Evidence that 3,3',5-triiodothyronine is concentrated in and delivered from the locus coeruleus to its noradrenergic targets via anterograde axonal transport.

Recent immunohistochemical studies of rat brain triiodothyronine reveal heaviest localization in locus coeruleus perikarya. The cellular distribution is similar to that observed in concomitant studies of tyrosine hydroxylase immunohistochemistry: heavy clumps of immunoreactive triiodothyronine are distributed within locus coeruleus cytosol and in cell processes, leaving cell nuclei unstained. At the same time, in locus coeruleus targets, cell nuclei as well as surrounding neuropil are prominently triiodothyronine labeled. These observations, combined with diverse evidence linking thyroid hormone with norepinephrine at many levels of physiological and pathophysiological function, led to the hypothesis that the locus coeruleus binds and accumulates triiodothyronine and delivers the hormone via anterograde axonal transport to postsynaptic locus coeruleus targets, where nuclear triiodothyronine receptors are densely concentrated. Furthermore, the hypothesis predicts that destruction of locus coeruleus nerve terminals would interrupt this neural route of triiodothyronine delivery and prevent or reduce triiodothyronine labeling of nuclear receptors in noradrenergic target cells. To test this formulation, we gave the specific locus coeruleus lesioning agent, N-(2-chloroethyl)-N-2-bromobenzylamine hydrochloride (DSP-4), to adult male rats and examined their brains three, five and seven days thereafter by triiodothyronine and, in alternate sections, tyrosine hydroxylase immunohistochemistry. Treatment with DSP-4 resulted in specific and selective reduction in tyrosine hydroxylase and triiodothyronine immunohistochemical labeling in cell nuclei and in nerve cell processes within the neuropil of the hippocampus and cerebral cortex at all time periods examined. The results demonstrate that full occupancy of locus coeruleus target cells by triiodothyronine requires the presence of intact locus coeruleus projections and supports the proposal that, like norepinephrine, triiodothyronine delivery to noradrenergic targets occurs through delivery by locus coeruleus terminals. These findings provide strong support for earlier proposals that triiodothyronine functions as a co-transmitter with norepinephrine in addition to or as part of its genomic role in the cells receiving noradrenergic innervation.

Immunohistochemical mapping of brain triiodothyronine reveals prominent localization in central noradrenergic systems.

Many lines of evidence support a close association between thyroid hormones and noradrenergic systems in peripheral tissues. However, there is little certainty regarding interactions of the two systems in brain. We now report that triiodothyronine is concentrated in both nuclei and projection sites of central noradrenergic systems. Immunohistochemical mapping of the hormone revealed the following: (1) Locus coeruleus and all other noradrenergic cell groups identified were the most prominently labeled neural centers in the brain. (2) The hormone was also concentrated in the widely dispersed targets of noradrenergic projections. (3) Triiodothyronine labeling in noradrenergic target cells was most prominent over the cell nuclei, indicating that the hormone was bound to its receptors. Therefore, targets of noradrenergic innervation should be responsive to triiodothyronine. (4) Unlike that in noradrenergic target cells, triiodothyronine staining was decidedly perikaryal in locus coeruleus (A-6) and the other A-1 to A-7 cell groups; the staining pattern in locus coeruleus cytosol and processes was heavy, clumped and similar to that seen in contiguous sections immunostained for tyrosine hydroxylase. Results of radio-immunoassay, immunoabsorption and pharmacological tests demonstrated the specificity of the antibody for triiodothyronine and ruled against cross-reactivity with norepinephrine or its metabolites as the basis for the staining reactions. Although other possibilities consistent with these new observations are given consideration, it appears that the structure and activity of central noradrenergic systems may be major determinants of triiodothyronine distribution patterns and actions in brain. If the noradrenergic system processes both triiodothyronine and norepinephrine and conducts them both to nerve cell groups receiving its terminal arborizations, specific postsynaptic receptors would be available for transduction of both sets of messages. The evidence provides a morphological basis for earlier proposals that triiodothyronine may play a neuromodulatory or neurotransmitter role in the adrenergic nervous system.

Calpain II-dependent solubilization of a nuclear protein kinase at micromolar calcium concentrations.

Incubation of isolated, Triton X-100 washed rat liver nuclei with purified bovine myocardial calpain II resulted in solubilization of a histone H1 kinase activity. The release of kinase from nuclei could be prevented by including the calpain inhibitors leupeptin or calpastatin in the incubation. Titration with Ca2+/EGTA buffers indicated that the calpain-dependent release of the kinase was half-maximal at approximately 3 microM Ca2+. In contrast, calpain II required at least 50 microM Ca2+ to produce detectable proteolysis of soluble or membrane-associated substrates. These results suggest that the cell nucleus is a site of calpain II activation and function.