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.

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.

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.

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.

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.

Potentiation, activation and blockade of GABAA receptors of clonal murine hypothalamic GT1-7 neurones by propofol.

1. The actions of GABA and the intravenous general anaesthetic propofol (2,6-diisopropylphenol) on GABAA receptors of self-replicating GT1-7 hypothalamic neurones were investigated by the patch clamp technique. 2. GABA (1 microM-1 mM) dose-dependently activated inward currents with an EC50 of 27 microM, recorded from whole cells voltage-clamped at -60 mV. GABA (100 microM)-activated currents reversed at the Cl-equilibrium potential. 3. Propofol (0.1-100 microM) dose-dependently potentiated GABA (100 microM)-evoked currents with an EC50 of 5 microM. 4. In the absence of GABA, propofol (10 microM-1 mM) activated small inward currents with a reversal potential similar to the Cl- equilibrium potential. The peak current amplitudes activated by propofol were only 31% of those activated by GABA in the same cells. 5. Like GABA (100 microM)-activated currents, propofol (100 microM)-activated currents were inhibited by the GABAA receptor antagonist, bicuculline (10 microM) and were abolished by Zn2+ (100 microM). 6. Propofol (10, 30 and 100 microM) dose-dependently activated currents in the absence of GABA. However, the peak amplitude of currents activated by propofol declined with concentrations > 100 microM. The cessation of application of a high dose of propofol (1 mM) was associated with a current 'surge'. 7. The surge current, seen after application of propofol (1 mM), had a reversal potential similar to the Cl- equilibrium potential. The ratio between peak current amplitude in the presence of propofol (1 mM) and surge current amplitude after propofol application, were not dependent on holding potential. Thus,it is unlikely that the surge current represents reversal of a voltage-dependent block of GABAA receptors by propofol.8. The amplitude of the surge current exceeded the amplitude of the initial propofol (1 mM)-evoked current following brief applications, but declined after prolonged applications of the drug.9. The observed modulatory actions of propofol may be due to separate potentiation, activation and inhibitory sites for this anaesthetic agent on GT1-7 cell GABAA receptors.

The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurones.

1. The interaction of the intravenous general anaesthetic propofol (2,6-diisopropylphenol) with the GABAA receptor has been investigated in voltage-clamped bovine chromaffin cells and rat cortical neurones in cell culture. Additionally, the effects of propofol on the glycine and GABAA receptors of murine spinal neurones were determined. 2. Propofol (1.7-16.8 microM) reversibly and dose-dependently potentiated the amplitude of membrane currents elicited by GABA (100 microM) applied locally to bovine chromaffin cells. Intracellular application of propofol (16.8 microM) was ineffective. In rat cortical neurones and murine spinal neurones, extracellular application of 8.4 microM and 1.7-16.8 microM propofol respectively produced a potentiation of GABA-evoked currents qualitatively similar to that seen in the bovine chromaffin cell. 3. The potentiation by propofol (1.7 microM) was not associated with a change in the reversal potential of the GABA-evoked whole cell current. On outside-out membrane patches isolated from bovine chromaffin cells, propofol (1.7 microM) had little or no effect on the GABA single channel conductances, but greatly increased the probability of the GABA-gated channel being in the conducting state. 4. The potentiation of GABA-evoked whole cell currents by propofol (1.7 microM) was not influenced by the benzodiazepine antagonist flumazenil (0.3 microM). A concentration of propofol (1.7 microM) that substantially potentiated GABA currents had little effect on currents induced by the activation of the GABAA receptor by pentobarbitone (1 mM). 5. Bath application of propofol (8.4-252 microM), to bovine chromaffin cells voltage clamped at -60 mV, induced an inward current associated with an increase in membrane current noise on all cells sensitive to GABA. Intracellular application of propofol (16.8 microM) was ineffective in this respect. Local application of propofol (600 microM) induced whole cell currents with a reversal potential dependent upon the Cl- gradient across the cell membrane. 6. On outside-out membrane patches formed from bovine chromaffin cells, propofol (30 microM) induced single channels with mean chord conductances of 29 and 12 pS. The frequency of propofol channels was greatly reduced by coapplication of 1 microM bicuculline. Under identical ionic conditions, GABA (1 microM) activated single channels with mean chord conductances of 33, 16 and 10pS. 7. Bath applied propofol (0.84-16.8 microM) dose-dependently potentiated strychnine-sensitive currents evoked by glycine (100 microM) in murine spinal neurones. 8. The relevance of the present results to the general anaesthetic action of propofol is discussed.

The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques.

