Pituitary intermediate lobe in dog: two cell types and high bioactive adrenocorticotropin content.

The pituitary intermediate lobe of most species is cytologically monotonous, but that of the dog is composed of two immunocytochemically distinct cell types. The predominant A cells are typical pars intermedia cells: they stain immunocytochemically for alpha-melanotropin and, more weakly, for adrenocorticotropin and beta-lipotropin. The B cells are like the corticotrophs of the anterior lobe: they stain intensely for adrenocorticotropin and beta-lipotropin but not for alpha-melanotropin. The B cells may account for the high concentration of bioactive adrenocorticotropin measured in the canine pars intermedia, and may explain why in dogs adenomas causing Cushing's disease through hypersecretion of adrenocorticotropin can arise from the intermediate as well as the anterior pituitary lobe.

Properties of cyclic AMP-dependent protein kinase in normal and goitrous rat thyroid gland.

Most of the cyclic AMP-dependent protein kinase activity in propylthiouracil-induced goiters and control rat thyroid glands was found in the soluble fraction. The activity in the particulate fractions was cyclic AMP-independent. Protein kinase activity was 2--3-fold higher in all the subcellular fractions of goitrous tissue than of control tissue. In the presence of Triton X-100, both groups showed a significant increase in kinase activity in all subcellular fractions, and the kinase activity in the particulate fractions could now be slightly stimulated by cyclic AMP. Again, enzyme activity in fractions from goiters was significantly higher than in control tissue. Two major peaks, Types I and II, of soluble cyclic AMP-dependent protein kinase activity could be separated by DEAE-cellulose chromatography. Chronic in vivo stimulation by TSH was associated with a selective increase in Type II isoenzyme activity. Elution and pH profiles, dissociation of subunits with 0.5 M NaCl, and activity ratios (-cyclic AMP/+cyclic AMP) for various substrates for Type II isoenzyme in goitrous and control tissue were similar. The elevated activity in goitrous tissue was manifested by an increase in V for histone, ATP, Mg2+ and cyclic AMP, with no change in the apparent Km.

Intrathyroidally generated iodide: the role of transport in its utilization.

Thyroid iodide labeled 72 h earlier with 131I was separated from organic iodine by polyacrylamide gel electrophoresis. The concentration of thyroid radioiodide was not significantly diminished 2 h after the administration of perchlorate (100 mg NaClO4) alone, but perchlorate reduced the rise in glandular radioiodide caused by simultaneously given TSH (1 IU). When propylthiouracil (PTU; 20 mg) was given along with it, perchlorate decreased thyroid radioiodide even in rats not treated with TSH. In rats given perchlorate, PTU caused a much slighter augmentation of the TSH-induced increase in the thyroid radioiodide concentration than in the absence of perchlorate. These results are interpreted as follows. Perchlorate-discharged iodide in rats not given TSH is largely transported iodide. Perchlorate can discharge intrathyroidally generated (internal) iodide too, but this is unequivocally reflected by a decrease in the thyroid iodide concentration only when the production of internal iodide is enhanced by TSH. Perchlorate in its own right interferes with organic binding of internal iodide, thereby partially preempting the effect whereby PTU causes accumulation of internal iodide in TSH-treated rats. We suggest that internal thyroid iodide is transported from its site of generation, the follicular cell, to its site of organic binding, the follicular lumen, by perchlorate-sensitive transport. It is also possible that internal iodide escaping from a follicle is conserved by perchlorate-inhibitable reuptake into follicular cells closer to the venous end of the capillary bed.

Intrathyroidally generated iodide: the role of propylthiouracil-sensitive processes in its production.

Thyroid radioiodide to serum radioiodide concentration (*T/*S) ratios 1 h after 131I administration were 3 times higher in rats pretreated with propylthiouracil (PTU) than in rats with binding thyroids. Although indications of a preponderance of transported iodide in the thyroid early after 131I injection were found, a reduction of the 1-h *T/*S ratio in TSH-treated rats by the inhibitor of iodotyrosine dehalogenation, mononitrotyrosine, points to an early contribution of intrathyroidally generated (internal) iodide to total thyroid iodide. In rat thyroids labeled 72 h earlier, there was no rise in electrophoretically separated radioiodide 2 h after the administration of PTU alone, but the elevation of thyroid iodide due to TSH was augmented by concurrently given PTU. On the assumption that PTU affects transported components of thyroid iodide identically whether they are labeled 1 or 72 h earlier, we conclude that PTU must severely depress the generation of internal iodide. This effect is no longer evident if TSH is given simultaneously. Further, our results are consistent with the hypothesis that internal iodide passes through a stage in which it is not available for organic binding before it mixes with transported iodide. TSH appears to facilitate the transfer of internal iodide into the pool which it shares with external iodide.

The effect of stress on the cytology and immunocytochemistry of pars intermedia cells in the rat pituitary.

