Absorption of angiotensin II antagonists in Ussing chambers, Caco-2, perfused jejunum loop and in vivo: importance of drug ionisation in the in vitro prediction of in vivo absorption.

The aims of this study were (i) to compare the absorption of three closely related inhibitors of angiotensin II, RU60018, RU60079 and HR720, in various in vitro and in vivo models, and (ii) to explain the differences in the results and to assess the importance of drug ionisation to predict absorption. Drug absorption was investigated in Ussing chambers, Caco-2 cell monolayers, perfused rat jejunum loops and in vivo after oral, intraduodenal or intravenous administration. In Ussing chambers, the analogues showed the same site-related absorption profile and a common mechanism involving the paracellular pathway. At pH 7.4 in Ussing chambers, perfused jejunum loop or Caco-2 transport studies, the three compounds exhibited low and comparable permeability values suggesting that a similar level of oral absorption may be expected for all three compounds. However, after oral or intraduodenal administration, only HR720 was significantly absorbed. The in vivo results can be explained by the ionic distribution profile which indicated that only HR720 possessed a significant amount of uncharged species at pH values close to that found in the upper part of intestinal tract. Hence, it is expected that in this part of the intestine, only HR720 absorption is favoured. This is supported by Caco-2 transport studies performed when the pH of the apical medium was lowered from 7.4 to 6.0, in which a dramatic increase in permeability was observed for HR720 compared to those of the other analogues. This study highlights the usefulness of different absorption models for drug screening and demonstrates that ionisation profiles must be carefully considered to avoid rejection of promising compounds.

Cholinergic receptors on cultured neurones from the central nervous system of embryonic cockroaches.

Cultured neurones from the cockroach, Periplaneta americana, have been used to investigate putative acetylcholine receptors. Ligand-binding experiments revealed that these neurones possessed an alpha-bungarotoxin binding site that was saturable, had an apparent affinity constant of 3.51 nM and was predominantly nicotinic in nature. An individual culture of 50,000 neurones had a maximum of 4200 pmol. binding sites per gram of protein. [I125]alpha-BTX autoradiography showed the binding sites to be distributed over both the neuronal cell bodies and their associated axonal processes. Both acetylcholine and nicotine applied by pressure ejection to the neuronal soma induced depolarizing responses and in the majority of cells tested the response was blocked by alpha-BTX at a concentration of 25 nM in a time dependent manner. Some of the neurones, however, were depolarized by acetylcholine and nicotine after 3 h incubation in alpha-BTX. These experiments suggest that two populations of cells possessing extrajunctional nicotinic receptors were present in these cultures. In the majority of cells these receptors were sensitive to alpha-BTX but in a subpopulation the receptors were unaffected by this toxin.

The ultrastructure of somatic neuromuscular junctions in the tick Amblyomma variegatum (Fabr.).

Peripheral nerve axons synapse with somatic muscle fibres in the tropical bont tick Amblyomma variegatum. Each fibre is innervated by numerous terminals; some of the axons synapse more than once. The nerve terminals on coxal muscle fibres contain agranular electron lucent vesicles 50 to 58 nm in size and have the specialised synaptic membranes characteristic of chemically transmitting nerve-muscle junctions. Some of the terminals on trochanteral muscle fibres additionally contain larger vesicles (90 nm) with electron dense cores, suggesting that these junctions operate with a different kind of neurotransmitter.

Effects of stimulation and rest on the ultrastructure of the excitatory neuromuscular junctions of Locusta migratoria L.

The terminals of the fast axon on extensor tibiae muscle fibres of Locusta were examined in untreated nerve-muscle preparations and in preparations stimulated electrically at frequencies varying from 0.5 to 100 Hz. The ultrastructure of the terminals in preparations stimulated at the lower range of these frequencies, which induce twitch contractions of the muscles, is similar to that of the controls. Stimulation at the higher frequencies induced tetanic muscle responses and rapid fatigue of the muscles after which they would not respond again to high frequency stimulation for about 1 h. This loss and recovery of the responses of the muscles is correlated with changes in the ultrastructural appearance of the terminals, in particular in the number and shape of the synaptic vesicles. The ultrastructure of these "recovering" axon terminals closely resembles that of the controls.

Changes in the distribution and size of synaptic vesicles in neuromuscular junctions of Locusta migratoria after stimulation and rest.

The distribution and size of synaptic vesicles in excitatory terminals of the extensor tibiae muscle were determined after stimulation at frequencies varying from 0.5 to 100 Hz and after subsequent rest. Only in preparations stimulated at the higher frequencies do these parameters differ from those in the controls. The synaptic vesicles in the nonsynaptic areas of these terminals are depleted in number, and the remaining vesicles are reduced in size. These effects are reversed after a 1 h rest.

Glutamate uptake after stimulation-induced depletion of vesicle numbers in neuromuscular junctions of Locusta migratoria L.

The distribution of radioactivity at branches and terminals of the fast axon in extensor tibiae muscle incubated in the radiolabelled putative neurotransmitter L-glutamate was determined by electron microscopic autoradiography. Quantitative analysis of the distribution of silver grains at the axon branches and terminals in preparations stimulated at a low frequency shows that most of the radioactivity is present in the glial cells. In preparations stimulated to the point of fatigue substantial radioactivity is present in both the glial cells and the axoplasm of the terminals. It is suggested that the uptake of L-glutamate into the axoplasm of the terminals is correlated with the depletion and recovery of vesicle numbers after stimulation.