COVID-19-related studies, this disparity grew further (3.4%-5.8% decrease), to a peak of almost 5,000 fewer ongoing trials than estimated for 2020 Q2. The initiated non-COVID-19 trials number was higher than predicted in 2020 Q4 (9.9%)., Conclusions: This pandemic has impacted clinical trials. Provided that current trends persist, clinical trial activities may soon recover to at least pre-COVID-19 levels., Competing Interests: All authors are employees of Creativ-Ceutical., (© 2022 Creativ-Ceutical. Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: Rivaroxaban and apixaban are the most widely used nonvitamin K oral anticoagulants (NOACs) in patients with venous thromboembolism (VTE). This meta-analysis evaluates the effectiveness and safety of both NOACs versus standard of care (SoC) in real-world practice., Methods: Real-world evidence (RWE) studies were identified through a systematic literature review conducted between January 2012 and July 2020, using Embase, MEDLINE, and the websites of cardiological, hematological, and oncological associations. Eligible RWE studies recruited adult patients with deep vein thrombosis and/or pulmonary embolism and presented a comparison between rivaroxaban and apixaban versus SoC, consisting either of vitamin K antagonists, heparins, or combinations thereof. Hazard ratios (HRs) for the comparison between NOACs and SoC were extracted from the relevant studies or estimated based on the reported binary data. The between-treatment contrasts were reported as HRs with associated 95% confidence intervals., Results: A total of 65 RWE studies were identified and considered relevant for the meta-analysis. Compared with SoC, both rivaroxaban and apixaban were associated with reduced risks of recurrent VTE and a lower rate of major bleeding events. Patients treated with rivaroxaban were at a lower risk of all-cause death compared with those receiving SoC (HR = 0.56 [0.39-0.80]), while evidence for apixaban from the identified studies was insufficient to demonstrate a statistically significant change in mortality (HR = 0.66 [0.30-1.47])., Conclusion: This analysis indicates that in real-world practice, rivaroxaban and apixaban are associated with a lower risk of recurrent VTE and major bleeding events compared with SoC. Survival benefit in patients treated with rivaroxaban was also observed., Competing Interests: OW, SM, TBL, and FS are external experts invited by Bayer AG to participate in the design of this analysis, consult for study selection and interpretation of the outcomes, and review the manuscript. AE, KB, and MH are the employees of Bayer Plc and/or Bayer AG, who participated in the design, interpretation of the results, and manuscript preparation. PW and WM are the employees of Creativ-Ceutical, who designed the analysis, managed the conduction of analyses and outcome reporting, interpreted the results, and actively participated in the preparation of the manuscript., (Copyright © 2022 Olivia Wu et al.)

