E. F. Adams

SECRETION OF LH, FSH AND PRL SHOWN BY CELL CULTURE AND IMMUNOCYTOCHEMISTRY OF HUMAN FUNCTIONLESS PITUITARY ADENOMAS

Hormone secretion by ten functionless human pituitary adenomas in cell culture has been measured, and compared with tissue immunocytochemistry and electron microscopy, as well as results with a normal pituitary. Patients presented following routine x‐ray and had no clinical or biochemical endocrine abnormality apart from one male with raised serum FSH and PRL, normal LH, and low testosterone. Of the ten adenomas, nine secreted both LH and FSH in cell culture and five of these also secreted PRL, one did not secrete any anterior pituitary hormones (ACTH was not measured). No GH or TSH was detectable in the cultures of the nine LH/FSH secretors excluding the possibility of contamination by normal anterior pituitary. The normal pituitary cells secreted all anterior pituitary hormones; the amounts of FSH/LH being comparable with those of the adenomas. Immunostaining confirmed the cell culture results and showed the adenoma FSH/LH cells to be scattered singly or in small groups of two to five cells with both hormones usually being in the same cell. PRL where found was in separate cells. Hormone granules were small (50–160 nm), round or irregular and scattered in the cytoplasm of rounded cells of low secretory activity. The negatively staining cells were not different ultrastructurally to those staining positively. It is concluded that a significant proportion of functionless pituitary adenomas have detectable low levels of LH/FSH secretion often accompanied by PRL when examined by cell culture or immunocytochemistry. Although these adenomas were endocrinologically quiescent activity could have been masked because of post‐menopausal secretion and one male probably had an FSH‐secreting adenoma.

GONADOTROPHIN AND ALPHA SUBUNIT SECRETION BY HUMAN ‘FUNCTIONLESS’ PITUITARY ADENOMAS IN CELL CULTURE: LONG TERM EFFECTS OF LUTEINIZING HORMONE RELEASING HORMONE AND THYROTROPHIN RELEASING HORMONE

The long‐term effects of LHRH and TRH on gonadotrophin alpha subunit, FSH and LH secretion by cell cultures of four human chromophobic pituitary tumours have been examined. The tumours derived from one male and three female patients who presented because of visual disturbance but had no evident endocrine symptoms. Subsequent serum hormone analysis showed the FSH to be high in the male but low or normal in the post‐menopausal females whereas LH levels were low in all patients. In culture, basal hormone secretion could be maintained for periods up to 63 d. All tumours secreted alpha subunit and FSH, but much lower amounts of LH. Addition of LHRH or TRH for a period of 12 to 41 d showed that alpha subunit, FSH and LH secretion were stimulated by LHRH from one tumour, by LHRH and TRH from two tumours. There was always a rapid decline in the LH secretion. The tumour which secreted FSH predominantly was stimulated by TRH. We conclude that human pituitary ‘functionless’ adenomas can secrete gonadotrophin alpha subunit and FSH in vitro and that secretion can be stimulated during long term releasing hormone experiments. LH secretion, however, cannot be maintained.

BROMOCRIPTINE SUPPRESSES ACTH SECRETION FROM HUMAN PITUITARY TUMOUR CELLS IN CULTURE BY A DOPAMINERGIC MECHANISM

Bromocriptine (0·13–13 μM) significantly inhibited ACTH secretion in a dose‐dependent manner when added to cell cultures of a human corticotrophic adenoma for 24 h. Haloperidol (13 μM), but not serotonin (13 μM), blocked this inhibition but had no significant effect when added alone. In addition, dopamine (10 μM) reduced ACTH secretion during a 4‐h incubation, whereas serotonin (0·01–10 μM) was ineffective. An ectopic ACTH secreting lung carcinoid was non‐responsive to doses of bromocriptine up to 13 μM. These results demonstrate a direct suppressive action of bromocriptine on a human pituitary corticotrophic adenoma through dopaminergic rather than sero‐toninergic mechanisms.

Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies.

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.

EFFECT OF PANCREATIC GROWTH HORMONE RELEASING FACTORS ON GH SECRETION BY HUMAN SOMATOTROPHIC PITUITARY TUMOURS IN CELL CULTURE

Human pancreatic growth hormone releasing factors (hpGRF(1–40) and hpGRF(1–44)) significantly stimulated GH secretion when added to cell cultures of human somatotrophic pituitary tumours for 2 h. There was little difference in potency between the two peptides, and the response of different tumours varied, ranging from 30% to 500% increases in GH secretion over control levels. This stimulatory effect was blocked by somatostatin (SRIF) and bromocriptine. The suppressive effect of bromocriptine, but not of SRIF, was overcome by high doses of hpGRF(1–44). TRH stimulated GH secretion by one of three somatotrophic tumours in cell culture, and it was found to potentiate the stimulatory effects of hpGRF(1–44). These results demonstrate that hpGRFs increase serum GH levels in man by a direct action at the pituitary somatotroph level.

