Demonstration of iodide transport defect but normal iodide organification in nonfunctioning nodules of human thyroid glands.

Benign and malignant nodules in human thyroid glands, which did not concentrate iodide in vivo, were also unable to accumulate iodide in vitro. The mean thyroid-to-medium ratio (T/M) in seven benign nodules was 0.8+/-0.2 compared with 7+/-2 in adjacent normal thyroid tissue. In four malignant thyroid nodules, the mean T/M was 0.5+/-0.1 compared with 11+/-4 in adjacent normal thyroid. Despite the inability of such nodules to concentrate iodide, iodide organification was present but was only one-half to one-third as active as in surrounding normal thyroid. Thyroid-stimulating hormone (TSH) increased iodide organification equally in both benign nodules and normal thyroid although it had no effect in three of the four malignant lesions. The reduction in organification is probably related to the absence of iodide transport, since incubation of normal thyroid slices with perchlorate caused similar diminution in iodide incorporation but no change in the response to TSH. Monoiodotyrosine (MIT) and di-iodotyrosine (DIT) accounted for most of the organic iodide in both the nodules and normal tissue. The MIT/DIT ratio was similar in normal and nodule tissue. The normal tissue contained much more inorganic iodide than the nodules, consistent with the absence of the iodide trap in the latter tissue. The thyroxine content of normal thyroid was 149+/-17 mug/g wet wt and 18+/-4 mug/g wet wt in the nodules. The transport defect in the nodules was not associated with any reduction in total, Na(+)-K(+)- or Mg(++)-activated ATPase activities or the concentration of ATP. Basal adenylate cyclase was higher in nodules than normal tissue. Although there was no difference between benign and malignant nodules, the response of adenylate cyclase to TSH was greater in the benign lesions. These studies demonstrate that nonfunctioning thyroid nodules, both benign and malignant, have a specific defect in iodide transport that accounts for their failure to accumulate radioactive iodide in vivo. In benign nodules, iodide organification was increased by TSH while no such effect was found in three of four malignant lesions, suggesting additional biochemical defects in thyroid carcinomas.

Analytical subcellular fractionation of rat pituitary homogenates, with special reference to prolactin proteolysis by lysosomes.

Prolactin proteolysis by rat pituitary homogenates was assayed by measuring the release of trichloroacetic acid-soluble peptides from 125I-labelled rat prolactin. There was a distinct optimum at pH 4.3, with only trace amounts of activity at neutral and alkaline pH. Rat pituitary homogenates were subjected to analytical subcellular fractionation by sucrose density gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their respective marker enzymes, including: cytosol (lactate dehydrogenase); plasma membrane (5'-nucleotidase); lysosomes (N-acetyl-beta-glucosaminidase, beta-glucuronidase); mitochondria (particulate malate dehydrogenase); endoplasmic reticulum (neutral alpha-glucosidase); prolactin granules (radioimmunoassayable prolactin). Acid prolactin protease had a similar distribution to the lysosomal marker enzymes. A localisation of the activity to lysosomes was confirmed by subcellular fractionation experiments in which the lysosomes were selectively disrupted with low concentrations of the membrane perturbant, digitonin. Experiments with specific inhibitors of the lysosomal cathepsins indicate that both cathepsins B and D are implicated in pituitary prolactin proteolysis.

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases.

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10-1.15 g . cm-3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5'-nucleotidase, had an apparent Km for 4-methylumbelliferyl phosphate of approx. 59 microM, and was inhibited by levamisole. The high-density (1.20-1.25 g . cm-3) peak was resistant to levamisole-inhibition, had an apparent Km of approx. 30 microM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.

Insulin resistance and insulin deficiency in diabetic retinopathy of non-insulin-dependent diabetes.

To assess the role of insulin resistance and insulin deficiency in the pathogenesis of diabetic retinopathy in non-insulin-dependent diabetes mellitus, 13 patients with and 12 patients without retinopathy were studied. The glucose clamp technique was used to measure insulin resistance and insulin response to glucose. During the euglycemic clamp, at comparable steady-state levels of glucose and insulin, the mean glucose infusion rate, which indicates the rate of glucose utilization, was lower in the retinopathy group than in the nonretinopathy group (6.1 +/- 0.5 versus 8.1 +/- 0.7 mg . kg-1 . min-1, P less than 0.02). Growth hormone (GH) concentrations were higher in the retinopathy group 8.4 +/- 2.5 versus 2.5 +/- 0.7 microIU/ml, P less than 0.05), but they did not correlate significantly with insulin resistance, expressed as mean glucose turnover. During the hyperglycemic clamp (+7 mmol/L above the fasting plasma glucose), the insulin response in the two groups of diabetics was similar. Increased insulin resistance represents an additional factor, which together with other factors, may be important in the pathogenesis of diabetic microvascular complications.