1 The characteristics of transmembrane currents evoked by 5-hydroxytryptamine (5-HT) in the neuroblastoma x Chinese hamster brain cell line NCB-20 and neuroblastoma clonal cell line N1E-115 have been studied under voltage-clamp conditions by the whole-cell recording and outside-out membrane patch modes of the patch-clamp technique. 2 In 73% of NCB-20 cells examined (n = 221), and all N1E-115 cells studied (n = 80), 5-HT (10 microM) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5-HT reversed in sign at a potential of approximately -2 mV and demonstrated inward rectification. 3 The reversal potential of 5-HT-induced currents (E5-HT) recorded from either NCB-20 or N1E-115 cells was unaffected by total replacement of internal K+ by Cs+. In N1E-115 cells, reducing internal K+ concentration from 140 to 20 mM produced a positive shift in E5-HT of approximately 28 mV, whereas reducing external Na+ from 143 to 20 mM was associated with a negative shift in E5-HT of about 37 mV. A large reduction in internal Cl- concentration (from 144 to 6 mM) had little effect on E5-HT. 4 5-HT-induced currents of NCB-20 cells were unaffected by methysergide (1 microM) or ketanserin (1 microM), but were reversibly antagonized by GR38032F (0.1-1.0 nM) with an IC50 of 0.25 nM. GR 38032F (0.3 nM) reduced 5-HT-induced currents in N1E-115 cells to approximately 26% of their control value. 5 On outside-out membrane patches excised from both NCB-20 and N1E-115 cells, 5-HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nM) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5-HT (1 microM) in N1E-115 cells suggests that 5-HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside-out membrane patches to 5-HT cannot at present be resolved into clear single channel events.

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.

Modulation of GABAA and glycine receptors by chlormethiazole.

The influence of chlormethiazole, on currents evoked by gamma-aminobutyric acid (GABA) and glycine, was investigated under voltage-clamp conditions, in bovine chromaffin cells and murine spinal neurones, respectively. Chlormethiazole (30 and 100 microM) dose dependently potentiated currents activated by either inhibitory neurotransmitter. The potentiation of the GABA-evoked response occurred without altering the reversal potential and was not influenced by the benzodiazepine receptor antagonist Ro 15-1788 (300 nM). GABA-gated channels, recorded from outside-out membrane patches, showed increased probability of being in the conducting state in the presence of chlormethiazole. High concentrations of chlormethiazole (3 mM) activated bicuculline (1 microM)-sensitive whole-cell currents with a reversal potential similar to the chloride equilibrium potential. Chlormethiazole potentiates GABA- and glycine-activated currents and at higher doses, directly activates the GABAA receptor.

Divalent cations modulate 5-HT3 receptor-induced currents in N1E-115 neuroblastoma cells.

The influence of extracellular calcium and magnesium ion concentrations upon 5-HT3 receptor-gated membrane currents in murine N1E-115 neuroblastoma cells has been studied under voltage-clamp conditions. A decrease in the concentration of either Ca2+ or Mg2+ from their standard values of 1.0 and 2.0 mM respectively augmented both the amplitude and duration of the 5-HT-induced current, whereas elevating the concentration of either divalent cation produced the opposite effect. Such modulation did not involve a change in the reversal potential of the response.

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).

Few cell lines with GABAA mRNAs have functional receptors.

In the preceding companion article (Tyndale et al., 1994) we used the PCR to investigate the occurrence of 13 GABAA receptor subunit mRNAs in several cell lines, including those derived from brain (B65, B104, and NB41A3), cerebellum (C17), glia (C6), pituitary (AtT-20), adrenal medulla (PC12), and the endocrine pancreas (RINm5F and beta TC3). In the present study we used the whole-cell configuration of the patch-clamp technique to determine which of these cell lines express functional GABAA receptors. All of the cell lines contain detectable levels of at least one GABAA receptor subunit mRNA (Tyndale et al., 1994); however, only RINm5F and beta TC3 cells exhibited GABA-evoked currents. GABA activated currents in all RINm5F cells, but currents were only barely detectable in 50% of beta TC3 cells tested. Many of the cell lines that failed to respond to GABA were derived from cell types with functional GABAA receptors. For example, the failure of PC12 cells to respond to GABA contrasts with the observation of GABA responses recorded from all primary cultured adrenomedullary chromaffin cells tested. GABA-evoked currents recorded from beta TC3 cells were too small (< 10 pA) to characterize pharmacologically. However, GABA activated robust currents recorded from RINm5F cells. These currents reversed at a holding potential similar to the equilibrium potential for Cl-, were blocked by the antagonist bicuculline methiodide (10 microM), and were potentiated by pentobarbital (100 microM). RINm5F cell GABAA receptors were insensitive to diazepam (10 microM) and were inhibited by Zn2+ (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT1-7) neurons.

