The induction of ret by Wnt-1 in PC12 cells is atypically dependent on continual Wnt-1 expression.

Wild type PC12 pheochromocytoma cells that had been infected with a Wnt-1-carrying virus and thus express Wnt-1 (PC12/Wnt-1) are known to acquire the same flat cell phenotype as that of spontaneously occurring PC12 flat cell variants except that the latter do not presently express Wnt-1. Flat cell variants of PC12 cells exhibit markedly altered morphology and gene expression. In order to assess the possibility that the spontaneously occurring flat cell variants could have been induced in wild type PC12 cells by previous transient expression of the cell's endogenous Wnt-1, we have isolated PC12/Wnt-1 cells expressing little or no Wnt-1. In spite of absent Wnt-1 expression, they retained their flat cell morphology, glutamate/aspartate transporter activity, increased neu mRNA levels and lack of both norepinephrine transporter activity and nerve growth factor-induced differentiation. Thus, Wnt-1 expression is not required to maintain the flat cell phenotype. However, we identified one gene, ret, whose mRNA level in PC12 was not only increased by Wnt-1 expression, but whose increased mRNA level was also dependent on continual Wnt-1 expression. This finding suggests that the induction of ret by Wnt-1 can be used to elucidate the Wnt-induced signalling pathway in mammalian cells.

Poor compliance is a major factor in unstable outpatient control of anticoagulant therapy.

Control of oral anticoagulant therapy in outpatients is often unsatisfactory. The contribution of poor compliance with prescribed warfarin to unstable anticoagulant control was investigated prospectively using low-dose phenobarbitone as an indicator of compliance in 30 out-patients, 15 with stable and 15 with unstable control. Following entry to the study, there was no significant change in anticoagulation (p = 0.36) in the group with stable control. In the group who previously had unstable control, there was a significant change in INR (p = 0.0045) and anticoagulant control greatly improved. It appears that the considerable fluctuation in INR seen in many of the latter patients before the study was due to poor compliance and that entering them into the study modified their behavior. Two patients in this group who continued to have unstable anticoagulant control were shown to be poorly compliant using the phenobarbitone indicator. The results suggest that, in outpatients, poor compliance is the major cause of unstable anticoagulation with warfarin.

Scopolamine and benzodiazepine models of dementia: cross-reversals by Ro 15-1788 and physostigmine.

The muscarinic antagonist scopolamine and the benzodiazepine lorazepam both produce transient impairments in memory and attention in normal volunteers. These impairments can be reversed by appropriate agents such as the cholinesterase inhibitor physostigmine in the case of scopolamine or the benzodiazepine antagonist Ro 15-1788 in the case of lorazepam. In this paper we investigated the pharmacological specificity of these reversals by examining the interactions of scopolamine and Ro 15-1788 and of lorazepam and physostigmine. There was no evidence that the effects of scopolamine and lorazepam on cognitive function could be attenuated by Ro 15-1788 and physostigmine, respectively. The results are discussed in terms of pharmacological models of Alzheimer's disease.

Pentagastrin induced panic attacks: enhanced sensitivity in panic disorder patients.

The effects of pentagastrin, a synthetic analogue of the cholecystokinin tetrapeptide (CCK4), were studied in 15 patients with panic disorder and 15 healthy controls. Three different intravenous dosages of pentagastrin (0.1, 0.3 and 0.6 microgram/kg) and saline were investigated. Subjects were randomly allocated to two of the four treatment groups and tested on two separate occasions, 1 week apart, using an unbalanced double-blind incomplete block design. The mean panic rate with pentagastrin was 55% (12/22) for patients and 5% (1/22) for controls. None of the subjects panicked with saline. The frequency of panic attacks between the three pentagastrin doses in patients was not different. One control subject had a panic-like attack at the highest dose of pentagastrin. These findings concur with previous studies on the panicogenic effect of CCK4 and pentagastrin and suggest a greater sensitivity for CCK receptor agonists in patients suffering from panic disorder than in healthy controls.

Indirect coupling of calcium transport in chromaffin granule ghosts to the proton pump.

In chromaffin granule ghosts, the Na+/Ca2+ exchanger in the granule membrane can provide a high affinity (Km 1-3 microM) and high capacity (Vmax 50-100 nmol mg min-2) mechanism for Ca2+ accumulation. The activity of the Na+/H+ antiporter can be used to couple Ca2+ uptake via Na+/Ca2+ exchange to ATP-dependent proton translocation via the granule membrane H(+)-ATPase. Therefore, Ca2+ uptake can be indirectly linked to the proton pump. However, under conditions designed to mimic the environment of a granule in the cytosol of a chromaffin cell, measured rates of Ca2+ uptake are low, a free Ca2+ concentration of about 5 microM in the ghost matrix being attained. Under such circumstances, the granules seem unlikely to play a major role in calcium homeostasis in the intact cell.

Acetylcholine active transport by rat brain synaptic vesicles.