Effects of bromocriptine on pituitary organelle marker enzyme activities in lactating and postlactating rats: selective activation of lysosomal prolactin proteolytic activity.

Effects of the dopamine agonist 2-bromo-alpha-ergocryptine (bromocriptine) on plasma and pituitary PRL and enzyme activities in lactating and postlactating rats have been investigated. Lactating rats which had been suckling their young for 3 days were given a single sc injection of bromocriptine or solvent. The treated and control animals were divided into 2 further groups. One group (lactating rats) was permitted to suckle their pups for a further 12 or 24 h; the young were removed from the other group (postlactating rats). Homogenates were prepared from the anterior pituitaries and assayed for organelle marker enzyme activities. When 0.5-500 micrograms bromocriptine were administered to lactating rats for 24 h, pituitary PRL was increased by all doses, but only the 500-micrograms dose significantly reduced plasma PRL. Total protein was unchanged, lysosomal acid PRL proteolytic activity increased 8-fold, N-acetyl-beta-glucosaminidase and beta-glucuronidase (lysosomes) were unchanged, acid phosphatase (lysosomes and endoplasmic reticulum) was increased by three of four doses, 5'-nucleotidase and alkaline phosphatase (plasma membrane) were increased 4-fold, neutral-alpha-glucosidase (endoplasmic reticulum) and malate dehydrogenase (mitochondria) were unchanged, and catalase (peroxisomes) was significantly increased. Bromocriptine (500 micrograms) administration to lactating and postlactating rats for 12 and 24 h significantly decreased the pituitary DNA but not the total protein content of the pituitaries in all animals. The lysosomal acid PRL proteolytic activity and the lysosomal enzyme activities, N-acetyl-beta-glucosaminidase and beta-glucuronidase, were increased by suckling withdrawal alone. Acid PRL proteolytic activity was further increased (to 18-fold) by coadministration of bromocriptine, whereas the increase in the activities of the other lysosomal marker enzymes was blocked. Malate dehydrogenase activity (mitochondria) was also increased by litter removal and blocked by bromocriptine. The activity of the plasma membrane markers 5'-nucleotidase and alkaline phosphatase were increased by litter removal, and bromocriptine further increased both enzyme activities. The activity of neutral-alpha-glucosidase (endoplasmic reticulum) was unchanged by any treatment. The results demonstrate that bromocriptine produces significant changes in the activities of lysosomal marker enzymes, particularly acid PRL proteolytic activity, as well as marker enzymes of plasma membranes and other organelles in pituitaries of lactating and postlactating rats.

Persistent pancreatic glucagon but not insulin response to arginine in pancreatectomized dogs.

Effects of total pancreatectomy on plasma glucagon, insulin and glucose responses to arginine were determined in 5 dogs. Portal vein and femoral artery samples were obtained in response to an arginine infusion (10 g/30 min) prior to, 1 h, 1 day and 1 week after pancreatectomy. Glucagon was measured using pancreatic-specific antiserum 30K (Unger, Dallas). Before pancreatectomy arginine significantly increased portal vein glucagon from 373 plus or minus 36 to 595 plus or minus 31 pg/ml and femoral artery levels from 233 plus or minus 28 to 342 plus or minus 74 pg/ml. Portal vein and femoral artery insulin concentrations of 74 plus or minus 21 and 17 plus or minus 3 muU/ml increased significantly to 173 plus or minus 64 and 31 plus or minus 7 muU/ml. Glucose levels did not change. One h after pancreatectomy, portal vein glucagon decreased to 121 plus or minus 15 pg/ml but increased to 230 plus or minus 42 pg/ml after arginine. Elevated blood glucose and the necessity for insulin treatment established the adequacy of pancreatectomy. Furthermore portal vein insulin levels were undetectable and unresponsive to arginine or a combination of glucose, glucagon, and tolbutamide 1 week after pancreatectomy. One day after pancreatectomy arginine significantly increased portal vein glucagon from 343 plus or minus 42 to 776 plus or minus 152 pg/ml. One week after pancreatectomy basal glucagon values were 374 plus or minus 30 in the portal vein and 360 plus or minus 49 in the femoral artery and responded to 1226 plus or minus 641 and 825 plus or minus 270 pg/ml, respectively, with arginine. Chromatography of plasma from one pancreatectomized dog on Sephadex G-50 after arginine stimulation revealed that much of the material cross-reacting with antibody 30K was eluted from the column earlier than either 125I-insulin or 125I-glucagon. In contrast, peak glucagon activity in plasma obtained from a normal human given arginine eluted from the column between the peak of 125I-insulin and 125I-glucagon; glucagon added to human plasma also was recovered in this same area between the 125I-insulin and 125I-glucagon peaks. These results suggest that some of the material that reacted with 30K antibody and which increased after pancreatectomy in response to arginine has a molecular weight greater than pancreatic glucagon. At autopsy no pancreatic tissue could be identified. Thus, after pancreatectomy, validated by absent insulin responses, the glucagon response to arginine was normal or increased. Since arginine is not thought to increase intestinal glucagon-like immunoreactive material, the source and nature of the material measured as glucagon after pancreatectomy is unknown, but may be important to any understanding of plasma glucagon measurements.