Introduction: Necrotising enterocolitis (NEC) is one of the most serious conditions in newborn infants, affecting up to 10% of very low birth weight (VLBW) infants. Mortality rates can rise as high as 60%.The suspected diagnosis is confirmed with typical findings on abdominal radiography (AR) such as pneumatosis intestinalis (PI), portal vein gas (PVG) and in extreme cases pneumoperitoneum. Abdominal ultrasound (AUS) can depict PI, PVG and pnuemoperitoneum (in some cases ahead of AR), but it also provides other crucial information such as bowel wall viability (thickness or thinning) and free abdominal fluid. These additional findings are helpful in diagnosing and managing NEC., Methods and Analysis: The hypothesis being tested is that preforming an AR in patients with clinical symptoms of NEC, but inconclusive/normal AR will enhance detection rates, and expedite treatment in infants born at <32 weeks. Additionally, the time needed to initiate treatment, according to decision made based on AR or AR and AUS will also be compared. The use of AUS together with AR as an add-on test may increase the accuracy of diagnosing NEC and expedite life-saving treatment. We plan to recruit 200 VLBW infants, who are most prone to NEC. It will also be the first multicentre study evaluating the use of AUS as an add-on test, enabling us to recruit a significantly higher number of patients compared with published studies., Ethics and Dissemination: The Bioethical Committee of the Medical University of Warsaw has approved the study (KB 130/2017). We plan to submit our findings to international peer-reviewed journals. Abstract will be submitted to local and international conferences., Trial Registration Number: NCT03188380; Protocol version: V.2.08.2019; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aim: This study evaluated whether practitioners from 70 countries used premedication for non-emergency neonatal intubation and identified attitudes and experience regarding the safety, side effects and efficiency of neonatal intubation., Methods: Invitations to take part in the survey were issued between December 18, 2018 and February 4, 2019 to the users of neonatal-based websites and Facebook groups, members of professional societies and the authors of relevant publications in the last five years., Results: We analysed 718 completed questionnaires from 40 European and 30 non-European countries. Most of the responses were from neonatologists (69.6%) and paediatric or neonatal trainees (10.3%). In units without a protocol (31.6%), more than half of the practitioners (60.4%) chose premedication according to personal preference and 37.0%-11.9% of the overall respondents did not use any drugs for non-emergency intubation. The most frequently reported drug combination was fentanyl, atropine and succinylcholine (6.8%). Most of the practitioners (78.5%) use the same drugs for term and preterm infants. Only 24.8% of the physicians were fully satisfied with their premedication practices., Conclusion: Nearly 12% of the respondents did not use premedication for non-emergency neonatal intubation. The wide-ranging policies and practices found among the respondents highlight the need for international consensus guidelines., (© 2019 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Introduction: Uncertainty exists regarding the optimal time for removal of central lines used to provide parenteral nutrition in preterm infants. The aim of this study is to determine whether earlier central line removal is non-inferior to its removal after reaching full enteral intake, in respect to growth outcome of preterm infants., Methods and Analysis: Very low birthweight premature infants will be recruited. Eligible infants will be randomised in equal proportions between two groups. In the intervention group central lines will be removed when infants reach 100 mL/kg/day of enteral intake. In the control group central lines will be removed when infants reach 140 mL/kg/day of enteral intake (full enteral intake). The primary outcome measure will be the difference between the two groups in weight at 36 weeks' postmenstrual age. Non-inferiority will be declared if the mean weight of children in the intervention group will be no worse than the mean weight of children from the control group, by a margin of -210 g., Ethics and Dissemination: The Bioethics Committee of the Medical University of Warsaw approved the study protocol prior to recruitment. The findings of this trial will be submitted to a peer-reviewed journal (neonatology, paediatrics or nutrition). Abstracts will be submitted to relevant national and international conferences., Trial Registration Number: NCT03730883., Protocol Version: Version 3. 14.08.2019., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The auxiliary α 2 δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α 2 and δ. We now show, using α 2 δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (Ca V 2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α 2 δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α 2 δ. Uncleaved α 2 δ does not support trafficking of Ca V 2.2 channel complexes into neuronal processes, and inhibits Ca 2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α 2 δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α 2 δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes., Competing Interests: The authors declare that no competing interests exist.

Gene deletion of the voltage-gated calcium channel auxiliary subunit α2δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α2δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α2δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α2δ-1 knockout are involved in setting the duration of individual APs and their firing frequency. Our main findings are that there is reduced Ca(2+) entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca(2+) buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca(2+)-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca(2+) elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'., (© 2016 The Authors.)