1. Individual immortalized gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons in semiconfluent cultures showed spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) as measured by video fluorescence microscopy and fura-2. In parallel experiments, GT1-7 neurons also showed spontaneous bursts of action potentials that were recorded as action currents from intact cells. The bursts of action currents occurred in characteristic patterns, suggesting an underlying rhythmic oscillation in membrane potential. 2. Depolarization with increased extracellular K+ evoked a concentration-dependent increase in the frequency of Ca2+ oscillations or a sustained plateau of increased [Ca2+]i in GT1-7 neurons. Increased extracellular K+ (30 mM) caused an initial increase in the frequency of action currents, after which they were reversibly abolished. 3. The Ca2+ channel blockers Ni2+ and nimodipine abolished Ca2+ oscillations, whereas nifedipine, gadolinium, omega-conotoxin and omega-agatoxin had no effect on Ca2+ oscillations. These results indicate that Ca2+ oscillations are generated by influx of Ca2+ through voltage-gated Ca2+ channels that are not sensitive to nifedipine and are not N-type or P-type channels. 4. Thapsigargin caused a small, transient rise in baseline [Ca2+]i but had no effect on Ca2+ oscillations. Caffeine and ryanodine had no effect on baseline [Ca2+]i or Ca2+ oscillations. These results indicate that the release of Ca2+ from inositol 1,4,5-trisphosphate (IP-3)-sensitive or caffeine sensitive intracellular stores does not play a major role in Ca2+ oscillations in GT1-7 neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

GABA has excitatory actions on GnRH-secreting immortalized hypothalamic (GT1-7) neurons.

The effects of gamma-aminobutyric acid (GABA) on clonal gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons were investigated using patch-clamp and fura-2 imaging techniques. Local application of GABA (100 microM) to GT1-7 cells voltage-clamped in the whole-cell configuration immediately increased membrane conductance and noise consistent with activation of the GABAA receptor-Cl- channel complex. Depolarization activated transient Na+ currents which were abolished by tetrodotoxin (TTX; 0.5 microM), and more sustained Ca2+ currents. Under constant current conditions, GT1-7 cells fired spontaneous action potentials, and depending on the Cl- equilibrium potential, GABA either depolarized cells, causing a rapid activation of action potentials, or hyperpolarized cells. In order to determine the effect of GABA on intact cells, the cell-attached patch configuration was used to record extracellularly. Under these conditions, application of GABA (100 microM), but not the GABAB receptor agonist baclofen (10 microM), immediately evoked multiple action potentials. Measurement of [Ca2+]i using fluorescence video microscopy and fura-2 revealed spontaneous, transient, repetitive increases in [Ca2+]i which had a periodicity ranging from 1 to 60 s. These Ca2+ oscillations were abolished by TTX (1 microM) and by the removal of extracellular Ca2+. Application of GABA (1 and 10 microM) induced an immediate increase in [Ca2+]i in all cells and increased the frequency of Ca2+ oscillations in a dose-dependent manner. The GABA-induced increase in [Ca2+]i was abolished by bicuculline and by the removal of extracellular Ca2+, and was inhibited by TTX. Baclofen (1 microM) had no effect on [Ca2+]i. These results suggest that activation of GABAA receptors has an excitatory action on GnRH-secreting immortalized hypothalamic neurons caused by a Cl(-)-dependent depolarization. GABA has been reported to increase GnRH secretion; a direct stimulatory action of the neurotransmitter on GABAA receptors of GnRH-secreting hypothalamic neurons may be responsible for this effect.

Functional analysis of cloned opioid receptors in transfected cell lines.

Opioids modulate numerous central and peripheral processes including pain perception neuroendocrine secretion and the immune response. The opioid signal is transduced from receptors through G proteins to various different effectors. Heterogeneity exists at all levels of the transduction process. There are numerous endogenous ligands with differing selectivities for at least three distinct opioid receptors (mu, delta, kappa). G proteins activated by opioid receptors are generally of the pertussis toxin-sensitive Gi/Go class, but there are also opioid actions that are thought to involve Gq and cholera toxin-sensitive G proteins. To further complicate the issue, the actions of opioid receptors may be mediated by G-protein alpha subunits and/or beta gamma subunits. Subsequent to G protein activation several effectors are known to orchestrate the opioid signal. For example activation of opioid receptors increases phosphatidyl inositol turnover, activates K+ channels and reduces adenylyl cyclase and Ca2+ channel activities. Each of these effectors shows considerable heterogeneity. In this review we examine the opioid signal transduction mechanism. Several important questions arise: Why do opioid ligands with similar binding affinities have different potencies in functional assays? To which Ca2+ channel subtypes do opioid receptors couple? Do opioid receptors couple to Ca2+ channels through direct G protein interactions? Does the opioid-induced inhibition of vesicular release occur through modulation of multiple effectors? We are attempting to answer these questions by expressing cloned opioid receptors in GH3 cells. Using this well characterized system we can study the entire opioid signal transduction process from ligand-receptor interaction to G protein-effector coupling and subsequent inhibition of vesicular release.