OVINE CORTICOTROPHIN RELEASING FACTOR STIMULATES ACTH RELEASE FROM HUMAN CORTICOTROPHINOMA CELLS IN CULTURE; INTERACTION WITH HYDROCORTISONE AND ARGININE VASOPRESSIN

We have investigated the effects of ovine corticotrophin releasing factor (oCRF) and its interaction with hydrocortisone (HC), and arginine vasopressin (AVP) on ACTH release from human corticotrophinoma cells in culture. Tumour tissue was obtained from six patients (three with active Cushings disease and three with Nelsons syndrome). Cultures were maintained for periods of up to six months. Ovine CRF (21 nmol) significantly (P>0·01) stimulated ACTH release from all tumours. Dose response (21 pmol‐21 nmol) effects were observed for the three tumours investigated over 2 and 4 h. Cortisol (20 μmol) significantly (P>0·01) inhibited basal ACTH release from one tumour (Nelsons syndrome) by 75% over 4 h, and completely prevented the stimulatory effects of oCRF. AVP directly stimulated ACTH release from two tumours (Nelsons syndrome), and also potentiated the stimulatory action of oCRF during 30 min incubations. These data show corticotrophinoma cells from subjects with Cushings disease and Nelsons syndrome can be directly stimulated by hypothalamic oCRF and may be potentiated by AVP. Cortisol and oCRF have been shown in one tumour to have antagonistic actions at the pituitary level.

Inappropriately low serum GH in an acromegalic: Lysosomal involvement in intracellular hormone degradation☆

A patient with galactorrhea, amenorrhae and severe acromegaly was found to have a large pituitary adenoma. In view of the severity of the disease the serum growth hormone (GH) level (22.5 mlU/liter) was considered inappropriately low. Tissue from the adenoma was obtained during successful treatment with interstitial irradiation. (90Yttrium). Trypsin-dispersed biopsy cells in culture for 12 days secreted low amounts of GH compared to the same number of adenoma cells from 5 other unselected acromegalics or a normal pituitary. No other hormones were secreted in culture. Immunocytochemical staining was positive only with GH antisera but showed low intracellular content. This was confirmed by direct analysis of the tumor tissue which showed the GH content to be only 20% of that found in 5 normal pituitaries and 4% of that found in 8 other adenomas from acromegalics. Electron microscopy showed a striking appearance, with GH secretory granules that were sparse in number, smaller than usual, and in the main arranged around numerous intracellular profiles with double membranes and low electron density that were tentatively identified as autophagic vacuoles (secondary lysosomes). Subcellular fractionation showed the distribution of the radioimmunoassayable GH in the gradient to be coincident with the peak of the lysosomes whereas in 2 other acromegalics the GH peak was clearly separated from the lysosomes. We conclude that the simultaneous appearance in our patient of the relatively low serum GH together with a large tumor and severe acromegaly can be explained biochemically by the striking finding of crinophagy - disposal of hormone secretory granules within the somatotroph cells themselves.

Ectopic Corticotrophin-Releasing-Factor and Growth Hormone Releasing Factor Secretion: Diagnosis Using Human Pituitary Cell Culture

Cell culture of human pituitary tissue has been used to diagnose a patient with Cushings syndrome due to ectopic secretion of corticotrophin-releasing factor (CRF; case 1) and a case of acromegaly associated with ectopic secretion of a growth-hormone releasing factor (GRF; case 2). In both patients a pituitary tumour was not detected. Case 1 had a small cell carcinoma and symptoms of the ectopic ACTH syndrome, but in culture the carcinoma failed to secrete detectable ACTH. However, the culture medium used to maintain this carcinoma in vitro was found to contain a substance which stimulated ACTH secretion by human pituitary corticotrophs in cell culture. Radioimmunoassays and HPLC indicated that this substance had similar elution characteristics to human CRF and cross-reacted with antiserum to ovine CRF. Case 2 was found to have a lung tumour, the removal of which led to regression of her acromegalic symptoms. In culture, this tumour did not secrete GH, but did secrete a GRF. We conclude that the Cushings syndrome and acromegaly, in cases 1 and 2, respectively, were due to ectopic secretion of CRF and GRF leading to hyperstimulation of the pituitary gland.

EFFECT OF NIVAZOL ON ACTH SECRETION BY HUMAN PITUITARY CORTICOTROPHIC TUMOURS IN CELL CULTURE

The effects of nivazol, a novel steroid which lacks glucocorticoid activity, on ACTH secretion by cell cultures of human pituitary corticotrophic tumours has been investigated. Nivazol (0·002‐20 μmol/1) inhibited ACTH secretion by 50–80%, after 4 and 24 h of incubation. (Trilostane (0·3–30 μmol/1) did not affect basal or stimulated ACTH secretion.) The potency of nivazol was comparable with that of hydrocortisone, and less than dexamethasone. The stimulatory effects of oCRF and AVP were reduced or completely blocked by nivazol in a similar manner to that previously described for hydrocortisone. It is concluded that nivazol may be of benefit in the direct treatment of Cushings disease, particularly since it lacks glucocorticoid activity.