As auxiliary subunits of voltage-gated Ca(2+) channels, the α2δ proteins modulate membrane trafficking of the channels and their localization to specific presynaptic sites. Following nerve injury, upregulation of the α2δ-1 subunit in sensory dorsal root ganglion neurons contributes to the generation of chronic pain states; however, very little is known about the underlying molecular mechanisms. Here we show that the increased expression of α2δ-1 in rat sensory neurons leads to prolonged Ca(2+) responses evoked by membrane depolarization. This mechanism is coupled to CaV2.2 channel-mediated responses, as it is blocked by a ω-conotoxin GVIA application. Once initiated, the prolonged Ca(2+) transients are not dependent on extracellular Ca(2+) and do not require Ca(2+) release from the endoplasmic reticulum. The selective inhibition of mitochondrial Ca(2+) uptake demonstrates that α2δ-1-mediated prolonged Ca(2+) signals are buffered by mitochondria, preferentially activated by Ca(2+) influx through CaV2.2 channels. Thus, by controlling channel abundance at the plasma membrane, the α2δ-1 subunit has a major impact on the organization of depolarization-induced intracellular Ca(2+) signaling in dorsal root ganglion neurons., (Copyright © 2015 the authors 0270-6474/15/355891-13$15.00/0.)

The α2δ-1 subunit of voltage-gated calcium channels is upregulated after sensory nerve injury and is also the therapeutic target of gabapentinoid drugs. It is therefore likely to play a key role in the development of neuropathic pain. In this study, we have examined mice in which α2δ-1 gene expression is disrupted, to determine whether α2δ-1 is involved in various modalities of nociception, and for the development of behavioral hypersensitivity after partial sciatic nerve ligation (PSNL). We find that naive α2δ-1(-/-) mice show a marked behavioral deficit in mechanical and cold sensitivity, but no change in thermal nociception threshold. The lower mechanical sensitivity is mirrored by a reduced in vivo electrophysiological response of dorsal horn wide dynamic range neurons. The CaV2.2 level is reduced in brain and spinal cord synaptosomes from α2δ-1(-/-) mice, and α2δ-1(-/-) DRG neurons exhibit lower calcium channel current density. Furthermore, a significantly smaller number of DRG neurons respond to the TRPM8 agonist menthol. After PSNL, α2δ-1(-/-) mice show delayed mechanical hypersensitivity, which only develops at 11 d after surgery, whereas in wild-type littermates it is maximal at the earliest time point measured (3 d). There is no compensatory upregulation of α2δ-2 or α2δ-3 after PSNL in α2δ-1(-/-) mice, and other transcripts, including neuropeptide Y and activating transcription factor-3, are upregulated normally. Furthermore, the ability of pregabalin to alleviate mechanical hypersensitivity is lost in PSNL α2δ-1(-/-) mice. Thus, α2δ-1 is essential for rapid development of mechanical hypersensitivity in a nerve injury model of neuropathic pain.

At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.

Synaptic neurotransmitter release is driven by Ca(2+) influx through active zone voltage-gated calcium channels (VGCCs). Control of active zone VGCC abundance and function remains poorly understood. Here we show that a trafficking step probably sets synaptic VGCC levels in rats, because overexpression of the pore-forming α1(A) VGCC subunit fails to change synaptic VGCC abundance or function. α2δs are a family of glycosylphosphatidylinositol (GPI)-anchored VGCC-associated subunits that, in addition to being the target of the potent neuropathic analgesics gabapentin and pregabalin (α2δ-1 and α2δ-2), were also identified in a forward genetic screen for pain genes (α2δ-3). We show that these proteins confer powerful modulation of presynaptic function through two distinct molecular mechanisms. First, α2δ subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells. Second, α2δs configure synaptic VGCCs to drive exocytosis through an extracellular metal ion-dependent adhesion site (MIDAS), a conserved set of amino acids within the predicted von Willebrand A domain of α2δ. Expression of α2δ with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. α2δs harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators. Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.