The purpose of this study was to determine if there were ultrastructural and immunocytochemical changes in intermediate lobe cells following stress. We found marked cytological changes in intermediate lobe cells after 30 min of stress with a buzzer and stroboscopic light (neurogenic). Following such treatment, the cytoplasm was filled with pale graules or empty vesicles. There was cytological evidence of increased secretory and synthetic activity. Innunocytochemical staining (for 17-39-ATCH) revelaed that there were fewer stained granules per cell in such rats although the individual granules stained as strongly as those in control rats. No cytological changes were observed following ether stress. These results correlated well with our bioassay and radioimmunoassay data which showed that only neurogenic stress resulted in depletion of ACTH from neuro-intermediate lobes (1). In agreement with the work of others, we found no obvious cytological changes in intermediate lobes from adrenalectomized or cortisol treated rats wheras anterior lobe ACTH cells hypertrophied following adrenalectomy and involuted following cortisol treatment. Anterior lobe ACTH cells from intact rats showed no degranulation following 30 min of buzzer stress or after 2 min of ether stress.

Plasma immunoreactive proopiomelanocortin peptides and cortisol in normal dogs and dogs with Addison's disease and Cushing's syndrome: basal concentrations.

We measured basal plasma concentrations of the immunoreactive (IR) proopiomelanocortin (POMC)-derived peptides ACTH, beta-lipotropin (beta LPH), beta-endorphin (beta END), and alpha MSH in 160 normal dogs, 32 dogs with Addison's disease, 42 dogs with adrenocortical tumors causing Cushing's syndrome, and 169 dogs with pituitary-dependent Cushing's disease. In normal dogs, plasma IR-POMC peptide levels were similar to those in man, except that IR-alpha MSH, a pars intermedia POMC product, was readily detected. In Addisonian dogs, plasma cortisol was decreased, and the IR-POMC peptides were increased, except for IR-alpha MSH, which was normal. In 7 Addisonian dogs given dexamethasone, elevated plasma IR-ACTH, beta LPH, and beta END levels fell dramatically. In dogs with Cushing's syndrome due to adrenal tumors, plasma IR-ACTH, beta LPH, and beta END were decreased, and cortisol was increased, but IR-alpha MSH was normal. Dogs with Cushing's disease due to pars distalis tumors had elevated plasma IR-ACTH, beta LPH, beta END, and cortisol, but normal IR-alpha MSH; their plasma cortisol was suppressed by dexamethasone. There appeared to be 2 types of pars intermedia tumors causing Cushing's disease: 1 dexamethasone nonsuppressible and with disproportionately high plasma IR-alpha MSH levels, the other relatively dexamethasone suppressible and with normal to slightly elevated IR-alpha MSH levels. These 2 pars intermedia tumor types may arise from 2 distinct normal canine pars intermedia cell types. Canine Cushing's disease may provide a useful model for variants of the disorder in man.

Ultrastructural-immunocytochemical localization of growth hormone and prolactin in human pituitaries.

Immunocytochemical staining using the unlabeled antibody peroxidase-antiperoxidase method was undertaken to localize and characterize in ultrathin sections of human pituitaries the cells responsible for the secretion of GH and PRL. Somatotrophs in seven pituitaries stained with human (h) PRL-absorbed antiserum to hGH, were abundant, round to ovoid, densely granulated cells, whose mean (+/-SD) granule diameter was 368 +/- 60 nm. Lactotrophs immunostained with antiserum to hPRL were less numerous, angular or branching cells, with fewer round to ovoid granules, the mean diameter (+/-SD) of which was 185 +/- 35 nm in six pituitaries. The somewhat larger PRL granules (up to a mean diameter of 360 nm) seen in two of three additional pituitaries may have been related to the previous therapeutic administration of estrogen. Whereas the immunostained GH-secreting cells resemble the presumed somatotrophs identified in other studies on the basis of nonimmunological staining, the immunostained PRL-secreting cells differ considerably from the cells with large (600--1000 nm) granules designated as lactotrophs by several previous investigators. The hazards of ultrastructural identification of human pituitary cell types on purely morphological (as opposed to immunocytochemical) grounds are emphasized.

Gonadotropin-producing pituitary adenoma in a man with long-standing primary hypogonadism.

Pituitary adenomas that secrete gonadotropins are generally believed to arise spontaneously rather than as a response to chronic primary gonadal failure. However, two women who were found to have gonadotroph adenomas several years after ovarian ablation have been reported. We describe a middle-aged man who developed bitemporal hemianopia and was found to have a large pituitary tumor 35 yr after castration. He had never received any replacement therapy. The tumor was considered to be a primary gonadotroph adenoma, rather than secondary gonadotroph hyperplasia, on the basis of its secretory capabilities, its reticulin patterns, and its specific immunostaining for human FSH beta, human LH beta, and alpha-subunit. Furthermore, the tumor did not decrease appreciably in size after 12 months of testosterone treatment, although plasma gonadotropin levels decreased. Unless the association of primary gonadal failure with a gonadotroph adenoma was coincidental, it suggests that some human gonadotroph adenomas may be secondary to failure of the gonads.

Immunostaining of growth hormone and prolactin in paraffin-embedded and stored or previously stained materials.

