Combined oil spill modelling and shoreline sensitivity analysis for contingency planning in the Irish Sea.

Offshore oil spills often result in severe environmental and socio-economic consequences. This work focuses on a busy, yet poorly studied part of NW Europe, the Irish Sea, to assess the impact of future oil spills on the nearby coast. By integrating numerical models and shoreline sensitivity analyses for two confined areas, Liverpool Bay and Milford Haven, this work acknowledges wind direction and speed as principal controls on the movement of oil under winter/storm conditions and in shallow waters. Ocean currents play a secondary role, but are significant in deeper waters and in low-wind summer conditions. The temporal elements used in the modelling thus stress that when the spill occurs is just as important as where. As a corollary, the fate of spilled oil is determined in this work for distinct scenarios and types. Response strategies are recommended to minimise the impact of future spills on coastal populations.

Modelling soil erosion responses to climate change in three catchments of Great Britain.

Simulations of 21st century climate change for Great Britain predict increased seasonal precipitation that may lead to widespread soil loss by increasing surface runoff. Land use and different vegetation cover can respond differently to this scenario, mitigating or enhancing soil erosion. Here, by means of a sensitivity analysis of the PESERA soil erosion model, we test the potential for climate and vegetation to impact soil loss by surface-runoff to three differentiated British catchments. First, to understand general behaviours, we modelled soil erosion adopting regular increments for rainfall and temperature from the baseline values (1961-1990). Then, we tested future climate scenarios adopting projections from UKCP09 (UK Climate Projections) under the IPCC (Intergovernmental Panel on Climate Change) on a defined medium CO2 emissions scenario, SRES-A1B (Nakicenovic et al., 2000), at the horizons 2010-39, 2040-69 and 2070-99. Our results indicate that the model reacts to the changes of the climatic parameters and the three catchments respond differently depending on their land use arrangement. Increases in rainfall produce a rise in soil erosion while higher temperatures tend to lower the process because of the mitigating action of the vegetation. Even under a significantly wetter climate, warmer air temperatures can limit soil erosion by enhancing primary productivity and in turn improving leaf interception, infiltration-capacity, and reducing soil erodibility. Consequently, for specific land uses, the increase in air temperature associated with climate change can modify the rainfall thresholds to generate soil loss, and soil erosion rates could decline by up to about 33% from 2070 to 2099. We deduce that enhanced primary productivity due to climate change can introduce a negative-feedback mechanism limiting soil loss by surface runoff as vegetation-induced impacts on soil hydrology and erodibility offset the effects of increased precipitation. The expansion of permanent vegetation cover could provide an adaptation strategy to reduce climate-driven soil loss.

Association of opioid prescribing practices with chronic pain and benzodiazepine co-prescription: a primary care data linkage study.

BACKGROUND: Opioid prescribing is increasing worldwide with associated increases in misuse and other harms. We studied variations in national opioid prescription rates, indicators of prescribing quality, co-prescribing of benzodiazepines and relationship with pain severity in Scotland. METHODS: Electronic linkages of opioid prescribing in Scotland were determined from: (i) national data from Information Services Division, NHS Scotland (2003-2012); and (ii) individual data from Generation Scotland: Scottish Family Health Study. Descriptive analyses were conducted on national data, multilevel modelling to examine factors associated with variations in prescribing rates. χ2 tests examined associations between individual pain severity and opioid prescriptions. RESULTS: The number of strong opioid prescriptions more than doubled from 474 385 in 2003 to 1 036 446 in 2012, and weak opioid prescribing increased from 3 261 547 to 4 852 583. In Scotland, 938 674 individuals were prescribed an opioid in 2012 (18% of the population). Patients in the most deprived areas were 3.5 times more likely to receive a strong opioid than patients in the least deprived. There was significant variation in prescribing rates between geographical areas, with much of this explained by deprivation. Of women aged 25-40 yr prescribed a strong opioid, 40% were also prescribed a benzodiazepine. There was significant association between pain severity and receipt of opioid prescription. Over 50% of people reporting severe pain were not prescribed an opioid analgesic. CONCLUSIONS: We found opioid prescribing in primary care to be common and increasing in Scotland, particularly for severe pain. Co-prescribing of opioids and benzodiazepines was common.