The pharmacology of G protein-coupled receptors can be influenced by factors such as constitutive receptor activation and Na(+) ions. In this study, we examined the coupling of natively and heterologously expressed nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors with voltage-dependent Ca(2+) channels after exposure to four high-affinity NOP receptor blockers [[Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-101), 1-[1-(cyclooctylmethyl)-1,2,3,6-tetrahydro-5-(hydroxymethyl)-4-pyridinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (Trap-101), 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl}pyrrolidine-2-carboxamide (compound 24), and N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride (JTC-801)] in sympathetic neurons. The enhanced tonic inhibition of Ca(2+) currents in the absence of agonists, indicative of constitutively active NOP receptors in transfected neurons, was abolished after pretreatment with pertussis toxin. In control neurons, the four antagonists did not exert any effects when applied alone but significantly blocked the N/OFQ-mediated Ca(2+) current inhibition. Exposure of transfected neurons to UFP-101 resulted in partial agonist effects. In contrast, Trap-101, compound 24, and JTC-801 exerted inverse agonism, as measured by the loss of tonic Ca(2+) current inhibition. In experiments designed to measure the N/OFQ concentration-response relationship under varying Na(+) concentrations, a leftward shift of IC(50) values was observed after Na(+) exposure. Although similar N/OFQ efficacies were measured with all solutions, a significant decrease of Hill coefficient values was obtained with increasing Na(+) concentrations. Examination of the allosteric effects of Na(+) on heterologously overexpressed NOP receptors showed that the tonic Ca(2+) current inhibition was abolished in the presence of the monovalent cation. These results demonstrate that constitutively active NOP receptors exhibit differential blocker pharmacology and allosteric regulation by Na(+). Data are also presented demonstrating that heterologously expressed mu opioid receptors in sympathetic neurons are similarly modulated.

Expression of the calcium channels Ca(V)2.1 and Ca(V)2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca(V)2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca(V)2.1 and Ca(V)2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg(52) and Arg(54)) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca(V)2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca(V)2.2 protein level, and this is also prevented by mutation of Arg(52) and Arg(54) to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg(52), Arg(54) motif are involved in the processes underlying dominant negative suppression.

The sphenopalatine ganglion (SPG) neurons represent the parasympathetic branch of the autonomic nervous system involved in controlling cerebral blood flow. In the present study, we examined the coupling mechanism between mu (mu) opioid receptors (MOR) and muscarinic acetylcholine receptors (mAChR) with Ca(2+) channels in acutely dissociated adult rat SPG neurons. Successful MOR activation was recorded in approximately 40-45% of SPG neurons employing the whole cell variant of the patch-clamp technique. In addition, immunofluorescence assays indicated that MOR are not expressed in all SPG neurons while M(2) mAChR staining was evident in all neurons. The concentration-response relationships generated with morphine and [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) showed IC(50) values of 15.2 and 56.1 nM and maximal Ca(2+) current inhibition of 26.0 and 38.7%, respectively. Activation of MOR or M(2) mAChR with morphine or oxotremorine-methiodide (Oxo-M), respectively, resulted in voltage-dependent inhibition of Ca(2+) currents via coupling with Galpha(i/o) protein subunits. The acute prolonged exposure (10 min) of neurons to morphine or Oxo-M led to the homologous desensitization of MOR and M(2) mAChR, respectively. The prolonged stimulation of M(2) mAChR with Oxo-M resulted in heterologous desensitization of morphine-mediated Ca(2+) current inhibition, and was sensitive to the M(2) mAChR blocker methoctramine. On the other hand, when the neurons were exposed to morphine or DAMGO for 10 min, heterologous desensitization of M(2) mAChR was not observed. These results suggest that in rat SPG neurons activation of M(2) mAChR likely modulates opioid transmission in the brain vasculature to adequately maintain cerebral blood flow.

The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Galphai/o subunits. However, the identification of the specific Galpha subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Galpha subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Galpha subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Galpha protein was examined by RT-PCR experiments. Fluorescence imaging showed that Galphai1, Galphai3, and Galphao were expressed in SG neurons. The transfection of Galphai1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Galphai3 or Galphao was without effect. Taken together, these results suggest that in SG neurons Galphai1 subunits selectively couple NOP receptors to N-type Ca2+ channels.

