Differentiating the role of gamma-aminobutyric acid type A (GABAA) receptor subtypes.

The inhibitory tone maintained throughout the central nervous system relies predominantly on the activity of neuronal GABAA (gamma-aminobutyric acid type A) receptors. This receptor family comprises various subtypes that have unique regional distributions, but little is known about the role played by each subtype. The majority of the receptors contain a gamma2 subunit and are sensitive to modulation by BZs (benzodiazepines), but differ with regard to alpha and beta subunits. Mutagenesis studies combined with molecular modelling have enabled a greater understanding of receptor structure and dynamics. This can now be extended to in vivo activity through translation to genetically modified mice containing these mutations. Ideally, the mutation should leave normal receptor function intact, and this is the case with mutations affecting the BZ-binding site of the GABAA receptor. We have generated mutations, which affect the BZ site of different alpha subunits, to enable discrimination of the various behavioural consequences of BZ drug action. This has aided our understanding of the roles played by individual GABAA receptor subtypes in particular behaviours. We have also used this technique to explore the role of different beta subunits in conferring the anaesthetic activity of etomidate. This technique together with the development of subtype-selective compounds facilitates our understanding of the roles played by each receptor subtype.

Time-dependent changes in NMDA receptor expression in neurones cultured from rat brain.

Several studies have shown marked changes in the regional expression pattern of N-methyl-D-aspartate (NMDA) receptor subunit composition in vivo in the developing brain. Similar developmental changes may also occur in vitro. Thus, displacement of [3H]MK-801 binding by the subunit-selective NMDA antagonist, ifenprodil, in membrane homogenates prepared from cultured neurones, has been shown to comprise two components, the relative proportion of which changed with time in culture [11]. In the present experiments we have used electrophysiological methods to determine the influence of time in culture on NMDA receptor subtype expression pattern in individual neurones. Shortly after plating (13 days in vitro (DIV)), approximately 70% of total NMDA-induced inward current in voltage-clamped rat cortical neurones is antagonised by ifenprodil with relatively high affinity (approximately 1 microM). By 65 DIV, however, the high-affinity component contributed to only approximately 20% of the overall antagonism. Cerebellar granule cells also appear to undergo a similar change in their NMDA receptor expression with the exception that, in general, they appear initially to show considerably less of the ifenprodil high-affinity component and this disappears completely by 15 DIV. These experiments suggest that individual cortical and granule cells express at least two different NMDA receptor subtypes and that their relative proportion changes with time in culture.

The effects of insulin-like growth factor analogues on survival of cultured cerebral cortex and cerebellar granule neurones.

Insulin and insulin-like growth factors (IGF-I, IGF-II) are closely related polypeptides which are found in the CNS and which promote neuronal survival and neurite outgrowth. They are each associated with specific cell surface receptors and several soluble binding proteins (IGFBPs) which are involved in regulating function and availability. Two analogues of IGF-I were produced by site directed mutagenesis: (Gln3, Ala4, Tyr15, (Leu16)IGF-1 (QAYL-IGF) and a B-chain mutant in which the first 16 amino acids of IGF-1 were replaced by the first 17 amino acids of insulin. These analogues have significantly reduced binding affinity for IGFBPs. Using glucose deprivation as a damaging stimulus and assaying lactate dehydrogenase released from cultures as a marker for cell death, we have investigated the effect of IGF analogues on cell death of cerebrocortical and cerebellar granule cell cultures. In the presence of IGF-I, QAYL-IGF or B-chain mutant, the amount of LDH released from cortical and cerebellar granule cell cultures was significantly reduced compared to control (no glucose), indicating that these molecules promote survival. Both QAYL and B-chain mutants, which have reduced affinity for IGFBPs, are as effective as IGF-I in promoting cell survival in conditions of glucose deprivation and their reduced affinity for IGFBPs has no apparent deleterious effect on their neuroprotective function. We also show that the neuroprotective effect of the IGF analogues is due to a direct effect on the neurones in these cultures and is independent of the presence of glia.

Electrophysiological characterisation of the antagonist properties of two novel NMDA receptor glycine site antagonists, L-695,902 and L-701,324.

