Microwave staining of enteric neurons using cuprolinic blue (Quinolinic phthalocyanin) combined with enzyme histochemistry and peroxidase immunohistochemistry.

Methods that visualize subsets as well as the entire enteric neuron population are not readily available or have proved to be unreliable. Therefore, we attempted to combine NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, techniques that mark subsets of enteric neurons, with a technique that appeared to visualize the entire enteric neuron population, the cuprolinic blue staining method. To guarantee representative staining results, the individual staining methods were modified by using microwaves. In addition, this preserved the characteristics of each of the individual techniques. The distribution of NADPH-d, AChE, and CGRP corresponded well with previous morphological and physiological reports. Consequently, the different combinations gave rise to rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons.

Calcitonin gene-related peptide receptors in pig spleen and the involvement of the CGRP(1)receptor in the splenocyte function.

Calcitonin gene-related peptide (CGRP)-positive nerve fibers have been found in the trabecula and parenchyme area of pig spleen. Receptor studies have demonstrated that the CGRP binding site in pig spleen membranes has an average K(d)2.24 +/- 0.48 nM and B(max)78 +/- 4.09 fmol/mg of protein. In the K(d)range demonstrated in the binding studies, the dose-dependent suppressive effect of CGRP on spleen T lymphocyte proliferation was found with the maximal effect in 10(-9)M concentration. The same effect, but in a different concentration, was found on peripheral blood T lymphocytes with the maximum in 10(-6)M concentration. Contrary to the results obtained through the simultaneous presence of CGRP and mitogen, preincubation with CGRP led to a stimulation of peripheral blood lymphocytes in response to ConA and had no effect on spleen T lymphocytes. These results illustrate the difference in CGRP effect between lymphocytes of different origins. Using CGRP(1)receptor antagonist CGRP(8-37), we established that the CGRP suppressive effect on spleen T lymphocyte proliferation is CGRP(1)-receptor mediated.

Ontogeny of epinephrine, norepinephrine, dopamine-beta-hydroxylase, and chromogranin A in the adrenal gland of pigs.

OBJECTIVE: To obtain data on the ontogeny of catecholamines and other chromaffin vesicle components, which could serve as a basis for the study of their role during fetal life in normal and pathologic conditions. DESIGN: Epinephrine, norepinephrine, dopamine-beta-hydroxylase, and chromogranin A contents were measured in the porcine adrenal gland during various stages of gestation. ANIMALS: 934 porcine fetuses representing 22 gestational ages between 43 and 108 days. PROCEDURE: Total homogenates of adrenal glands were extracted and contents of different neurochemical markers were measured, using high-performance liquid chromatography, immunoassays, and western blotting. Immunohistochemical studies also were performed. RESULTS: Epinephrine and norepinephrine contents as a function of gestational age can be represented by a sigmoidal curve. Norepinephrine content rises early in gestation, whereas epinephrine content increases later. Maximal increase was significantly higher for epinephrine content. A progressive appearance of separate epinephrine- and norepinephrine-storing cells was documented. Dopamine-beta-hydroxylase content as a function of gestational age can be adequately represented by a parabolic curve. No quantitative changes in chromogranin A concentration were observed, but western blotting revealed qualitative changes with progressing gestational age. CONCLUSIONS: Important changes occur in catecholamine formation around day 60 of gestation. The sharp increase in epinephrine/norepinephrine contents and the appearance of separate epinephrine- and norepinephrine-storing cells may be related to the progressive splanchnic innervation of the adrenal gland. The presence of chromogranin A early in gestation may indicate its necessity for catecholamine storage.