The sphenopalatine ganglion (SPG) is the main parasympathetic ganglion that is involved in regulating cerebral vascular tone and gland secretion. SPG neurons have been implicated in some types of migraine headaches but their precise role has yet to be determined. In addition, very little information is available regarding ion channel modulation by neurotransmitters that are involved in the parasympathetic drive of SPG neurons. In this study, acute isolation of adult rat SPG neurons was developed in order to begin the electrophysiological characterization of this ganglion. Under our dissociation conditions, the average number of neurons obtained per ganglion was greater than 1200. Immunofluorescence imaging results showed positive labeling with acetylcholinesterase (AChE), confirming the parasympathetic nature of SPG neurons. On the other hand, weak tyrosine hydroxylase immunostaining was observed in these neurons. Whole-cell patch-clamp recordings revealed that most of the Ca(2+) current is carried by N-type (53%) and SNX-482 resistant R-type (30%) Ca(2+) channels. In addition, Ca(2+) currents were inhibited in a voltage-dependent manner following exposure to oxotremorine-M (Oxo-M), norepinephrine and ATP via muscarinic acetylcholine receptor 2 (M(2) AChR) subtype, adrenergic and P2Y purinergic receptors, respectively. The peptides VIP and angiotensin II failed to modulate Ca(2+) currents, suggesting that these receptors are not present on the SPG soma or do not couple to Ca(2+) channels. In summary, our data suggest that the Ca(2+) current inhibition mediated by Oxo-M, NE and ATP in adult rat SPG neurons plays an integral part in maintaining parasympathetic control of cranial functions.

The most common single-nucleotide polymorphism (SNP) of the human mu-opioid receptor (hMOR) gene occurs at position 118 (A118G) and results in substitution of asparagine to aspartate at the N-terminus. The purpose of the present study was to compare the pharmacological profile of several opioid agonists to heterologously expressed hMOR and N-type Ca(2+) channels in sympathetic neurons. cDNA constructs coding for wild-type and mutant hMOR were microinjected in rat superior cervical ganglion neurons and N-type Ca(2+) channel modulation was investigated using the whole cell variant of the patch-clamp technique. Concentration-response relationships were generated with the following selective MOR agonists: DAMGO, morphine, morphine-6-glucuronide (M-6-G), and endomorphin I. The estimated maximal inhibition for the agonists ranged from 52 to 64% for neurons expressing either hMOR subtype. The rank order of potencies for estimated EC(50) values (nM) in cells expressing wild-type hMOR was: DAMGO (31) >> morphine (76) congruent with M-6-G (77) congruent with endomorphin I (86). On the other hand, the rank order in mutant-expressing neurons was: DAMGO (14) >> morphine (39) >> endomorphin I (74) congruent with M-6-G (82), with a twofold leftward shift for both DAMGO and morphine. The DAMGO-mediated Ca(2+) current inhibition was abolished by the selective MOR blocker, CTAP, and by pertussis toxin pretreatment of neurons expressing either hMOR subtype. These results suggest that the A118G variant MOR exhibits an altered signal transduction pathway and may help explain the variability of responses to opiates observed with carriers of the mutant allele.

Receptor distributions in the brain are studied by autoradiographic mapping in brain slices, which is a labor-intensive and expensive procedure. To keep track of the results of such studies, we have designed CoReDat, a multi-user relational database system that is available for download from www.cocomac.org/coredat. Here, we describe the data model and provide an architectural overview of CoReDat for the neuroscientist who wants to use this database, adapt it for related purposes, or build a new one.