Potent inhibition by ropivacaine of metastatic colon cancer SW620 cell invasion and NaV1.5 channel function.

BACKGROUND: Metastatic breast and colon cancer cells express neonatal and adult splice variants of NaV1.5 voltage-activated Na(+) channels (VASCs). Block of VASCs inhibits cell invasion. Local anaesthetics used during surgical tumour excision inhibit VASC activity on nociceptive neurones providing regional anaesthesia. Inhibition of VASCs on circulating metastatic cancer cells may also be beneficial during the perioperative period. However, ropivacaine, frequently used to provide analgesia during tumour resection, has not been tested on colon cancer cell VASC function or invasion. METHODS: We used reverse transcription-polymerase chain reaction and sequencing to identify NaV1.5 variants in the SW620 metastatic colon cancer cell line. Recombinant adult and neonatal NaV1.5 variants were expressed in human embryonic kidney cells. Voltage-clamp recordings and invasion assays were used to examine the effects of ropivacaine on recombinant NaV1.5 channels and the metastatic potential of SW620 cells, respectively. RESULTS: SW620 cells expressed adult and neonatal NaV1.5 variants, which had similar steady-state inactivation profiles, but distinctive activation curves with the neonatal variant having a V1/2 of activation 7.8 mV more depolarized than the adult variant. Ropivacaine caused a concentration-dependent block of both NaV1.5 variants, with IC50 values of 2.5 and 3.9 µM, respectively. However, the reduction in available steady-state current was selective for neonatal NaV1.5 channels. Ropivacaine inhibited SW620 invasion, with a potency similar to that of inhibition of NaV1.5 channels (3.8 µM). CONCLUSIONS: Ropivacaine is a potent inhibitor of both NaV1.5 channel activity and metastatic colon cancer cell invasion, which may be beneficial during surgical colon cancer excision.

Novel structural determinants of single-channel conductance in nicotinic acetylcholine and 5-hydroxytryptamine type-3 receptors.

Nicotinic ACh (acetylcholine) and 5-HT3 (5-hydroxytryptamine type-3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Numerous lines of evidence indicate that the channel lining domain of such receptors is formed by the alpha-helical M2 domain (second transmembrane domain) contributed by each of five subunits present within the receptor complex. Specific amino acid residues within the M2 domain have accordingly been demonstrated to influence both single-channel conductance (gamma) and ion selectivity. However, it is now clear from work performed on the homomeric 5-HT3A receptor, heteromeric 5-HT3A/5-HT3B receptor and 5-HT3A/5-HT3B receptor subunit chimaeric constructs that an additional major determinant of gamma resides within a cytoplasmic domain of the receptor termed the MA-stretch (membrane-associated stretch). The MA-stretch, within the M3-M4 loop, is not traditionally thought to be implicated in ion permeation and selection. Here, we describe how such observations extend to a representative neuronal nicotinic ACh receptor composed of alpha4 and beta2 subunits and, by inference, probably other members of the Cys-loop family. In addition, we will attempt to interpret our results within the context of a recently developed atomic scale model of the nicotinic ACh receptor of Torpedo marmorata (marbled electric ray).

A lithospheric instability origin for Columbia River flood basalts and Wallowa Mountains uplift in northeast Oregon.

Flood basalts appear to form during the initiation of hotspot magmatism. The Columbia River basalts (CRB) represent the largest volume of flood basalts associated with the Yellowstone hotspot, yet their source appears to be in the vicinity of the Wallowa Mountains, about 500 km north of the projected hotspot track. These mountains are composed of a large granitic pluton intruded into a region of oceanic lithosphere affinity. The elevation of the interface between Columbia River basalts and other geological formations indicates that mild pre-eruptive subsidence took place in the Wallowa Mountains, followed by syn-eruptive uplift of several hundred metres and a long-term uplift of about 2 km. The mapped surface uplift mimics regional topography, with the Wallowa Mountains in the centre of a 'bull's eye' pattern of valleys and low-elevation mountains. Here we present the seismic velocity structure of the mantle underlying this region and erosion-corrected elevation maps of lava flows, and show that an area of reduced mantle melt content coincides with the 200-km-wide topographic uplift. We conclude that convective downwelling and detachment of a compositionally dense plutonic root can explain the timing and magnitude of Columbia River basalt magmatism, as well as the surface uplift and existence of the observed melt-depleted mantle.

