The Prevalence of Seizures in Brain Metastasis Patients on Anticonvulsant Prophylaxis: A Systematic Review and Meta-Analysis.

BACKGROUND: Brain metastasis (BM) prognosis is incredibly poor and is often associated with considerable morbidity. Seizures are commonly present in these patients, and their biopsychosocial impact can be dangerous. The use of antiepileptic drugs (AEDs) as primary prophylaxis remains controversial. This systematic review and meta-analysis aim to evaluate the efficacy of AED prophylaxis in patients with BM. METHODS: MEDLINE via PubMed, Web of Science, EMBASE, and Cochrane were searched for articles pertinent to AED prophylaxis use in patients with BM. Patients with BM previously treated for cancer who were seizure naive at the time of inclusion were included. Data regarding patient characteristics, type of AED, prior treatments, and groups at a high risk of seizure were extracted. Seizure prevalence was obtained. RESULTS: Eight studies were included in this systematic review and meta-analysis; 1902 total patients with BM were included, with 381 receiving antiepileptic prophylaxis, and 1521 receiving no prophylaxis. Although the odds of a seizure in the treatment group was found to be 1.158 times the odds of a seizure in the control group, the odds ratio was not statistically significant (t-statistic = 0.62, P value = 0.5543). CONCLUSIONS: There was no significant difference in the odds of seizure development in control groups compared to patients receiving prophylactic antiepileptic therapy. As patients with BM present with heterogeneity in tumor characteristics and receive various treatment modalities, future research is needed to identify groups that may benefit more significantly from AED prophylaxis.

Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore.

The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.

Potent and orally efficacious benzothiazole amides as TRPV1 antagonists.

Benzothiazole amides were identified as TRPV1 antagonists from high throughput screening using recombinant human TRPV1 receptor and structure-activity relationships were explored to pinpoint key pharmacophore interactions. By increasing aqueous solubility, through the attachment of polar groups to the benzothiazole core, and enhancing metabolic stability, by blocking metabolic sites, the drug-like properties and pharmokinetic profiles of benzothiazole compounds were sufficiently optimized such that their therapeutic potential could be verified in rat pharmacological models of pain.

Stereoselective effects of 4-hydroxynonenal in cultured mouse hepatocytes.

4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 µM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. Caspase-3-dependent cleavage of the glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 h. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 h after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms.

Pain interference in individuals in driver intervention programs for driving under the influence offenders.

Pain-related problems among individuals in court-mandated Driver Intervention Programs (DIPs) for "driving under the influence" (DUI) offenders have not been well studied. This project examines 3,189 individuals from a DIP in Dayton, Ohio. Over 11% of participants reported significant pain-related interference in the past 4 weeks. Pain was significantly more likely in those with depression, more childhood conduct problems, and recent use of multiple illicit drugs. Many individuals seen in court-mandated DIP programs for DUI offenders also report difficulties with pain. DIP programming should address pain in relation to substance use and mental health issues.

Medical Degree Disparity Among Authors of Original Research in Pediatric Journals: A Four-Year Follow-Up.

INTRODUCTION AND OBJECTIVE: Scholarly activity is a major component of residency training and the accreditation process for graduate medical education. In 2014, Accreditation Council for Graduate Medical Education and the American Association of Colleges of Osteopathic Medicine announced a single accreditation system with the transition beginning July 1, 2015. Previous data before the transition had shown that osteopathic physicians rarely published original research in three high-impact pediatric journals. The objective of this study is to determine if there is a degree disparity between osteopathic and allopathic physicians among authors who publish original research manuscripts in three high-impact pediatric journals after the beginning of the transition to a single graduate medical education accreditation system.  Methods: Degree designation for the first and senior authors of original research manuscripts was reviewed for the Journal of Pediatrics (J Pediatr), Pediatrics, and JAMA Pediatrics (JAMA Pediatr) for the years 2016, 2017, 2018, and 2019. Inter-rater reliability was calculated by the kappa coefficient, and data were analyzed with descriptive statistics and simple linear regression.  Results: A total of 3,252 manuscripts and 4,068 authors were reviewed with 0.98% of all authors being osteopathic physicians. A total of 1.65% of first authors and 0.41% of senior authors were osteopathic physicians. For those with a dual degree, a total of 1.03% of first, and 0.41% of senior authors were osteopathic physicians. No statistical trend could be established for increased first, senior, dual-degree first, or dual-degree senior osteopathic physician authorship. CONCLUSION: Osteopathic physicians continue to be underrepresented as first and senior authors in original publications in the three high-impact pediatric journals as compared to their allopathic counterparts.