Uptake of acetylcholine was studied in a synaptic vesicle fraction isolated from rat brain. Hyposmotically treated P3 vesicles took up acetylcholine (ACh) in the presence of MgATP, and the uptake was inhibited by low temperature, ammonium ions, the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) and bafilomycin A1, a specific inhibitor of the vacuolar H(+)-ATPase. Uptake was also inhibited by drugs that bind allosterically to the ACh transporter, namely vesamicol (IC50 value of 170 nM) and 4-aminobenzovesamicol (IC50 value of 25 nM). KM and Vmax values for ACh active transport were estimated to be 5 mM and 4 nmol min-1 mg-1 of cholinergic vesciles, respectively. Active transport of ACh by synaptic vesicles partially purified from brain is mediated by a vesamicol-sensitive transporter and is dependent on a proton gradient generated by the vesicular H(+)-ATPase.

RO 16-6028, a benzodiazepine receptor partial agonist, does not exhibit anticonvulsant tolerance in mice.

The development of anticonvulsant tolerance with RO 16-6028, a benzodiazepine receptor partial agonist, was assessed in mice using an i.v. infusion of pentylenetetrazol as the convulsive stimulus. In contrast to other benzodiazepines tested previously in this seizure model the anticonvulsant protection afforded by RO 16-6028 did not change significantly during 10 days treatment (2 mg/kg b.i.d.). This result supports the hypothesis that partial agonists at the benzodiazepine receptor may induce less tolerance and/or dependence than full agonists.

Development of tolerance to the anticonvulsant effects of clobazam.

The anticonvulsant properties of the 1,5-benzodiazepine clobazam were studied in mice during and after chronic treatment at two different dose levels. Pentylenetetrazol given by slow intravenous infusion was used as the convulsant stimulus. Tolerance to the anticonvulsant effects was observed; this was rapid in onset and could be overcome by increasing the dose of clobazam.

Benzodiazepine cross-tolerance in mice extends to sodium valproate.

Slow intravenous infusion of pentylenetetrazol was used to measure the convulsive threshold in mice. The anticonvulsant effects of clobazam, clonazepam, diazepam, lorazepam, sodium phenobarbitone and sodium valproate were assessed in naive animals and compared with the effects of the same compounds in animals which had been pretreated (twice daily for 3 days) with one of the benzodiazepines or sodium valproate. Cross-tolerance was observed between all the benzodiazepines but not between benzodiazepines and sodium phenobarbitone. Animals pretreated with the benzodiazepines were cross-tolerant to valproate, but the converse was not true; nor did sodium valproate induce tolerance to itself.

Can benzodiazepines be classified by characterising their anticonvulsant tolerance-inducing potential?

The development of anticonvulsant tolerance with three benzodiazepines was assessed in mice using a slow intravenous infusion of pentylenetetrazol as the convulsive stimulus. Chlordiazepoxide (12.5 mg/kg b.d.) and midazolam (0.75 mg/kg b.d.) induced a slowly evolving tolerance over 15 days whereas nitrazepam (0.6 mg/kg b.d.) induced a very marked rapid tolerance which developed no further during 6 days treatment. Tolerance appeared to be incomplete with all three benzodiazepines. Possible explanations for the differences in tolerance profile are discussed and an alternative basis for the classification of benzodiazepines is suggested.

Differences between the tolerance characteristics of two anticonvulsant benzodiazepines.

Clobazam (10 mg/kg) and clonazepam (0.25 mg/kg) were administered to mice twice daily by the intraperitoneal route. The development of tolerance to their anticonvulsant effect was compared using a slow intravenous infusion of pentylenetetrazole as the convulsant stimulus. Tolerance to clonazepam developed gradually throughout a 72 h study and did not become significant until the fifth dose. In contrast, tolerance to clobazam occurred extremely rapidly, after only one dose; it was manifested as a single step and no further significant change in protection was observed. Recovery from benzodiazepine tolerance was also studied and seen to occur rapidly with both these compounds; following cessation of dosing, protection was restored to initial levels within 36-48 h.

Plasma concentrations of clobazam and its N-desmethyl metabolite; protection against pentetrazol-induced convulsions in mice.

The anticonvulsant effects of acute administration of clobazam and its principal metabolite N-desmethylclobazam were studied in mice. Pentetrazol, given by slow intravenous infusion 1 or 2 h after the anticonvulsant dose, was used as the convulsant stimulus. Log dose response relationships for both clobazam and N-desmethylclobazam appeared linear, but there was no correlation between plasma concentrations of clobazam and protection. However, correlation between plasma concentrations of N-desmethylclobazam and protection was significant in both cases.

Tolerance to the anticonvulsant effect of clonazepam in mice: no concurrent change in plasma concentration.

