An inhibitory effect of atropine on responses of the vas deferens of the mouse to field stimulation.

1 Atropine is shown to impair responses of the vas deferens of the mouse to field stimulation by acting at a site proximal to the smooth muscle cells. 2 The inhibitory effect of atropine is prevented by desmethylimipramine and reversed by dexamphetamine, and appears similar to the adrenergic-neurone blockade of guanethidine. 3 Electronmicroscopical studies show the presence in vas of presumptive noradrenergic axons which have acetylcholinesterase reaction product associated with their axolemmae. 4 These results are discussed in relation to the controversial hypothesis of a 'cholinergic link' in noradrenergic transmission.

Noradrenaline and motor transmission in the vas deferens of the mouse.

1. A comprehensive investigation of the innervation of the vas deferens of the mouse was made using pharmacological, histochemical and electronmicroscopical techniques. 2. Guanethidine inhibited the response of the vas to transmural stimulation and potentiated the response to noradrenaline (NA). Phentolamine abolished responses to NA and to transmural stimulation. 3. After chemical sympathectomy degenerative changes were seen in presumptive noradrenergic axons; histochemical fluorescence due to catecholamines was absent. The vas failed to respond to transmural stimulation, and a 10-fold increase in sensitivity of the vas to exogenous NA was observed. 4. NA is shown to diffuse slowly through this tissue whose muscle cells are densely packed. This is disscussed in relation to the apparent "insensitivity" of the vas to exogenous NA. 5. A cholinergic component was identified histochemically which did not contribute significantly to the motor response of the vas as chemical sympathectomy abolished completely the motor response elicited by transmural stimulation. 6. It is concluded that NA is the motor trnasmitter for the smooth muscle of the vas deferens of the mouse.

Neostigmine augments responses of the rat anococcygeus muscle to field stimulation.

1 The effects of neostigmine on noradrenergic transmission have been studied in the field stimulated, isolated anococcygeus muscle of the rat. 2 In those muscles where the excitatory response to field stimulation was not completely inhibited by guanethidine (5 X 10(-6) to 10(-5) M) or phentolamine (10(-6) M), atropine (5 X 10(-8) M) gave no further inhibition of the response. 3 The shape of the response to field stimulation was altered in a dose-dependent manner by neostigmine (5 X 10(-7) to 5 X 10(-6) M), such that a 'shoulder' appeared during the relaxation phase. The 'shoulder', present at all stimulation frequencies tested between 3 and 40 Hz, was abolished by atropine (5 X 10(-8) M) and unaffected by tubocurarine (10(-6) M). 4 Neostigmine (2.5 X 10(-6) M), whether alone or in the presence of atropine (5 X 10(-8) M), had no effect on the uptake or stimulation-induced release of [3H]-noradrenaline. 5 Using electron microscopy, small Schwann/axon bundles close to smooth muscle cells rarely showed cholinesterase staining, whereas larger bundles at the outer serosal aspects of the muscle exhibited dense staining. 6 It is concluded that the observed effects of neostigmine are not due to a presynaptic effect on noradrenergic transmission.

The effect of morpine dependence on the vesicular content of adrenergic nerves in relation to arteriolar smooth muscle in the pancreas of the rat.

1. Sibling male Wistar rats were kept in groups of three under identical conditions. Two groups were made morphine dependent in 28 days and the third served as control. The state of dependence was established by recording the development of tolerance to the analgesic action of the drug and the effect of acute withdrawal of morphine and injection of nalorphine on one test group.2. Animals in the other test group and the control group were killed and specimens of pancreas collected and prepared for examination by electron microscopy. The adrenergic innervation of pancreatic arterioles was located and photographed and the effect of morphinization on the percentage of granular and agranular small vesicles in the axons determined.3. Morphinization produces a significant reduction in the granular or dense core vesicle population of these adrenergic axons.

Separation of putative alpha 1A- and alpha 1B-adrenoceptor mediated components in the tension response of the rat vas deferens to electrical field stimulation.