A Comparative Analysis of Long-Term Survival of Robotic Versus Thoracoscopic Lobectomy.

BACKGROUND: Minimally invasive lobectomy can be performed robotically or thoracoscopically. Short-term outcomes between the 2 approaches are reported to be similar; however, the comparative oncological effectiveness is not known. We sought to compare long-term survival after robotic and thoracoscopic lobectomy. METHODS: We performed a propensity-matched analysis of SEER (Surveillance, Epidemiology and End Results)-Medicare patients with non-small cell lung cancer from 2008 to 2013 who underwent minimally invasive lobectomy using either a thoracoscopic (n = 3881) or a robotic-assisted (n = 426) approach. Patients in the 2 groups were propensity matched 1:1 based on demographics, comorbidities, treatment, and tumor characteristics. We compared the overall survival (OS) and cancer-specific mortality (CSM) between the 2 groups. RESULTS: Within the matched cohort (n = 409 per group), the median age at surgery was 73 (range, 65-91) years, with a median follow-up of 35 months postsurgery. There was no difference in OS or CSM between the thoracoscopic and robotic-assisted groups (OS: 71.4% vs 73.1% at 3 years, overall P = .366; CSM: 16.6% vs 14.9% at 3 years, overall P = .639). CONCLUSIONS: Our propensity-matched analysis demonstrates that patients undergoing robotic-assisted lobectomy have similar OS and CSM compared with those patients undergoing thoracoscopic lobectomy. Oncologic outcomes are similar between the 2 minimally invasive approaches. These results demonstrate that further investigation is needed in the form of a randomized control trial, its variations, or additional large-scale registry analyses to verify these results.

Novel temperature activation cell-based assay on thermo-TRP ion channels.

The precise temperature control of the ABI Prism 7900HT Sequence Detection System designed for detection of fluorescence of a biological sample in real-time PCR assays (TaqMan assays) was used to activate Thermo-TRP ion channels, enabling a novel 384-/96-well plate-based assay. Functional pharmacology was verified against the temperature activation using intracellular calcium fluorescence as a measure of ion channel activity. The assay is applicable to both heterologous expression systems and dorsal root ganglia primary cells. This will benefit several analgesic drug discovery programs searching for new Thermo-TRP modulators.

Pharmacological characteristics of the inhibition of nondepolarizing neuromuscular blocking agents at human adult muscle nicotinic acetylcholine receptor.