Clonazepam was administered for 10 or more days on three different dose regimens (0.5, 0.25 and 0.08 mg kg-1 twice daily) to mice given pentetrazol by slow intravenous infusion. Plasma concentrations of clonazepam were assayed by high performance liquid chromatography. Tolerance developed to the anticonvulsant effect of clonazepam at all doses but was incomplete and could be overcome by increasing the dose. With the 0.5 and 0.25 mg kg-1 regimens there was no significant change in the drug plasma concentrations during development of tolerance; on the lowest dose, levels were below the limits of accurate detection. Anticonvulsant tolerance does not seem to be the result of a disturbance in clonazepam metabolism.

A sodium/proton antiporter in chromaffin-granule membranes.

Chromaffin granules, the secretory vesicles of the adrenal medulla, have a Na+/H+ exchange activity in their membranes which brings their proton gradient into equilibrium with a Na+ gradient. This explains why Na+ is mildly inhibitory to amine transport (which is driven by the H+ gradient) The activity can be demonstrated by using accumulation of 22Na+ in response to a pH gradient that is either imposed by diluting membrane 'ghosts' into alkaline media, or generated by ATP hydrolysis. It can also be monitored indirectly by fluorescence measurements in which the pH inside 'ghost' is monitored by quenching of a fluorescent weak base. This method has been used to monitor Na+ entry into acid-loaded 'ghosts' of H+ entry into methylamine accumulation. The exchanger appears to be reversible and non-electrogenic, with a stoichiometry of 1:1. Using an indirect assay we measured an apparent Km for Na+ of 4.7 mM, and a Ki for amiloride, a competitive inhibitor, of 0.26 mM. Direct assays using 22Na+ suggested a higher Km. Ethylisopropylamiloride was not inhibitory.

Phenolics production by encapsulated Nicotiana tabacum cells.

Plant cells of tobacco (Nicotiana tabacum L.) were grown for several generations in suspension cultures. Cells were immobilized in continuous bioreactors in calcium alginate (Ca Alg) beads or in poly-L-lysine (PLL) encapsulated calcium alginatehydrogels. In each case, the cells were fed continuously a modified Linsmaier-Skoog plant cell culture medium. The bioreactor effluent was analyzed for total phenolic compounds. The net specific productivity of phenolics was calculated on a daily basis for several test runs. For comparison, productivity in suspension cultures was monitored. Productivity of suspended cells declined to zero within 9 d; both immobilized and encapsulated cells remained productive for 16 d following inoculation. Specific productivity by encapsulated cells was higher than that by immobilized cells; in both types similar rates of decline in productivity occurred.

Free concentrations of sodium, potassium and calcium in chromaffin granules.

We have measured the contents of Na+ and K+ in isolated chromaffin granules. Total contents varied between 227 and 283 nmol/mg of protein, equivalent to matrix concentrations of 53-66 mM. The value found depended on the isolation buffer used, and the ratio of the two ions reflected the composition of the buffer. We then measured the free concentration of each of these ions, and of Ca2+, in the matrix, by using a null-point method with acridine-fluorescence quenching. This monitored H+ fluxes induced by an ionophore in the presence of known concentrations of the ion in the supporting medium. In contrast with organic constituents of the matrix, which have low activity coefficients, Na+ and K+ were found to have activity coefficients around 0.8 Ca2+, however, was strongly bound: its free concentration was only 0.03% of the total.

Pharmacokinetics of N-desmethylclobazam in healthy volunteers and patients with epilepsy.

1. The single dose pharmacokinetics of N-desmethylclobazam (NDMC) and clobazam were studied in eight healthy male volunteers. 2. Steady-state pharmacokinetic data are described from four healthy male volunteers and eight epileptic patients taking NDMC. 3. A single 30 mg dose of NDMC produced a greater Cmax (P less than 0.001) and AUC0-infinity (P less than 0.005) and a shorter tmax (P less than 0.05) and t1/2 (P less than 0.01) for NDMC than did 30 mg clobazam. 4. Mean steady-state NDMC concentrations were greater in male patients than in female patients and also in male patients compared with male volunteers. The differences between patients and volunteers might be explained by concomitant antiepileptic medication.

Lipids and platelet function in runners.

The effect of distance running on the fatty acid composition of platelet and erythrocyte membranes has been investigated, together with platelet aggregation and levels of high density lipoprotein-cholesterol (HDLc), low density lipoprotein cholesterol (LDLc), total cholesterol and triglycerides in runners (n = 11) and healthy age-matched non-running controls (n = 12). Platelet aggregation and fatty acid composition of membrane lipids in both platelets and erythrocytes are similar in both groups with the following exceptions: in platelets docosahexaenoic acid (C22:6 omega 3) is significantly higher in runners; in erythrocytes the fatty acids C20:3 omega 6/C22:1 omega 9 and C22:5 omega 3 are significantly higher in runners. There were no significant differences between the levels of HDLc, LDLc and total cholesterol in runners and controls although triglycerides were significantly lower in runners. Possible beneficial effects of running are probably mediated through effects on serum lipid and lipoprotein concentrations, and probably not due to any antithrombotic effect of platelets.