Follicle-stimulating hormone-secreting pituitary adenomas.

This retrospective study concerns 40 patients with an apparently nonsecretory pituitary adenoma who were operated on during an 11-yr period from 1971 to 1981. Among them, 6 men had elevated serum FSH levels. LH levels were normal in 5 and slightly elevated in 1. Testosterone levels were low in 2 patients and within normal limits in 2 others. Sexual impotency had developed from 6 months to 1 yr before surgery in all patients. Primary hypogonadism could be eliminated on clinical grounds (recent onset of hypogonadism, previous fertility of 5 of the 6, and postoperative improvement). After transsphenoidal adenomectomy, FSH levels returned to normal values in all, and clinical recovery occurred in most patients. Tumor tissue obtained at operation stained positively for the gonadotropins, but was negative for other pituitary hormones in all patients. The most probable explanation for these findings was that the tumors were responsible for the elevated FSH secretion. This explanation is supported by the immunocytochemical identification of gonadotropin-containing cells in the tumors. We conclude that these 6 men from a series of 40 patients who presented with pituitary tumor but no GH, PRL, or ACTH hypersecretion had primary gonadotropinomas.

Vasoactive intestinal peptide stimulates adrenocorticotropin release from human corticotropinoma cells in culture: interaction with arginine vasopressin and hydrocortisone.

The effect of vasoactive intestinal peptide (VIP), cholecystokinin octapeptide (CCK), bombesin, arginine vasopressin (AVP), and hydrocortisone (HC) on ACTH release from human corticotropinoma cells in culture has been studied. Tumor tissue was obtained from 6 patients with pituitary corticotropinomas. Eleven to 21 cultures yielding 0.7-2.0 X 10(6) cells/culture, were obtained from each tumor and maintained for periods of 4 weeks to longer than 6 months. VIP (500 ng/ml) significantly (P less than 0.005) stimulated ACTH release from all tumors studied, and a dose (5-500 ng/ml)-response effect was observed in 3 of 5 tumors. Stimulation by VIP was seen at 2,4, and 24 h and was maximal at 4 h. CCK and bombesin were without effect on ACTH release from 4 tumors studies at 4 h. AVP (1-10 mU/ml) stimulated ACTH from 4 tumors studied at 60 min or 4 h. Coincubation of cultures with VIP (50-500 ng/ml) and AVP (1-10 mU/ml) resulted in at least an additive effect. HC (100 ng/ml) significantly (P less than 0.025) inhibited basal ACTH secretion from 2 of 4 tumors at 4 h and from 3 of 4 (P less than 0.005) at 24 h. Simultaneous coincubation of cultures with VIP (50 ng/ml) and HC (100 ng/ml) resulted in an attenuation or blockade of the VIP-stimulated ACTH release, whereas prior incubation of cultures with HC for 28 h before exposure to VIP did not. The results demonstrate that VIP is a potent ACTH secretagogue from human corticotropinoma cells in culture; its effects are additive to those of AVP and modulated by HC.

Adrenocorticotropin and lipotropin secretion by dispersed cell cultures of a human corticotropic adenoma: effect of hypothalamic extract, arginine vasopressin, hydrocortisone, and serotonin.