BACKGROUND: Nondepolarizing neuromuscular blocking agents (NMBAs) are classic competitive-inhibitors at the muscle nicotinic acetylcholine receptor (nAChR). Although the fetal subtype muscle nAChR has been extensively studied at a molecular level, less is known about the interaction between nondepolarizing NMBAs and the human adult muscle nAChR. The aim of this study was to investigate the effect of clinically used nondepolarizing NMBAs at human adult muscle nAChRs and the mechanisms behind the inhibition. METHODS: Human subunits for the adult alpha(1)beta(1)delta(epsilon) muscle nAChR were cloned and expressed into Xenopus oocytes and thereafter studied with two-electrode voltage clamp. The effect of the clinically used nondepolarizing NMBAs, including atracurium, cis-atracurium, mivacurium, pancuronium, rocuronium, vecuronium, and d-tubocurarine, on acetylcholine-induced and dimethylphenylpiperazinium-induced currents were investigated. RESULTS: All nondepolarizing NMBAs tested inhibited acetylcholine- and dimethylphenylpiperazinium-induced currents in human adult alpha(1)beta(1)delta(epsilon) muscle nAChRs, and no receptor activation was seen. Interestingly, acetylcholine desensitized the human adult alpha(1)beta(1)delta(epsilon) muscle type receptor and attenuated the inhibition caused by nondepolarizing NMBAs, as evident by lack of increase in IC(50) values for the nondepolarizing NMBAs with increased concentrations of acetylcholine. In contrast, dimethylphenylpiperazinium-induced currents were competitively inhibited by the nondepolarizing NMBAs. CONCLUSIONS: This study demonstrates that nondepolarizing NMBAs inhibit human adult muscle nAChRs expressed in Xenopus oocytes by mixed mechanisms. When using the nondesensitizing agonist dimethylphenylpiperazinium, inhibition by the NMBA is competitive, whereas activation with high concentrations of acetylcholine in combination with NMBA induces a noncompetitive inhibition, which the authors speculate can involve receptor desensitization similar to that observed in the neuromuscular junction.

Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1.

Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.

The role of mitochondrial and oxidative injury in BDE 47 toxicity to human fetal liver hematopoietic stem cells.

The polybrominated diphenyl ethers (PBDEs) are a group of flame retardants whose residues have markedly increased in the environment and in human tissues during the last decade. Of the various congeners, BDE 47 (2,2',4,4'-tetrabromodiphenyl ether) is typically the predominant congener observed in fish and wildlife samples, as well as in human tissues. Several studies indicate in utero transfer of PBDEs during pregnancy with residues accumulating in fetal tissues, and thus the potential for BDE 47-mediated injury in utero is of concern. In this study, we examined the mechanisms of BDE 47-mediated injury to primary human fetal liver hematopoietic stem cells (HSCs), which comprise a large proportion of fetal hepatic cells and play a key role in hematopoiesis during fetal development. Incubation of fetal liver HSCs with BDE 47 led to a loss of mitochondrial membrane potential and the onset of apoptosis. These effects were observed in the low micromolar range of BDE 47 exposures. At higher concentrations, BDE 47 elicited a loss of viability, which was accompanied by the generation of reactive oxygen species and peroxidation of HSC lipids. Preincubation of fetal liver HSCs with N-acetylcysteine, a glutathione (GSH) precursor, caused an increase in cellular GSH concentrations, restored mitochondrial redox status, and ameliorated the toxicity of BDE 47. BDE 47-mediated cytotoxicity or oxidative injury was not evident at the lower concentrations (< 1microM). Collectively, these data support a role for oxidative stress in the cytotoxicity of BDE 47 and indicate that oxidative stress-associated biomarkers may be useful in assessing the sublethal effects of BDE 47 toxicity in other models. However, the fact that BDE 47 undergoes a concentration-dependent accumulation in other primary cells in media that can underestimate cellular concentrations (W. R. Mundy et al., 2004, Toxicol. Sci. 82, 164-169) suggests that the HSC cell injury observed in our study may be of less relevance to human in utero PBDE exposures.

Co-expression of ß Subunits with the Voltage-Gated Sodium Channel NaV1.7: the Importance of Subunit Association and Phosphorylation and Their Effects on Channel Pharmacology and Biophysics.

The voltage-gated sodium ion channel NaV1.7 is crucial in pain signaling. We examined how auxiliary ß2 and ß3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human NaV1.7α subunit was co-expressed with either ß2 or ß3 subunits in HEK-293 cells. The ß2 subunits and the NaV1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the ß2 subunits did not change the biophysical properties of the channel. In contrast, ß3 subunit was clearly associated with NaV1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated ß3 subunit was associated with the NaV1.7α. Electrophysiological characterisation revealed that the ß3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the ß3 subunit reduced the susceptibility of NaV1.7α to several sodium channel blockers. In addition, we assessed the functional effect of NaV1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of ß subunits with NaV1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with ß2. The ß3 subunit significantly influences the function of NaV1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of NaV1.7 channel in pain signaling.