The pharmacological effects of two novel N-methyl-D-aspartate (NMDA) receptor glycine site antagonists, L-701,324 and L-695,902 were examined on whole-cell voltage-clamped cells and compared to a prototypic antagonist, 7-chlorokynurenic acid. Both L-701,324 and L-695,902 non-competitively antagonised NMDA responses elicited in rat cultured cortical neurones, this was shown to be due to a competitive interaction at the glycine co-agonist site on the receptor complex (Kb values: 19 nM and 2.6 microM, respectively). Inhibition curves for the antagonism of responses to combined applications of NMDA and glycine showed that both antagonists were devoid of any intrinsic activity i.e. "full" antagonists and were, therefore, capable of completely abolishing inward currents. Despite this fact, both of these novel antagonists apparently modulated glutamate affinity for its recognition site-a property hitherto associated only with glycine site partial agonists. Human recombinant NMDA receptors comprising NR1a/NR2A and NR1a/NR2B subunits expressed in mouse fibroblast cells were also used to determine whether L-701,324 and L-695,902 were capable of discriminating between subtypes of NMDA receptor. Inhibition curves to each antagonist showed no difference in affinity for either of these subunit assemblies (mK1 values L-701,324 = 0.005 microM on both assemblies; L-695,902 = 4.37 and 3.7 microM on NR1a/NR2A and NR1a/NR2B, respectively). Kinetic analysis of the off-rates of antagonism with L-701,324 revealed that the high affinity of this compound compared to 7-chlorokynurenic acid were attributable to an exceptionally slow dissociation of the antagonist from the receptor.

The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response. 6. This expression system produced functional receptors that behaved in a fashion analogous to those found endogenously in other preparations. Thus, this system appears to provide a useful and versatile preparation for the analysis of sub-unit regulation of GABAA receptor pharmacology.

Psychological aspects of 211 female patients attending a urodynamic unit.

Psychological aspects of 211 female patients attending a Urodynamic Unit were assessed by means of standardized questionnaires. Patients with genuine stress incontinence (GSI) showed changes comparable to other patients with longstanding physical complaints. Patients with sensory urgency (SU) lacked self esteem and were more anxious than those with GSI. Patients with detrusor instability (DI) were as anxious and lacking in self esteem as patients with sensory urgency and additionally had morbid thoughts and worries and higher scores on the hysteria sub-scale of the Crown-Crisp Experiential Index. Roughly a quarter of all the patients reported that their urinary symptoms rendered life intolerable and they were as anxious, depressed and phobic as psychiatric inpatients.

Chlamydia trachomatis infections in the female rectums.

One hundred and fifteen consecutive new women patients were examined in a department of genitourinary medicine for evidence of infection with Chlamydia trachomatis in the rectum, in addition to the routine screening tests performed. An impression smear of the rectal mucosa was made as a semiquantitative assessment of the degree of proctitis, and details of bowel habit and symptoms and of sexual practice were noted. Chlamydial infection was found in the cervices of 15 (13%) and the rectums of six (5%). Rectal infection was significantly associated with rectal bleeding and microscopic evidence of proctitis, but not with diarrhoea or macroscopic proctitis.

Micturition and the mind: psychological factors in the aetiology and treatment of urinary symptoms in women.

The mental state of 211 women attending a urodynamic clinic was assessed using questionnaires. Patients with genuine stress incontinence had scores comparable with other patients with longstanding physical complaints. Patients with sensory urgency were more anxious than those with genuine stress incontinence. Patients with detrusor instability were as anxious as patients with sensory urgency and in addition had higher scores on the hysteria scale. A subset of patients (roughly a quarter of the total) was identified, comprising members of all three diagnostic groups, for whom urinary symptoms rendered life intolerable. These patients were as anxious, depressed, and phobic as psychiatric inpatients, emphasising the serious psychological morbidity experienced by patients with urinary symptoms. Fifty patients with detrusor instability or sensory urgency entered a randomised trial comparing psychotherapy, bladder drill, and propantheline. The psychotherapy group significantly improved on measures of urgency, incontinence, and nocturia, though not on frequency. Bladder training was an effective treatment for frequency and patients became less anxious and depressed. There was a modest improvement in frequency of micturition in patients given propantheline. Frequency may be a learnt disorder which responds to the direct symptom oriented approach of bladder training. Patients with urgency and nocturia predominating might derive more benefit from psychotherapy.

The relationship between nocturnal motility and the enuresis alarm device.

The mode of action of the enuresis alarm is not clear. Earlier work suggested that the wire mesh itself may alter sleep and in this way affect nocturnal enuresis. Seven healthy males have been studied within a two-period cross-over design in which their nocturnal motility, on and off an enuresis wire mesh, was recorded using a motility bed. There were no order, treatment or day effects in any of the variables studied. However, the mean hourly motility for each subject averaged over the three nights shows a substantial trend towards increased motility on the mesh. This lends some support to the hypothesis and merits further study.

Chlamydia trachomatis infection of the cervix: the need for a diagnostic service.

Chlamydia trachomatis was isolated from the cervix in 20.4 per cent (104/511) of women attending a department of genito-urinary medicine. Isolation rates ranged from 43.3 per cent in women with gonorrhoea to 4.2 per cent in women with no STD and who required no treatment. There was no association between the presence of chlamydia in the cervix and any symptom complex. Although high isolation rates were also noted in women with signs of cervicitis and in women whose partners had non-gonococcal urethritis, the accuracy of such clinical and epidemiological criteria in predicting cervical infection was low. In the absence of a screening service the chlamydial infection of 81 women (those who were not NGU contacts) would have been undiagnosed and untreated. The need for a screening service is discussed.