The 5-hydroxytryptamine type 3 (5-HT3) receptor reveals a novel determinant of single-channel conductance.

5-HT3 (5-hydroxytryptamine type 3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been characterized in detail, although additional putative 5-HT3 subunit genes (HTR3C, HTR3D and HTR3E) have recently been reported. 5-HT3 receptors function as homopentameric assemblies of the 5-HT3 subunit, or heteropentamers of 5-HT3A and 5-HT3B subunits of unknown stoichiometry. The single-channel conductances of human recombinant homomeric and heteromeric 5-HT3 receptors are markedly different, being <1 and approx. 16 pS respectively. Paradoxically, from the results of studies performed on the closely related nicotinic acetylcholine receptor, the channel-lining M2 domain of the 5-HT3A subunit is predicted to enhance cation conduction, whereas that of the 5-HT3B subunit would not. The present study describes a novel determinant of single-channel conductance, out with the M2 domain, which accounts for this anomaly. Utilizing a panel of chimaeric 5-HT3A and 5-HT3B subunits, a profound determinant of single-channel conductance was traced to a putative amphipathic helix (the 'HA stretch') within the large cytoplasmic loop of the receptor. Replacement of three arginine residues (R432, R436 and R440) unique to the HA stretch of the 5-HT3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT3B subunit increased the single-channel conductance 28-fold. Significantly, from ultrastructural studies of the Torpedo nicotinic acetylcholine receptor, the key residues may be components of narrow openings within the inner vestibule of the channel, located in the cytoplasm, which contribute to the permeation pathway. Our findings indicate an important and hitherto unappreciated function for the HA stretch in the Cys-loop family of transmitter-gated ion channels.

Bacillus anthracis contamination and inhalational anthrax in a mail processing and distribution center.

AIMS: Four inhalational anthrax cases occurred in a large mail processing and distribution center in Washington, DC, after envelopes containing Bacillus anthracis spores were processed. This report describes the results of sampling for B. anthracis spores during investigations conducted in October and December 2001. METHODS AND RESULTS: Wet swabs, wet wipes, vacuum sock, and air-filter samples were collected throughout the facility to characterize the extent of building contamination. The results showed widespread contamination of B. anthracis spores, particularly associated with one delivery bar code sorter (DBCS) machine that had sorted the spore-containing envelopes and an area where the envelopes were handled by postal workers. Spore concentrations decreased as distance from the DBCS machine increased, but spores were widely dispersed into surrounding areas. CONCLUSION: The spatial distribution of culture positive samples was closely related to the work areas of the inhalational anthrax cases and supported epidemiological evidence that the workers became ill from exposure to B. anthracis spores in areas where the contaminated envelopes had travelled. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this investigation were used to guide decontamination efforts and provided baseline spore concentrations for follow-up measurements after the building had been cleaned. Implementing methods to reduce aerosolization and dispersion of dust within the facility would reduce postal workers' potential exposures to bioterrorism agents.

Introduction of the 5-HT3B subunit alters the functional properties of 5-HT3 receptors native to neuroblastoma cells.

The identification of a second 5-HT(3) (5-HT(3B)) subunit provides an explanation for 5-HT(3) receptor heterogeneity. We investigated whether introduction of recombinant 5-HT(3B) subunits would alter the functional properties of mouse neuroblastoma 5-HT(3) receptors. RT-PCR analysis revealed that NB41A3 cells contain mRNAs encoding 5-HT(3A) and 5-HT(3B) subunits. 5-HT increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and caused the concentration-dependent activation of inward currents recorded at -60 mV. Both actions of 5-HT were antagonized by ondansetron. The 5-HT concentration-response relationship of NB41A3 cells was indistinguishable from that of the related NG108-15 cell line. The selective 5-HT(3)-receptor agonist mCPBG also elevated [Ca(2+)](i) and activated inward currents. 2-M-5HT was less efficacious than 5-HT as an activator of 5-HT(3) receptors in NB41A3 cells and did not significantly increase [Ca(2+)](i). The 5-HT induced increase in [Ca(2+)](i) did not involve caffeine- or thapsigargin-sensitive intracellular Ca(2+) stores. The introduction of the 5-HT(3B) subunit by transient transfection of NB41A3 cells caused 5-HT to become less potent as an activator of 5-HT(3) receptors and altered the kinetics of 5-HT activated currents so that they resembled currents mediated by 5-HT(3AB) receptors. The 5-HT(3B) subunit also abolished the 5-HT induced [Ca(2+)](i) increase seen in untransfected NB41A3 cells. These data are consistent with the hypothesis that NB41A3 cells predominantly express homomeric 5-HT(3A) receptors that become heteromeric 5-HT(3AB) receptors upon introduction of the recombinant 5-HT(3B) subunit.