Determining direct binders of the Androgen Receptor using a high-throughput Cellular Thermal Shift Assay.

Androgen Receptor (AR) is a key driver in prostate cancer. Direct targeting of AR has valuable therapeutic potential. However, the lack of disease relevant cellular methodologies capable of discriminating between inhibitors that directly bind AR and those that instead act on AR co-regulators has made identification of novel antagonists challenging. The Cellular Thermal Shift Assay (CETSA) is a technology enabling confirmation of direct target engagement with label-free, endogenous protein in living cells. We report the development of the first high-throughput CETSA assay (CETSA HT) to identify direct AR binders in a prostate cancer cell line endogenously expressing AR. Using this approach, we screened a pharmacology library containing both compounds reported to directly engage AR, and compounds expected to target AR co-regulators. Our results show that CETSA HT exclusively identifies direct AR binders, differentiating them from co-regulator inhibitors where other cellular assays measuring functional responses cannot. Using this CETSA HT approach we can derive apparent binding affinities for a range of AR antagonists, which represent an intracellular measure of antagonist-receptor Ki performed for the first time in a label-free, disease-relevant context. These results highlight the potential of CETSA HT to improve the success rates for novel therapeutic interventions directly targeting AR.

Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes.

Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1ß1δε, α3ß2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1ß1δε, α3ß2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses.

The novel diazoxide analog 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide is a selective Kir6.2/SUR1 channel opener.

ATP-sensitive K(+) (K(ATP)) channels are activated by a diverse group of compounds known as potassium channel openers (PCOs). Here, we report functional studies of the Kir6.2/SUR1 Selective PCO 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216). We recorded cloned K(ATP) channel currents from inside-out patches excised from Xenopus laevis oocytes heterologously expressing Kir6.2/SUR1, Kir6.2/SUR2A, or Kir6.2/SUR2B, corresponding to the beta-cell, cardiac, and smooth muscle types of the K(ATP) channel. NNC 55-9216 reversibly activated Kir6.2/SUR1 currents (EC(50) = 16 micromol/l). This activation was dependent on intracellular MgATP and was abolished by mutation of a single residue in the Walker A motifs of either nucleotide-binding domain of SUR1. The drug had no effect on Kir6.2/SUR2A or Kir6.2/SUR2B currents. We therefore used chimeras of SUR1 and SUR2A to identify regions of SUR1 involved in the response to NNC 55-9216. Activation was completely abolished and significantly reduced by swapping transmembrane domains 8-11. The reverse chimera consisting of SUR2A with transmembrane domains 8-11 and NBD2 consisting SUR1 was activated by NNC 55-9216, indicating that these SUR1 regions are important for drug activation. [(3)H]glibenclamide binding to membranes from HEK293 cells transfected with SUR1 was displaced by NNC 55-9216 (IC(50) = 105 micromol/l), and this effect was impaired when NBD2 of SUR1 was replaced by that of SUR2A. These results suggest NNC 55-9216 is a SUR1-selective PCO that requires structural determinants, which differ from those needed for activation of the K(ATP) channel by pinacidil and cromakalim. The high selectivity of NNC 55-9216 may prove to be useful for studies of the molecular mechanism of PCO action.

Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers.

According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl(-) channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1ß. This aptamer potentiated whole-cell Cl(-) currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.

Combining an amyloid-beta (Aß) cleaving enzyme inhibitor with a γ-secretase modulator results in an additive reduction of Aß production.

A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-ß (Aß) peptides in amyloid plaques. Aß peptides are produced by sequential cleavage of the amyloid precursor protein by the ß amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aß42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aß42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aß42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aß production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.

Glutamate-cysteine ligase attenuates TNF-induced mitochondrial injury and apoptosis.