1. The effects of the putative alpha 1B-adrenoceptor antagonist, chloroethylchlonidine (CEC), on tension responses of the rat isolated whole vas deferens to single and multiple pulses of electrical field stimulation have been evaluated by use of a microcomputer system which enables the averaging of like-responses throughout their time course. 2. CEC (10(-7) to 3 x 10(-6) M) selectively and in a concentration-dependent manner blocked the noradrenergic component of the response to a single field stimulus in the absence or presence of nifedipine (10(-5) M, which blocked the purinergic but not the noradrenergic component of the response). The concentration-response curve of the vas to exogenously-applied noradrenaline (NA) was unaffected by CEC (10(-6) M) but was flattened by nifedipine (10(-5) M). 3. The tension response to 10 Hz trains of pulses was biphasic, with an early (less than 2 s) and a plateau (greater than 4 s) phase. We deduce from our pharmacological analysis that the early phase contains a putative alpha 1B-adrenoceptor component (susceptible to CEC or prazosin but not to nifedipine) and a P2-purinoceptor component (susceptible to suramin or nifedipine) whereas the plateau phase contains an alpha 1A-adrenoceptor component (susceptible to prazosin or nifedipine but not to CEC) and a P2-purinoceptor component (susceptible to suramin or nifedipine). 4. We suggest that the putative alpha 1B-adrenoceptors may be functionally confined to the synaptic region whereas the putative alpha 1A-adrenoceptors are excluded from this region. Trains of pulses would allow NA to accumulate and spill out beyond the synaptic region to reach and activate the putative alphalA-adrenoceptors.

3 H-noradrenaline-accumulating ability of cholinergic nerve terminals in cat sympathetic ganglia.

1. After incubation of pieces of cat posterior mesenteric ganglion in [(3)H]-noradrenaline solution, the localization of the isotope was assessed by electron-autoradiography.2. Significantly higher concentrations of [(3)H]-noradrenaline were found over acetylcholinesterase-positive axons terminal on ganglion cells than in adjacent background areas.3. [(3)H]-Noradrenaline was not accumulated by ganglion cells under these circumstances.4. The significance of these findings in relation to previous physiological and pharmacological investigation is discussed.

In pursuit of the alpha-adrenoceptor: a fine-structural and electron autoradiographic study using 3H-phenoxybenzamine and smooth muscle from the cat and guinea-pig.

1. Electron microscopical evaluation of sets of serial fine sections of pancreatic arteriolar muscle of the cat showed no significant difference between the number of pinocytotic vesicles per unit length of smooth muscle cell membrane in ;synaptic' and ;non-synaptic' regions.2. With high resolution autoradiography, (3)H-phenoxybenzamine was found to be distributed over the cytoplasm and nuclei of smooth muscle cell profiles of guinea-pig vas deferens, although no localization in relation to discrete morphological organelles was evident.3. ;Specific protection' of alpha-adrenoceptors using 606 muM noradrenaline resulted in a significant diminution in the binding of (3)H-phenoxybenzamine as evidenced by a decrease in silver grain concentrations over smooth muscle cell profiles in autoradiographs.4. The significance of these findings in relation to the site and distribution of alpha-adrenoceptors is discussed.

The effects of neostigmine on the response of the rat anococcygeus muscle to field stimulation are not a consequence of cholinesterase inhibition.

Neostigmine (5 X 10(-7) to 5 X 10(-6) M) and physostigmine (10(-5) M) each augment the responses of the rat anococcygeus muscle to field stimulation whereas iso-OMPA (5 X 10(-6) to 5 X 10(-4) M) or BW 62c47 (5 X 10(-7) to 5 X 10(-5) M) do not. At a concentration of 10(-5) M, neostigmine. BW 62c47 and iso-OMPA respectively produced a 48, 50 and 68% inhibition of cholinesterase activity in homogenates of anococcygeus muscle. The ED50 for cholinesterase inhibition by neostigmine (1.4 X 10(-5) M) is approximately 15 fold greater than the ED50 for the augmentation of the response to field stimulation (9.5 X 10(-7) M). It is concluded that the action of neostigmine in augmenting the response of the rat anococcygeus muscle to field stimulation is not a consequence of cholinesterase inhibition even though stimulation of muscarinic receptors is implicated.