Basal and modulated secretion of ACTH and lipotropin (LPH) by cultures of trypsin-dispersed cells of a biopsy of a human corticotropic adenoma have been examined. ACTH secretion was detectable throughout the period of culture (13 days) but declined steadily from an initial production rate of 238 +/- 124 ng/3 X 10(5) cells/12 h. The time course of secretion showed a slower phase over the first 4 h, with increases up to 12 h. An extract of rat stalk median eminence caused a significant (P less than 0.005) dose-dependent increase in both ACTH and LPH secretion during 30 min. The patterns of response for ACTH and LPH were very similar; both exhibited a decline in the basal release of peptide subsequent to the period of stimulation. The addition of hydrocortisone (0.2 micrograms/ml) did not suppress basal ACTH secretion during 30 min but significantly (P less than 0.05) inhibited stimulation produced by rat stalk median eminence extract. Arginine vasopressin (dose range, 1-9 ng/ml) significantly (P less than 0.025) stimulated both ACTH and LPH secretion during 30 min. The patterns of response were again very similar. Serotonin (dose range, 0.01-10 micrograms/ml) did not affect ACTH secretion during incubations of 30 min to 4 h. The results obtained with the cell cultures of a human corticotropic cell adenoma concur with in vivo findings of incomplete autonomy of secretion, parallel secretion of ACTH and LPH in response to provocative stimuli, and suppression by corticosteroids. The technique has potential for exploring the cellular mechanisms controlling secretion by human corticotropic adenomas as well as the nature of the hormones produced.

Long term follow-up of patients with Cushing's disease treated by interstitial irradiation.

The first 86 patients with Cushing's disease treated with interstitial irradiation (by needle implantation) as the sole therapy were reviewed. In the 82 patients who were reassessed 1 yr after treatment 63 (77%) achieved remission. This study comprises the outcome and complications in the 54 patients who had a remission and whom we were able to follow. The follow-up period ranged from 3-26 yr (mean, 10.5) from the time of remission. No instance of clinical or radiological relapse has occurred. Of these 54 patients, yttrium-90 alone was used in 32, of whom 12 (37%) required corticosteroid or T4 replacement therapy in a mean time of 3.5 months; in 7 of these 12 we elected to give an ablative dose. Gold-198 alone was used in 15 patients, of whom 7 (47%) developed hypopituitarism in a mean time of 76 months. Both isotopes were used in 7 patients. A diurnal serum cortisol rhythm was found in 28 of the 31 patients who were not receiving corticosteroid therapy. In 5 of the 7 patients with an initially abnormal pituitary fossa, serial radiological studies revealed remodelling in 3. There have been no complications in the last 17 years. Pituitary implantation with yttrium-90 is an effective alternative to transsphenoidal hypophysectomy, with a high remission rate, no recurrence (as yet), no operative complications, and avoidance of hormone replacement in the majority.

Bombesin: action on gut hormones and calcium in man.

Bombesin, a peptide with widespread biological actions, has been demonstrated in human tissues by immunological methods. To investigate its effect in man, synthetic bombesin was infused at low doses in six male volunteers. Bombesin at 2.4 pmol kg-1 min-1 produced significant rises in plasma insulin, glucagon, pancreatic polypeptide, gastrin, cholecystokinin, motilin, glucose-dependent insulinotropic polypeptide, neurotensin, enteroglucagon, vasoactive intestinal polypeptide, and serum calcium. In contrast, bombesin caused a profound fall in parathyroid hormone levels and reduced plasma glucose concentrations. A late rise in plasma calcitonin was also observed. Bombesin had no significant effect on the pituitary hormones, TSH, GH, PRL, or cortisol. No hormonal changes or alterations in calcium were noted during saline infusions. Bombesin has a marked stimulatory effect on gastrointestinal hormones, which is unique and opposite to the effect of somatostatin, a potent inhibitor of gut hormone release. Bombesin also influences calcium-regulating hormones, either directly or through its action on gut hormones. The bombesin concentrations achieved with the dosages used were low enough to indicate a possible physiological role for the endogenous peptide.

Purification and properties of the subunits of human pituitary follicle-stimulating hormone.

Highly purified human pituitary FSH was partially dissociated by treatment with 8M-urea, and alpha- and beta-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated alpha-subunit had less than 1%, and beta-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with thepurified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the beta-subunit.

Bromocriptine treatment of acromegaly.