Alternative transcripts of the GABA(A) receptor epsilon subunit in human and rat.

In both human and rat tissues, complex patterns of transcripts are derived from the genes that encode the gamma-aminobutyric acid (GABA)(A) receptor epsilon subunit. An epsilon subunit transcript (approximately 3.6 kb) is expressed at relatively high levels in regions of the human brain and heart, but is not detected in most other major tissues. The encoded human epsilon subunit (epsilon (h)) confers distinctive properties to receptors into which it assembles. A distinct transcript of the gene (6.2 kb) is expressed abundantly in a variety of human tissues. This alternative transcript (ET2) appears to originate from within the epsilon subunit gene. It is possible that this transcript encodes a truncated subunit (epsilon (hS)), containing all of the transmembrane and intracellular domains. However, a combination of biochemical and electrophysiological analyses does not support this hypothesis. A distinct transcript of the epsilon subunit gene, encoding a large extracellular pro/glx domain, is expressed abundantly in rat and mouse brain. Functional analyses also failed to provide evidence for incorporation of this subunit (epsilon (rL)) into recombinant receptors. However, a shorter rat epsilon subunit (epsilon (r)), which lacks the pro/glx domain, conferred epsilon (h)-like properties to recombinant receptors, providing evidence for a functional rat epsilon subunit. In common with its human orthologue, incorporation of the epsilon (r) subunit into recombinant GABA(A) receptors confers several distinctive properties, including a reduced modulation by the anesthetic propofol and the appearance of spontaneous current.

Evidence for the formation of functionally distinct alphabetagammaepsilon GABA(A) receptors.

1. We transiently introduced the human GABA(A) receptor epsilon subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing alpha1beta3gamma2 receptors (WSS-1 cells) to establish whether the subunit competes with the gamma2 subunit for assembly into receptors. GABA-evoked currents were recorded using the patch-clamp technique from cells transfected with cDNA encoding green fluorescent protein (GFP) alone or in combination with the epsilon subunit cDNA. 2. The epsilon subunit did not change the potency of GABA: the GABA EC(50) was 34 +/- 6 microM in control WSS-1 cells and 37 +/- 6 microM in cells expressing the epsilon subunit. The introduction of the epsilon subunit reduced the peak current amplitude activated by GABA (1 mM) from 1.8 +/- 0.2 nA in control cells to 0.9 +/- 0.2 nA in cells expressing the epsilon subunit (P < 0.05). 3. The epsilon subunit caused the appearance of leak currents recorded in the absence of GABA. Outside-out patches excised from epsilon subunit-containing WSS-1 cells exhibited spontaneously opening GABA(A) channels not seen in patches excised from control GFP-expressing WSS-1 cells. Introduction of the epsilon subunit did not alter the GABA-evoked single-channel cord conductance. 4. The anaesthetic 2,6-diisopropylphenol (propofol, 3 microM) and the benzodiazepine flunitrazepam (1 microM) potentiated GABA-evoked currents recorded from control cells labelled with GFP. The epsilon subunit reduced potentiation by both agents 48-96 h after transfection. 5. The introduction of the epsilon subunit had no effect on the ability of propofol (3-30 microM) relative to GABA (1 mM) to activate GABA(A) receptors in WSS-1 cells. High concentrations of propofol (> or = 100 microM) produced a more marked desensitization of GABA(A) receptor activity in WSS-1 cells transfected with cDNA for the epsilon subunit than in control cells. 6. There was no difference in the potency of Zn(2+) as an inhibitor of currents recorded from control cells (IC(50) = 165 +/- 34 microM) or cells expressing the epsilon subunit (IC(50) = 179 +/- 11 microM). 7. GABA-activated currents recorded both from control cells and cells expressing the epsilon subunit reversed in sign at the Cl- equilibrium potential and exhibited outward rectification. 8. The introduction of the epsilon subunit changes the functional properties of GABA(A) receptors in WSS-1 cells. The resulting receptors have a unique combination of properties indicative of the co-assembly of alpha, beta, gamma and epsilon subunits.

