Influence of membrane cholesterol on modulation of the GABA(A) receptor by neuroactive steroids and other potentiators.

1. The influence of membrane cholesterol on some pharmacological properties of the GABA(A) receptor was investigated in acutely dissociated rat hippocampal neurones with whole cell patch clamp recording. The cholesterol levels were varied between 56% and 235% control using methyl-beta-cyclodextrin as the cholesterol carrier. 2. Enrichment of neurones with cholesterol increased the effects of the non-steroidal GABA potentiators propofol, flunitrazepam and pentobarbitone. A similar result was obtained after pre-incubation of neurones with epicholesterol, the 3alpha-hydroxy isomer of cholesterol. 3. In contrast, the effects of the steroidal GABA potentiators pregnanolone and alfaxalone were reduced by cholesterol enrichment, but not by epicholesterol. Depletion of membrane cholesterol increased the potentiation of GABA by pregnanolone and alfaxalone but did not affect the non-steroidal potentiators. 4. The steroidal antagonist of GABA, pregnenolone sulphate, reduced the maximum response to GABA. This effect, also, was diminished in cholesterol-enriched neurones and enhanced in cholesterol-depleted neurones. 5. The effects of the cholesterol manipulations that were selective for the steroidal modulators of GABA are suggested to arise from direct interactions between membrane cholesterol and the GABA(A) receptor. The separate effects on the non-steroidal potentiators of GABA of cholesterol-enrichment or addition of epicholesterol to the neurones are suggested to be due to changes in membrane fluidity. 6. In view of the likely physiological modulation of GABA(A) receptors by endogenous neuroactive steroids and evidence of the in vivo lability of membrane cholesterol, the present observations may have physiological as well as pharmacological relevance.

Effects of membrane cholesterol on the sensitivity of the GABA(A) receptor to GABA in acutely dissociated rat hippocampal neurones.

The effects of membrane cholesterol on the GABA(A) receptor were investigated in acutely dissociated rat hippocampal neurones, using the whole-cell patch clamp technique. Neuronal cholesterol was manipulated within the range 56-250% control by incubation with methyl-beta-cyclodextrin for depletion and a complex of cholesterol and methyl-beta-cyclodextrin for enrichment. Manipulation over a narrower range was achieved with cholesterol + phosphatidylcholine liposomes. A complex of epicholesterol and methyl-beta-cyclodextrin was used to insert epicholesterol. Cholesterol enrichment reduced the potency of GABA, as did cholesterol depletion, with increases in EC(50) of up to 4-fold. Cholesterol enrichment reduced the potency of the competitive antagonist bicuculline but did not affect that of the non-competitive antagonist picrotoxinin. Cholesterol depletion did not affect the potencies of either antagonist. Epicholesterol substituted functionally for cholesterol with respect to the effects of enrichment. In cholesterol-depleted neurones, however, only incubation with cholesterol was able to restore GABA potency to normal. These results suggest a specific requirement for cholesterol at control levels to maintain optimal GABA potency, which may involve specific binding of cholesterol to the GABA(A) receptor. The reduction in GABA potency by enrichment with cholesterol or epicholesterol is more likely to be due to reduced plasma membrane fluidity.

Extrusion of niosomes from capillaries: approaches to a pulsed delivery device.

We describe an early prototype of a pulsatile delivery system for drug containing vesicles. Nonionic surfactant vesicles (niosomes) of average diameter 4-30 microm are extruded from glass capillaries (exit diameter, 5-10 microm), using air pressures of 0.5-5 p.s.i. The formulation of the vesicles is vital. Extrusions were affected by the size, shape, and membrane composition of the niosomes used. Spherical or polyhedral niosomes, formed by polyoxyethylene alkyl ethers with and without cholesterol, respectively, with diameters larger than the exit diameter of the capillary do not retain their membrane integrity on extrusion and were sheared to form new ultrastructures. The expulsion of single or groups of intact polystyrene microspheres or tetradecyl-beta-D-maltoside niosomes with sizes smaller than the exit diameter can be achieved readily. The stepwise release profile of luteinizing hormone releasing hormone (LHRH) obtained after pulsatile expulsion of groups of niosomes entrapping LHRH indicates the feasibility of this system for pulsatile delivery of vesicles, although it requires miniaturization.

Increased membrane cholesterol reduces the potentiation of GABA(A) currents by neurosteroids in dissociated hippocampal neurones.

The effects of increased membrane cholesterol on the potentiation of GABA(A) currents by pregnanolone (5beta-pregnan-3alpha-ol-20-one), allopregnanolone (5alpha-pregnan-3alpha-ol-20-one), alphaxalone (5alpha-pregnan-3alpha-ol-11,20-dione) and propofol were investigated in acutely dissociated rat hippocampal neurones using the whole-cell patch clamp technique. Cholesterol enrichment of the neurones, isolated from 10 to 16-day-old Wistar rat brains, was achieved by incubation with cholesterol + phosphatidylcholine liposomes. Cholesterol enrichment (25.8+/-3.4%) reduced the potentiation of GABA(A) currents by pregnanolone (0.3 and 1 microM), allopregnanolone (1 microM) and alphaxalone (1 microM) but the potentiation of GABA(A) currents by propofol (5 microM) was not affected. Acute application of cholesterol (1 microM) did not significantly change the potentiation of GABA(A) currents by pregnanolone (1 microM). These results suggest that cholesterol within the neuronal membrane may compete with neurosteroids for their sites of action on the GABA(A) receptor or modulate the potentiating effect of the neurosteroids in some other ways.

Preliminary evaluation of the anticonvulsant activity of a valproic acid analog: N-(2-propylpentanoyl) urea.

Anticonvulsant activity, lethality and neurotoxicity of a valproic acid (VPA) analog, N-(2-propylpentanoyl) urea (VPU) in comparison to its parent compound were investigated in mice. Intraperitoneally administered VPU demonstrated a higher protection than VPA in both the maximal electroshock seizure (MES) and the pentylenetetrazole (PTZ) tests exhibiting a median effective dose (ED50) of 66 and 57 mg/kg, respectively. VPU weakly blocked the effect of bicuculline and was ineffective in strychnine test. Furthermore, VPU was also active orally demonstrating an ED50 approximately 6 times higher than its ED50 by the intraperitoneal route. Based on the relatively high median lethal dose (LD50), 1553 mg/kg, VPU possesses a greater margin of safety (LD50/ED50) than did VPA. Unwanted (side) effects in terms of impairment of motor activity and neurotoxicity were assessed by the rotorod test, locomotor activity test as well as potentiation of barbiturate sleeping time. The median neurotoxic dose (TD50) as measured by rotorod test were 625 mg/kg for intraperitoneally given VPU. This finding results in higher protective index (PI = TD50/ED50) of VPU (PI = 9.5) than that of VPA (PI = 1.1) implying that, in therapeutic dose, VPU may produce less neurological side effects than did VPA. Superiority of VPU in terms of higher potency in parallel with minimal neurological deficit as assessed by rotorod test was evident throughout the observation period of 12 hours. Similar results on locomotor activity as well as potentiation of barbiturate sleeping time were obtained with VPU and VPA. Thus, VPU is preferably expected to exert minor degree of CNS depression. Taken altogether, our findings demonstrate greater anticovulsant activity for VPU than for VPA. In addition, this compound is also orally active and seems to offer a greater safety margin in parallel with lower unwanted effects in relation to its parent compound. As indicated by the animal data obtained, VPU is an attractive anticonvulsant candidate for further investigation.