Bromocriptine at a dose of 7.5-30 mg/day was given to 12 acromegalics for 6 mo. Mean serum growth hormone (GH) levels during a glucose tolerance test (GTT) were significantly lowered by the drug. In four patients the serum GH response during a GTT was suppressed to normal (i.e. less than or equal to 5 mlU/liter). If bromocriptine had not brought the serum GH response to a GTT to normal at a dose of 20 mg/day, this effect was not achieved by raising the dose to 30 mg/day. Bromocriptine was effective for the duration of treatment. On discontinuing therapy there was an increase in serum GH levels. No obvious clinical changes in the acromegalic features were noted. One patient with impaired glucose tolerance and one with established diabetes had normal glucose tolerance while on bromocriptine and another two patients with impaired glucose tolerance showed no obvious changes while on the drug. Side effects were minor. X-rays of the pituitary fossa before starting and at the end of treatment showed no significant change. We conclude that although bromocriptine is the most promising form of medical treatment for acromegaly to date, it is fully effective only in a minority of patients.

Serum T4, T3, and TSH levels in primary hypothyroidism during replacement therapy with thyroxine.

Forty-five patients with primary hypothyroidism were studied during the first 4 to 36 months of replacement therapy with thyroxine. All became clinically euthyroid (23 patients while taking 0.1 mg/d, 14 patients while taking 0.15 mg/d, 7 patients while taking 0.2 mg/d, and 1 patient while taking 0.25 mg/d) over a period of three to six months. The patients were then divided for data analysis into two groups. Group I had normal serum T3 levels, normal (or elevated) serum T4 levels, and normal serum TSH levels. Group II had normal serum T3 levels, normal (or elevated) serum T4 levels, but high serum TSH levels. Group II was subdivided further into a group of 13 patients (group IIa) whose dose of thyroxine was deliberately increased until the serum TSH level was normalized; five of these patients became clinically and biochemically hyperthyroid. Group IIb consisted of eight patients with normal serum T3 and T4 levels and high serum TSH levels who were followed up without attempting to normalize their serum TSH levels. None became thyrotoxic, and their serum TSH levels showed little change. These findings suggest that serum TSH levels alone are not adequate to assess the required dose of thyroxine replacement therapy.

Deoxycorticosterone and aldosterone excretion in Cushing's syndrome.

We have used specific radioimmunoassays to measure urinary free deoxycorticosterone (DOC) and total aldosterone excretion in 15 patients with Cushing's syndrome. Free DOC excretion was increased in 6 of 8 patients with pituitary-dependent adrenal hyperplasia, in 2 of 3 patients with adrenal adenoma, and in all 4 cases with adrenal carcinoma. The most marked increase was noted in the adrenal carcinoma cases. Aldosterone excretion was high, normal, or low in each of the three types of Cushing's syndrome. The free DOC response to metyrapone in Cushing's syndrome due to adrenal adenoma was markedly different from that in patients with adrenal hyperplasia (pituitary-dependent) and may serve as a test to ascertain the etiology of the disorder. Correlations of free DOC and aldosterone excretion with free cortisol excretion, and their responses to the administration of metyrapone and dexamethasone were compatible with ACTH dependency in adrenal hyperplasia, autonomous production of steroids in adrenal adenomas and a chaotic steroidogenesis in adrenal carcinoma.

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.

Production and storage of (125-I) thyroxine and (125-I) triiodothyronine of high specific activity.

High specific activity [125-I] triiodothyronine and [125-I] thyroxine have been produced regularly by the chloramine T radioiodination method. Simultaneous production of [125-I] triiodothyronine and [125-I] thyroxine is usual when diiodothyronine or triiodothyronine are employed as the starting materials. Specific activities reached vary with the starting compound (diiodothyronine, triiodothyronine, thyroxine) used, as both substitution and, less readily, exchange of iodine atoms take place. Starting with diiodothyronine specific activities of approximately 2400 and 5200 Ci/g were achieved for [125-I]triidothyronine and [125I] thyroxine, respectively, and, similarly, specific activities of approximately 1200 and 4000 Ci/g for [125I] triiodothyronine and [125I]-thyroxine, respectively, were reached when triiodothyronine was the starting material. [125-I] Triiodoacetic acid and [125I] tetraiodoacetic acid have been produced in the same way from triiodoacetic acid. By column chromatography on Sephadex G-25 (fine), eluting with alkaline phosphate buffer, good separation of the radioiodinated products has been readily achieved. Studies on the stability of the radioiodinated hormones showed that 50% methanol, ethanol, propanol and propylene glycol were all equivalent as preserving agents and, further, that the stability of the radioiodinated hormones was linearly related to the concentration of these preserving agents.