Reboxetine (REB) is a member of a new class of antidepressant drugs, which selectively inhibit the neuronal reuptake of noradrenaline. It is devoid of any affinity for neurotransmitter receptors nor does it inhibit monoamine oxidases A or B. Since our earlier studies have shown that antidepressant drugs administered repeatedly increase the responsiveness of alpha1-adrenergic receptors and induce the up-regulation of postsynaptic dopamine D2/D3 receptors in the rat brain, we designed the present experiments to determine whether repeated administration of REB evokes similar effects. The experiments were carried out on male Wistar rats. REB was administered at a dose of 10 mg/kg (or 30 mg/kg in some cases) once or repeatedly (twice daily for 14 days). The obtained results show that REB administered repeatedly increased exploratory behavior induced by phenylephrine and potentiated the hyperlocomotion induced by D-amphetamine. These behavioral effects indicate the hyperresponsiveness of alpha1-adrenergic receptors. Biochemical studies did not show any changes in the binding parameters of [3H]prazosin (Bmax, or Kd), but the ability of the alpha1-adrenergic receptor agonist, phenylephrine, to compete for these sites was significantly increased upon repeated administration of REB. Locomotor activity induced by quinpirole was not changed, although there was a potentiation of 7-OH-DPAT-induced locomotor hyperactivity in rats receiving repeated administration of REB. At the same time no significant changes in the binding of [3H]quinpirole and [3H]7-OH-DPAT, or at the level of mRNA coding for dopamine D2 receptors in the rat brain were observed. Enhanced responsiveness to 7-OH-DPAT observed in the behavioral studies might, therefore, result from alterations at the postreceptor level. The above results indicate that repeated administration of REB induces the adaptive changes in the alpha1-adrenergic receptors, especially it enhances their functional responsiveness. However, the question whether this functional responsiveness is important for the clinical antidepressant efficacy, remains to be elucidated.

Antisense strategies have a potential to specifically block the production of a given protein, e.g. receptor subtype, thus may help to uncover its behavioral and/or biochemical function. In the present study we demonstrated the utility of this approach for studying the role of dopamine D2 receptors in the anti-immobility effect of imipramine in the forced swimming test. Following intracerebroventricular (i.c.v.) administration of phosphorothioate oligonucleotide complementary to mRNA encoding for dopamine D2 receptors (D2 antisense ODN; 1 nmol/1 microl H2O, twice a day for 5 days) to the rats, the decrease in the locomotor activity (shortened total distance travelled and decrease in vertical activity, without differences in the stereotypic movements of animals), as well as the decrease of specific binding of [3H]raclopride in the striatum and limbic forebrain were observed. At the same time, i.c.v. administration of D2 antisense ODN reversed the effect of imipramine in the forced swimming test, what may indicate that the dopamine D2 receptors play a significant role in the behavioral anti-immobility effects of imipramine.

Milnacipran (MIL) is a representative of a new class of antidepressants (SNRIs) which inhibit the reuptake of serotonin and noradrenaline, but, in contrast to tricyclics, show no affinity for neurotransmitters receptors. The present study was aimed at determining whether repeated MIL administration (given at doses of 10 or 30 mg/kg, twice daily for 14 days) induced the adaptive changes in the dopaminergic system similar to those reported by us earlier for tricyclic antidepressants. The obtained results showed that MIL administered repeatedly did not change the responsiveness of dopamine D1 receptors since it did not change the SKF 38393-induced grooming. Repeated MIL treatment increased the hyperlocomotion induced by D-amphetamine and 7-OH-DPAT, but did not affect the D-amphetamine and apomorphine stereotypies. The binding parameters (Bmax and Kd) to dopamine D1 and D2 receptors in the limbic forebrain were not affected by repeated MIL treatment when [3H]SCH 23390 and [3H]spiperone, respectively, were used as ligands. On the other hand, the increased density of dopamine D2 receptors (Bmax) was observed in the striatum after repeated treatment with MIL. MIL administered acutely or repeatedly did not change the binding of [3H]7-OH-DPAT to dopamine D3 receptors in the islands of Calleja and the shell region of the nucleus accumbens septi. The above results indicate that repeated MIL administration induces the adaptive changes in the dopaminergic system, especially it enhances the functional responsiveness of dopamine D2 and D3 receptors. However, the question whether this increased functional responsiveness is important for the clinical antidepressant efficacy, remains open.