Functional properties of Cav1.3 (alpha1D) L-type Ca2+ channel splice variants expressed by rat brain and neuroendocrine GH3 cells.

Ca(2+) enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca(2+) entry through activation of pertussis toxin-sensitive G proteins leading to activation of K(+) channels and voltage-sensitive inhibition of L-type channel activity. Despite the cloning and functional expression of several Ca(2+) channels, those involved in regulating hormone release remain unknown. Using reverse transcription-polymerase chain reaction we identified mRNAs encoding three alpha(1) (alpha(1A), alpha(1C), and alpha(1D)), four beta, and one alpha(2)-delta subunit in rat pituitary GH(3) cells; alpha(1B) and alpha(1S) transcripts were absent. GH(3) cells express multiple alternatively spliced alpha(1D) mRNAs. Many of the alpha(1D) transcript variants encode "short" alpha(1D) (alpha(1D-S)) subunits, which have a QXXER amino acid sequence at their C termini, a motif found in all other alpha(1) subunits that couple to opioid receptors. The other splice variants identified terminate with a longer C terminus that lacks the QXXER motif (alpha(1D-L)). We cloned and expressed the predominant alpha(1D-S) transcript variants in rat brain and GH(3) cells and their alpha(lD-L) counterpart in GH(3) cells. Unlike alpha(1A) channels, alpha(1D) channels exhibited current-voltage relationships similar to those of native GH(3) cell Ca(2+) channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced alpha(1D) transcripts form functional Ca(2+) channels that exhibit voltage-dependent, G protein-independent facilitation. Furthermore, the QXXER motif, located on the C terminus of alpha(1D-S) subunit, is not sufficient to confer sensitivity to inhibitory G proteins.

Advances in random matrix theory, zeta functions, and sphere packing.

Over four hundred years ago, Sir Walter Raleigh asked his mathematical assistant to find formulas for the number of cannonballs in regularly stacked piles. These investigations aroused the curiosity of the astronomer Johannes Kepler and led to a problem that has gone centuries without a solution: why is the familiar cannonball stack the most efficient arrangement possible? Here we discuss the solution that Hales found in 1998. Almost every part of the 282-page proof relies on long computer verifications. Random matrix theory was developed by physicists to describe the spectra of complex nuclei. In particular, the statistical fluctuations of the eigenvalues ("the energy levels") follow certain universal laws based on symmetry types. We describe these and then discuss the remarkable appearance of these laws for zeros of the Riemann zeta function (which is the generating function for prime numbers and is the last special function from the last century that is not understood today.) Explaining this phenomenon is a central problem. These topics are distinct, so we present them separately with their own introductory remarks.

delta-Opioid receptors are more efficiently coupled to adenylyl cyclase than to L-type Ca(2+) channels in transfected rat pituitary cells.

Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which delta-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH(3) cells with cDNAs encoding for delta-opioid receptors. In cells transfected with a relatively low delta-receptor density of 0.55 pmol/mg of protein (GH(3)DOR), activation of delta-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca(2+) current. In contrast, activation of delta-receptors in a clone that contained a higher density of delta-receptors (2.45 pmol/mg of protein) and was also coexpressed with mu-opioid receptors (GH(3)MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca(2+) current. The purpose of the present study was to determine whether these observations resulted from differences in delta-opioid receptor density between clones or interaction between delta- and mu-opioid receptors to allow the activation of different G proteins and signaling to Ca(2+) channels. Using the delta-opioid receptor alkylating agent SUPERFIT, reduction of available delta-opioid receptors in GH(3)MORDOR cells to a density similar to that of delta-opioid receptors in the GH(3)DOR clone resulted in abolishment of coupling to Ca(2+) channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by delta-opioid receptors in GH(3)MORDOR cells, delta-opioid receptor activation in GH(3)DOR cells resulted in coupling to the identical pattern of G proteins seen in GH(3)MORDOR cells. These findings suggest that different threshold densities of delta-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that delta-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca(2+) channels.

