In brain stem slices from neonatal (postnatal days 0-4) CD-1 mice, muscarinic ACh receptors (MAChRs) increased rhythmic inspiratory-related and tonic hypoglossal nerve discharge and depolarized single hypoglossal motoneurons (HMs) via an inward current without changing input resistance. These responses were blocked by the MAChR antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; 100 nM). MAChRs shifted voltage-dependent activation of the hyperpolarization-activated cation current to more positive levels. MAChRs increased the HM repetitive firing rate and decreased rheobase, with both effects being blocked by 4-DAMP. Muscarinic agonists reduced the afterhyperpolarization of single action potentials (APs), suggesting that small-conductance Ca(2+)-dependent K(+) current inhibition increased the HM firing rate. Muscarinic agonists also reduced the AP amplitude and slowed its time course, suggesting that MAChRs inhibited voltage-gated Na(+) channels. To compare muscarinic excitation of single HMs to muscarinic excitatory effects on motor output in thicker brain stem slices requiring higher extracellular K(+) for rhythmic activity, we tested the effects of muscarinic agonists on single HM excitability in high-K(+) artificial cerebrospinal fluid (aCSF). In high-K(+) aCSF, muscarinic agonists still depolarized HMs and altered AP size and shape, as in standard aCSF, but did not increase the steady-state firing rate, decrease afterhyperpolarization, or alter threshold potential. These results indicate that the basic cellular response of HMs to muscarinic receptors is excitatory, via a number of distinct mechanisms, and that this excitatory response will be largely preserved in rhythmically active brain stem slices.
SUMMARY
The distribution of neurophysin in subcellular fractions obtained by differential centrifugation of bovine neurohypophyses is essentially similar to the distribution of the hormones. The neurosecretory particle fraction contained more neurophysin than other fractions and neurophysin comprised half of its water soluble protein. Estimates were derived for the concentrations of oxytocin, vasopressin and neurophysin in neurosecretory granules.
In experiments in which neurosecretory granules were suspended in media at pH values between 6·2 and 8·0 it was found that release of hormones increased with increasing pH.
The role of neurophysin in the transport and release of neurohypophysial hormones is discussed.
SUMMARY
Association of oxytocin and arginine vasopressin with protein in bovine and rabbit neurohypophysial extracts has been studied by co-precipitation of the hormones with protein on addition of NaCl, by gel filtration and by dialysis. Although precipitates formed by addition of NaCl (2·5–20 g./100 ml.) to bovine neurohypophysial extracts at pH 3·1 contain both hormones, oxytocin and arginine vasopressin were partially separated at 5·0 g. NaCl/100 ml. when the proportion of oxytocin precipitated was approximately double that of arginine vasopressin. No precipitate formed on addition of NaCl (15 g./100 ml.) to bovine neurohypophysial extract at pH 5.8.
Experiments by gel filtration and dialysis showed that the binding of oxytocin and vasopressin to protein in neurohypophysial extracts is pH dependent and is maximal in the range pH 5·2–5·8. Dilution of a solution containing neurohypophysial hormones and protein results in dissociation of the complexes and this could account for differences observed in experiments with bovine and rabbit neurohypophysial extracts. It is suggested that the mode of binding between the hormones and protein is ionic association between the cationic free terminal NH2 of the cystine residue in the hormones and free carboxyl groups in protein.
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