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).

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.

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.

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.

GABA(A) receptors mediate inhibition of T cell responses.

We describe the presence of functional GABA(A) receptors on T cells. GABA inhibited anti-CD3 and antigen-specific T cell proliferation in vitro in a dose-dependent manner that was 1) mimicked by the GABA(A) receptor agonist muscimol (but not the GABA(B) receptor agonist baclofen), 2) blocked by GABA(A) receptor antagonists and a GABA(A) receptor Cl- channel blocker (picrotoxin) and 3) enhanced by pentobarbital. These data suggest that GABA(A) receptors mediate this immune inhibition and that these receptors can be modulated in a similar fashion to their neuronal counterparts. Finally, GABA inhibited DTH responses in vivo. Thus, pharmacological modulation of GABA(A) receptors may provide new approaches to modulate T cell responses in inflammation and autoimmune disease.

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells.

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.

The 5-HT3B subunit is a major determinant of serotonin-receptor function.

The neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) mediates rapid excitatory responses through ligand-gated channels (5-HT3 receptors). Recombinant expression of the only identified receptor subunit (5-HT3A) yields functional 5-HT3 receptors. However, the conductance of these homomeric receptors (sub-picosiemens) is too small to be resolved directly, and contrasts with a robust channel conductance displayed by neuronal 5-HT3 receptors (9-17 pS). Neuronal 5-HT3 receptors also display a permeability to calcium ions and a current-voltage relationship that differ from those of homomeric receptors. Here we describe a new class of 5-HT3-receptor subunit (5-HT3B). Transcripts of this subunit are co-expressed with the 5-HT3A subunit in the amygdala, caudate and hippocampus. Heteromeric assemblies of 5-HT3A and 5-HT3B subunits display a large single-channel conductance (16 pS), low permeability to calcium ions, and a current-voltage relationship which resembles that of characterized neuronal 5-HT3 channels. The heteromeric receptors also display distinctive pharmacological properties. Surprisingly, the M2 region of the 5-HT3B subunit lacks any of the structural features that are known to promote the conductance of related receptors. In addition to providing a new target for therapeutic agents, the 5-HT3B subunit will be a valuable resource for defining the molecular mechanisms of ion-channel function.

A conditionally immortalized glial cell line that expresses mature myelin proteins and functional GABA(A) receptors.

We have isolated and characterized a conditionally immortalized glial cell line that expresses mature myelin proteins, as well as functional GABA(A) receptors. Glial cells were isolated from postnatal day 1 H-2Kb-tsA58 transgenic mice that contain the temperature-sensitive SV40 large T antigen oncogene under the control of an interferon-gamma-inducible promoter. A clonal line was isolated that grew rapidly under permissive conditions (33 degrees C in the presence of interferon-gamma), but not under nonpermissive conditions (37 degrees C in the absence of interferon-gamma). Cells expressed mRNAs of mature myelin proteins (myelin basic proteins and proteolipid protein) when grown under either permissive or nonpermissive conditions, but myelin basic proteins were detected only when cells were maintained at 37 degrees C. GABA induced an increase in intracellular calcium concentration, indicating the presence of functional receptors for this transmitter. Characterization of the GABA-evoked calcium response and whole-cell currents indicated that these responses were mediated by GABA(A) receptors. Reverse transcription-polymerase chain reaction revealed differential expression of mRNAs for specific GABA(A) receptor subunits. Messages for the alpha2, alpha4, beta1, and delta subunits were readily detected by reverse transcription-polymerase chain reaction, whereas messages for the alpha1, alpha3, alpha5, alpha6, beta2, beta3, and gamma1-3 subunits were not. As this cell line can be easily propagated and differentiated, it should provide a valuable tool in studying not only the development of oligodendroglia, but also the function of their transmitter receptors and myelin proteins.

Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells.

1. Radioligand binding and patch-clamp techniques were used to study the actions of gamma-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5 alpha-pregnan-3 alpha-ol-20-one on rat alpha 1 and beta 3 GABAA receptor subunits, expressed either alone or in combination. 2. Membranes from HEK293 cells after transfection with alpha 1 cDNA did not bind significant levels of [35S]-tert-butyl bicyclophosphorothionate ([35S]-TBPS) (< 0.03 pmol mg-1 protein). GABA (100 microM) applied to whole-cells transfected with alpha 1 cDNA and clamped at -60 mV, also failed to activate discernible currents. 3. The membranes of cells expressing beta 3 cDNAs bound [35S]-TBPS (approximately 1 pmol mg-1 protein). However, the binding was not influenced by GABA (10 nM-100 microM). Neither GABA (100 microM) nor picrotoxin (10 microM) affected currents recorded from cells expressing beta 3 cDNA, suggesting that beta 3 subunits do not form functional GABAA receptors or spontaneously active ion channels. 4. GABA (10 nM-100 microM) modulated [35S]-TBPS binding to the membranes of cells transfected with both alpha 1 and beta 3 cDNAs. GABA (0.1 microM-1 mM) also dose-dependently activated inward currents with an EC50 of 9 microM recorded from cells transfected with alpha 1 and beta 3 cDNAs, clamped at -60 mV. 5. Propofol (10 nM-100 microM), pentobarbitone (10 nM-100 microM) and 5 alpha-pregnan-3 alpha-ol-20-one (1 nM-30 microM) modulated [35S]-TBPS binding to the membranes of cells expressing either alpha 1 beta 3 or beta 3 receptors. Propofol (100 microM), pentobarbitone (1 mM) and 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) also activated currents recorded from cells expressing alpha 1 beta 3 receptors. 6. Propofol (1 microM-1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing beta 3 homomers. In contrast, application of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) failed to activate detectable currents. 7. Propofol (100 microM)-activated currents recorded from cells expressing either alpha 1 beta 3 or beta 3 receptors reversed at the Cl- equilibrium potential and were inhibited to 34 +/- 13% and 39 +/- 10% of control, respectively, by picrotoxin (10 microM). 5 alpha-Pregnan-3 alpha-ol-20-one (100 nM) enhanced propofol (100 microM)-evoked currents mediated by alpha 1 beta 3 receptors to 1101 +/- 299% of control. In contrast, even at high concentration 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) caused only a modest facilitation (to 128 +/- 12% of control) of propofol (100 microM)-evoked currents mediated by beta 3 homomers. 8. Propofol (3-100 microM) activated alpha 1 beta 3 and beta 3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 microM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a "surge' current on cessation of propofol (300 microM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) to cells expressing alpha 1 beta 3 receptors. 9. These observations demonstrate that rat beta 3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABAA receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.