Evidence for expression of heteromeric serotonin 5-HT(3) receptors in rodents.

The gene and cDNAs that encode a novel subunit of rodent serotonin 5-HT(3) receptors were isolated from mouse and rat tissues. Each of the new rodent subunits shares 40% amino acid identity with the rat 5-HT(3A) subunit and 73% identity with the human 5-HT(3B) subunit. Despite a relatively low level of structural conservation, sequence analysis and functional studies suggest that the new rodent subunits are orthologues of the human 5-HT(3B) subunit. In common with homologous human receptors, rat heteromeric 5-HT(3) receptors displayed a substantially larger single-channel conductance than homomeric 5-HT(3A) receptors. In addition, the rat heteromeric receptors were less sensitive to antagonism by tubocurarine. However, in contrast to human heteromeric receptors, those of the rat displayed pronounced inward rectification of both the whole-cell and single-channel current amplitudes. Transcripts of the mouse 5-HT(3A) and 5-HT(3B) subunits are coexpressed in several cell lines that possess endogenous 5-HT(3) receptors. In addition, treatment of rat PC12 cells with nerve growth factor induced expression of both subunit mRNAs, with a similar time course for accumulation of each transcript. The combination of functional data and expression patterns is consistent with the existence of heteromeric 5-HT(3) receptors in rodent neurons.

Cloned delta-opioid receptors in GH(3) cells inhibit spontaneous Ca(2+) oscillations and prolactin release through K(IR) channel activation.

Opioid receptors can couple to K(+) and Ca(2+) channels, adenylyl cyclase, and phosphatidyl inositol turnover. Any of these actions may be important in the regulation of neurotransmitter and hormone release from excitable cells. GH(3) cells exhibit spontaneous oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) and prolactin release. Activation of cloned delta-opioid receptors stably expressed in GH(3) cells inhibits both spontaneous Ca(2+) signaling and basal prolactin release. The objective of this study was to examine a possible role for K(+) channels in these processes using the patch-clamp technique, fluorescence imaging, and a sensitive ELISA for prolactin. The selective delta receptor agonist [D-Pen(2), D-Pen(2)]enkephalin (DPDPE) inhibited [Ca(2+)](i) oscillations in GH(3) cells expressing both mu and delta receptors (GH(3)MORDOR cells) but had no effect on control GH(3) cells or cells expressing mu receptors alone (GH(3)MOR cells). The inhibition of [Ca(2+)](i) oscillations by DPDPE was unaffected by thapsigargin pretreatment, suggesting that this effect is independent of inositol 1,4,5-triphosphate-sensitive Ca(2+) stores. DPDPE caused a concentration-dependent inhibition of prolactin release from GH(3)MORDOR cells with an IC(50) of 4 nM. DPDPE increased inward K(+) current recorded from GH(3)MORDOR cells but had no significant effect on K(+) currents recorded from control GH(3) cells or GH(3)MOR cells. The mu receptor agonist morphine also had no effect on currents recorded from control cells but activated inward K(+) currents recorded from GH(3)MOR and GH(3)MORDOR cells. Somatostatin activated inward currents recorded from all three cell lines. The DPDPE-sensitive K(+) current was inwardly rectifying and was inhibited by Ba(2+) but not TEA. DPDPE had no effect on delayed rectifier-, Ca(2+)-, and voltage-activated or A-type K(+) currents, recorded from GH(3)MORDOR cells. Ba(2+) attenuated the inhibition of [Ca(2+)](i) and prolactin release by DPDPE, whereas TEA had no effect, consistent with an involvement of K(IR) channels in these actions of the opioid.

The influence of an endogenous beta3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells.

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of gamma-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant alpha1 and gamma2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of alpha1 and gamma2 subunits and suggest the existence of an endogenous beta3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the gamma2 subunit, WSS-1 cell receptors also contain beta2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain beta3 mRNA. We examined the contribution of the beta3 subunit in the function of receptors formed by expression of alpha1 and gamma2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with alpha1 and gamma2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of alpha1 and gamma2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks beta subunit mRNAs, failed to produce functional receptors. These data reaffirm that alpha1 and gamma2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain alpha1, beta3 and gamma2S subunits.