Glutathione (GSH) is important in free radical scavenging, maintaining cellular redox status, and regulating cell survival in response to a wide variety of toxicants. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits. To determine whether increased GSH biosynthetic capacity enhances cellular resistance to tumor necrosis factor-alpha- (TNF-alpha-) induced apoptotic cell death, we have established several mouse liver hepatoma (Hepa-1) cell lines overexpressing GCLC and/or GCLM. Cells overexpressing GCLC alone exhibit modest increases in GCL activity, while cells overexpressing both subunits have large increases in GCL activity. Importantly, cells overexpressing both GCL subunits exhibit increased resistance to TNF-induced apoptosis as judged by a loss of redox potential; mitochondrial membrane potential; translocation of cytochrome c to the cytoplasm; and activation of caspase-3, caspase-8, and caspase-9. Analysis of the effects of TNF on these parameters indicates that maintaining mitochondrial integrity mediates this protective effect in GCL-overexpressing cells.

Toward tissue-selective pancreatic B-cells KATP channel openers belonging to 3-alkylamino-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides.

3-(Alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides were synthesized, and their activity on rat-insulin-secreting cells and rat aorta rings was compared to that of the K(ATP) channel activators diazoxide and pinacidil. Structure-activity relationships indicated that an improved potency and selectivity for the pancreatic tissue was obtained by introducing a fluorine atom in the 7-position and a short linear (preferably ethyl) or cyclic (preferably cyclobutyl) hydrocarbon chain on the nitrogen atom in the 3-position. By contrast, strong myorelaxant activity was gained by the introduction of a halogen atom different from the fluorine atom in the 7-position and a bulky branched alkylamino chain in the 3-position. Thus, 3-(ethylamino)-7-fluoro-4H-1,2,4-benzothiadiazine 1,1-dioxide (11) expressed a marked inhibitory activity on pancreatic B-cells (IC(50) = 1 microM) associated with a weak vasorelaxant effect (ED(50) > 300 microM), whereas 7-chloro-3-(1,1-dimethylpropyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide (27), which was only slightly active on insulin-secreting cells (IC(50) > 10 microM), was found to be very potent on vascular smooth muscle cells (ED(50) = 0.29 microM). Radioisotopic and electrophysiological investigations performed with 7-chlorinated, 7-iodinated, and 7-fluorinated 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides confirmed that the drugs activated K(ATP) channels. The present data revealed that subtle structural modifications of 3-(alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides can generate original compounds activating K(ATP) channels and exhibiting different in vitro tissue selectivity profiles.

Characterization of two novel forms of the rat sulphonylurea receptor SUR1A2 and SUR1BDelta31.

1. The ATP-sensitive potassium channel (K(ATP)) of pancreatic beta-cells is composed of the sulphonylurea-binding protein, SUR1, and the inwardly rectifying K(+) channel subunit, Kir6.2. We have characterized two novel isoforms of rat SUR1 in the RINm5F insulin-secreting cell line. 2. SUR1A2 is an allelic variant with a single amino acid change in the first nucleotide-binding domain. Coinjection of SUR1A2 plus Kir6.2 into Xenopus oocytes or expression of a SUR1A2-Kir6.2 tandem in HEK-293 cells yielded large currents with characteristics similar to the wild-type K(ATP) channel. 3. SUR1BDelta31, detected in several human tissues, is a splice variant of the rat SUR1 gene that lacks exon 31 of the corresponding human SUR1 gene. SUR1BDelta31 lacks the TM16-TM17 transmembrane-spanning helices leading to a protein with a different transmembrane topology. Coinjection of SUR1BDelta31 plus Kir6.2 into Xenopus oocytes or expression of the Kir6.2/SUR1BDelta31 tandem construct in HEK-293 cells did not result in any current, and a surface expression assay indicated that this channel does not reach the plasma membrane. 4. SUR1A2 and SUR1A1 proteins expressed in HEK-293 cells display similar binding affinities for [(3)H]-glibenclamide, while SUR1BDelta31 shows a 500-fold lower affinity. 5. These findings confirm that TM16-TM17 of SUR1 are important for high-affinity glibenclamide binding and that their deletion impairs trafficking of the K(ATP) channel to the surface membrane.