Neuropeptide y- and alpha-adrenergic receptors in pig spleen: Localization, binding characteristics, cyclic amp effects and functional responses in control and denervated animals

Abstract

The localization of neuropeptide Y binding sites in the pig spleen, as revealed by [ 125I]Bolton-Hunter-labelled porcine neuropeptide Y and alpha 1-adrenergic receptor binding sites, as revealed by [ 125I](2-beta/4-hydroxy-phenyl/-ethylaminomethyl)-tetralone as radioligand, was compared with the distribution of neuropeptide Y and noradrenaline nerves, the latter revealed by tyrosine hydroxylase and dopamine-beta-hydroxylase, using immunohistochemistry. A large degree of codistribution was obtained between [ 125I]neuropeptide Y and alpha 1-binding sites in the capsule, trabeculae, blood vessels and the red pulp of the spleen. Neuropeptide Y and tyrosine hydroxylase as well as dopamine-beta-hydroxylase-positive nerves were identical in the spleen and had a similar gross distribution pattern as the [ 125I]neuropeptide Y and alpha 1 binding sites. In functional studies using the isolated blood-perfused spleen from pentobarbital-anaesthetized pigs, neuropeptide Y, noradrenaline and the alpha 1-selective agonist phenylephrine contracted the capsule and induced vasoconstriction in the spleen in vivo. However, the selective alpha 2-adrenoceptor agonists clonidine and azepexole had no effects on blood flow or perfusion pressure, suggesting that postjunctional alpha-receptors were of the alpha 1 type. Neuropeptide Y inhibited the forskolin-evoked, cyclic adenosine monophosphate formation in vitro. The [ 125I]neuropeptide Y binding, with an equilibrium-dissociation constant of 503 ± 73pM and a maximal number of specific binding sites of 23 ± 3fmol/mg protein, the neuropeptide Y-induced perfusion-pressure increase in vivo and the inhibition of forskolin-evoked cyclic adenosine monophosphate formation in vitro were dependent on the amidation of the C-terminal portion of the peptide molecule. Furthermore, the effects of neuropeptide Y were not changed by alpha- and beta-adrenoceptor blockade using prazosin and propranolol. Two weeks after postganglionic denervation the neuropeptide Y and the noradrenaline contents of the pig spleen were reduced by 97% and 99%, respectively. These changes were associated with a selective supersensitivity for the noradrenaline-induced perfusion-pressure increase in vivo compared with the effect of neuropeptide Y. However, a similar potentiation of the noradrenaline effect was induced by the monoamine-uptake blocker desipramine in the absence of denervation and there was no change in the functional response to phenylephrine after denervation. This indicated that the denervation supersensitivity for noradrenaline was of prejunctional rather than of postjunctional origin, although a significant increase in the maximal number of specific binding sites of the [ 3H]prazosin binding (75%) was observed in vitro. No significant changes in the equilibrium-dissociation constant or in the maximal number of specific binding sites were observed after denervation for the [ 125I]neuropeptide Y binding sites. Furthermore, the neuropeptide Y-induced inhibition of forskolin-induced cyclic adenosine monophosphate formation was not changed after denervation. It is concluded that alpha 1-adrenoceptor and neuropeptide Y binding sites are co-distributed and can be activated in parallel to induce muscle contraction in support of a co-transmitter role for noradrenaline and neuropeptide Y in the sympathetic nervous control of the pig spleen. Denervation was associated with a selective up-regulation of the alpha 1-adrenoceptor binding sites, but no changes in the binding characteristics were observed for neuropeptide Y, suggesting the absence of homeostatic responses in the regulation of neuropeptide Y receptors of the spleen.