In attempts to evaluate immunocytochemically autopsy and biopsy material previously obtained and processed for conventional histologic staining, we had to resort to immunostaining of tissues embedded years ago or even sections already stained with hematoxylin-eosin or aldehyde thionin-PAS-orange G. Hypophysial growth hormone and prolactin proved remarkably resistant to such prior treatment with regard to their antigenic properties, and could be readily immunostained in tissue embedded in paraffin 3-4 years earlier, and after destaining of sections prepared up to 7 years earlier. The results of such "retrospective" immunocytochemical evaluation of autopsy and biopsy materail is illustrated with the staining of "pregnancy cells" for prolactin in the hypophysis of a woman postpartum, the immunostaining for prolactin in the cells of adenomas associated with marked hyperprolactinemia, the staining for growth hormone in adenomas removed from children with gigantism, and the immunostaining for prolactin, growth hormone or both in several adenomas that were discovered at autopsy and not associated with a known clinical history of endocrine aberrations.

Immunoperoxidase staining of primate pituitaries with antibodies against the beta subunits of human pituitary glycoprotein hormones.

The immunocytochemical specificity of antibodies against the beta subunits of human pituitary glycoprotein hormones was tested on human and monkey hypophyses. Anti-luteinizing hormone (LH) beta stained only gonadotrophic cells in both species, and anti-thyroid-stimulating hormone (TSH) beta only thyrotrophic cells. In monkeys, anti-follicle-stimulating hormone (FSH) beta demonstrated only gonadotrophs, but in man the antibody stained thyrotrophs equally well. Staining of the two cell types was diminished to a similar degree by dilution of the anti-FSH beta antibody. Human gonadotroph heterogeneity was revealed by absorption of anti-FSH beta with LH beta or TSH beta, which abolished immunostaining of a subset of gonadotrophs. Limitations in the immunocytochemical specificity of antibodies against beta subunits of pituitary glycoprotein hormones may be a consequence of incomplete purity of the generating antigens, structural overlap among the beta subunit of the three hormones, or a combination of these.

The cells of origin of ACTH in man.

The evidence from ACTH-producing tumors, from the morphologic effects of excess glucorticoids on the hypophysis, and from immunocytochemistry all points to the predominant "basophils" of the human anterior lobe, the intensely PAS-possitive beta(R) cells, as the source of ACTH. Similar cells often also occur, sometimes in large numbers, in the pars nervosa. beta(R) cells can be immunostained with antibodies against alphaMSH and betaMSH, but it is likely that they actually contain the precursor molecule(s) of betaMSH, betaLPH (and may be gammaLPH). Once this is satisfactorily documented, the functional term corticolipotrops should replace the provisional name beta(R) cells. Electron microscopically, these cells contain randomly arranged granules with a maximum diameter of 300-500 nm, and occasionally small amounts of filamentous material, which increases dramatically in response to excess glucocorticoids. In the beta (R) cells of adults, a single set of granules stains for ACTH and with antibodies to betaMSH. In fetal life, ACTH cells appear early and material reactive with anti-betaMSH accumulates in them (and possibly also in cells not containing ACTH) only later. The posterior lobe beta(R) cells can be immunostained with both anti-betaMSH and antibodies against the COOH-terminal portion of ACTH, but the presence of bioactive ACTH in them remains to be shown.

Long-term immunization against the beta-subunit of ovine luteinizing hormone (oLH beta) has no adverse effects on pituitary function in rhesus monkeys.

One postulated safety hazard of contraceptive methods based on immunization against gonadotropic hormones is the possibility that circulating antibodies which crossreact with pituitary hormones may impair pituitary function through the deposition of immunoglobulin and/or complement suggesting immune complexes. In order to evaluate this possibility in rhesus monkeys actively immunized against the beta-subunit of ovine luteinizing hormone (oLH beta), we used three approaches to study the effects of long-term immunization on pituitary function: a) evaluation of pituitary responsiveness to challenge with a GnRH-agonist; b) examination of pituitary histology and immunostaining with gonadotropin antisera; and c) examination of pituitary cells for deposition of immune complexes. Our results indicate that circulating anti-oLH beta antibodies did not result in significant impairment of pituitary function in rhesus monkeys.

'Acidophilic' pituitary tumors: a reappraisal with differential staining and immunocytochemical techniques.

The cells of pituitary adenomas classified as acidophilic on PAS-light green-orange G staining could be further differentiated with the Brookes technique: they stained nonspecifically bluish-gray, orange with orange G, or red with carmoisine. On immunostaining for growth hormone and prolactin, the gray cells were either negative or reactive for prolactin, the orange cells contained growth hormone, and the red cells contained prolactin. Of 28 tumors, eight showed no immunostaining, 11 stained only for prolactin, three stained only for growth hormone, five contained mostly growth hormone cells and some prolactin cells, and one contained predominantly prolactin cells but also numerous growth hormone cells. Immunoreactive growth hormone granules in adenoma cells were usually arranged randomly; prolactin granules were often concentrated along one nuclear pole. This study emphasizes the tinctorial and immunocytochemical heterogeneity of "acidophilic" adenomas.