Evidence for a postsynaptic action of neostigmine on muscarinic receptors in the anococcygeus muscle of the rat.

In the rat anococcygeus muscle neostigmine induces atropine-sensitive, significant leftward displacements of the log concentration-response curves to both noradrenaline and 5-hydroxytryptamine. Responses to high K+ were also potentiated by neostigmine. However, high K+ elicited only small and irregular overflows of tritium from [3H]noradrenaline pre-incubated tissues, in contrast to the large overflow elicited by field stimulation. In addition guanethidine blocked responses to field stimulation but not those to high K+. This is consistent with the dose-related responses to high K+ being elicited postsynaptically. These results indicate that postsynaptic muscarinic receptors are involved in the potentiation by neostigmine of rat anococcygeus muscle responses to field stimulation or exogenous agonists, possibly by an action on receptor operated ion channels. Additional support for a postsynaptic site of action comes from the failure of neostigmine to potentiate tension responses to nerve or field stimulation in the chick expansor secundariorum, a muscle which is devoid of postsynaptic muscarinic receptors.

Observations on the production of frozen-dried thin sections for electron microscopy using unfixed fresh liver, fast-frozen without cryoprotectants.

Thin sections of unfixed liver, fast-frozen without cryoprotectants, have been cut using conditions under which momentary thawing of the sections is unlikely to occur. A transfer stage which facilitates this procedure is described. Sections show hole damage probably due to ice-crystal formation during the freezing process and have well defined edges, but despite hole damage, some morphological features of the cell are discernible. Presumptive mitochondria appear smaller in frozen sections than in conventional Araldite sections. Sections devoid of hole damage have indistinct edges and are presumed to have undergone transient thawing. Carbon coating of freeze-dried sections to exclude atmospheric moisture during transference of sections to the electron microscope (EM) appear unnecessary as regards preservation of morphological structure. The results are discussed in relation to the limitations of the method and the potential value of the technique.

Evolution of the proteinase inhibitor I family and apparent lack of hypervariability in the proteinase contact loop.

A protein phylogenetic tree was constructed from 24 homologous proteinase inhibitor I sequences identified in the EMBL/Genbank and Swiss-Prot databases and from translated amino acid data from four constitutive cDNA clones of proteinase inhibitor I characterized from potato tuber mRNA. The tree suggests that divergence of at least four paralogous proteins with functional specialization occurred at different times during the evolutionary history of the proteinase inhibitor I family. Five distinct regions in the primary structure, earlier identified by structural studies, were used to analyze the inhibitor family for hypervariability (Creighton and Darby, Trends Biochem Sci 14:319-324, 1989). Mutations did not occur with higher-than-random frequency within the proteinase binding region. When isoinhibitor, orthologous, or paralogous data subsets were subsequently analyzed the same results were obtained. Comparison of the amino acid sequences for all the known potato proteinase isoinhibitor I proteins identified ten highly variable sites. These also were distributed randomly. Thus hypervariability, which has been observed in all other serine proteinase inhibitor families to date, appears to be lacking in the proteinase inhibitor I family.

The effects of low doses of diazepam on human performance in group administered tasks.

1. The effects of diazepam in 5 mg dosage were assessed on a range of psychological tasks. Seventy-eight healthy subjects were tested in an independent groups design; subjects were randomly assigned to either control, placebo or drug group. Treatments were administered orally under double blind conditions. 2. Auditory vigilance performance was unimpaired, in terms of (a) correct detections, (b) false alarms or (c) the subjects' estimates of the duration of the task. 3. The short term retention of digit strings was impaired by diazepam (P less than 0.05), especially for those digits presented in the middle of the sequence. 4. Searching for a few letters among many was significantly impaired by diazepam (P less than 0.01). 5. Diaepam had no effect on performance at a mental arithmetic task; neither was there a placebo effect. 6. Results were discussed in the light of the characteristics of drug sensitive tasks. It was concluded that characteristics such as feedback of results, monotony, and memory load are more likely to be drug sensitive when in combination than in isolation.