Licorice - or more?

A 57 yr old man presented to endocrinology clinic with a six year history of poorly controlled hypertension which was treated with Metoprolol 200 mg/day and Enalapril 20 mg/day. He was asymptomatic but incidentally hypokalaemia was detected while having cholecystectomy, two years prior to his clinic appointment. He had never been on diuretics or laxatives. He was started on potassium supplements (120 mmol/d) and advised to increase dietary potassium by the surgical team. A detailed personal history revealed ingestion of 300-500 g licorice per day. Physical examination was unremarkable apart from increased blood pressure of 180/105 mmHg. Following the initial visit, his serum electrolyes (K+3.7 mmol/l) were normal with potassium supplementation and as were morning cortisol, ACTH, 11-deoxycortisol and plasma metanephrines. 17 OH-P, DHEAS and androstenedione were normal but testosterone was low. Morning ambulant aldosterone was slightly increased at 801 pmol/L and renin activity was undetectable. Urinary 24 h aldosterone excretion was significantly increased at 162 ng/24 h with normal cortisol and catecholamine excretion. Four weeks following advice to stop licorice, serum potassium decreased to 3.4 mmol/L despite continuous supplementation. Morning plasma aldosterone increased to 1 449 pmol/ml, renin activity remained undetectable but 24 h urine aldosterone excretion increased to 434 ng/24 h with a reduction in urinary cortisol excretion. Interestingly 17 OH-P and androstenedione levels, although within the reference range, were slightly higher compared to the levels whilst on licorice. Testosterone level had significantly increased to be within normal range. Abdominal imaging with US and MRI showed a 2.7 cmx2.2 cmx1.7 cm left adrenal mass. He underwent laparoscopic left adrenalectomy and histology confirmed aldosterone producing adrenal adenoma. Post-operatively his aldosterone and serum potassium levels normalized and he became normotensive without any antihypertensive medication.

Bioengineering of human thyrotropin superactive analogs by site-directed "lysine-scanning" mutagenesis. Cooperative effects between peripheral loops.

We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the alphaL3 loop of the common alpha-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent alphaL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the alphaL3 loop analog compared with both the alphaL1 loop analog and the hTSH-betaL3 loop analog was observed. We demonstrated that spatially distinct regions of the common alpha-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date.

Human thyroid-stimulating hormone: structure-function analysis.

This article provides the reader with an overview of methodological strategies to investigate structure-function relationships of human thyroid-stimulating hormone (hTSH). Various aspects of hTSH production, purification, and characterization described here in more detail are not only relevant to studies on other members of the glycoprotein hormone family, but also applicable to studies of other glycosylated proteins. Knowledge of structure-function relationships of specific hTSH domains is important for a better understanding of the molecular mechanisms of its action. New insights from such studies permit the design of glycoprotein hormone analogs with specific pharmacological properties and potential clinical applications.

A rational design strategy for protein hormone superagonists.

By combining evolutionary considerations, sequence comparisons and homology modeling we have designed recombinant human thyroid-stimulating hormone (hTSH) analogs with increased receptor binding and activity. The introduction of seven basic residues into the peripheral loops of hTSH resulted in up to a 50,000-fold increase in receptor binding affinity and 1300-fold increase in intrinsic activity. Such analogs are not only of potential clinical interest but can be tools to explore molecular aspects of conventional as well as nonclassical actions of glycoprotein hormones. These design strategies should be applicable to the development of novel analogs of other related hormones and growth factors with a variety of therapeutic and basic science applications, particularly for proteins that have undergone evolutionary decrease in bioactivity.

Generation of repetitive Ca2+ transients by bombesin requires intracellular release and influx of Ca2+ through voltage-dependent and voltage independent channels in single HIT cells.

In the present study, the bombesin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were investigated in single Fura-2 loaded SV-40 transformed hamster beta-cells (HIT). Bombesin (50-500 pM) caused frequency-modulated repetitive Ca2+ transients. The average frequency of the Ca2+ transients induced by bombesin (200 pM) was 0.58 +/- 0.02 min-1 (n = 121 cells). High concentrations of bombesin (> or = 2 nM) triggered a large initial Ca2+ transient followed by a sustained plateau or by a decrease to basal levels. In Ca(2+)- free medium, bombesin caused only one or two Ca2+ transients and withdrawal of extracellular Ca2+ abolished the Ca2+ transients. The voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (50 microM) and nifedipine (10 microM), reduced amplitude and frequency of the Ca2+ transients and stopped the Ca2+ transients in some cells. Thapsigargin caused a sustained rise in [Ca2+]i in the presence of extracellular Ca2+ while in its absence the rise in [Ca2+]i was transient. Verapamil (50 microM) inhibited the thapsigargin-induced increase in [Ca2+]i by about 50%. Depletion of intracellular Ca2+ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca(2+)-free medium caused an agonist-independent increase in [Ca2+]i when extracellular Ca2+ was restored, which was larger than in control cells that had been incubated in Ca(2+)-free medium for the same period of time. This rise in [Ca2+]i and the thapsigargin-induced increase in [Ca2+]i were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca2+ pool enhances Ca2+ influx through VDCC and voltage-independent Ca2+ channels (VICC). In conclusion, the bombesin-induced Ca2+ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca2+ transients. Intracellular Ca2+ is mobilized during each Ca2+ transient, but Ca2+ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca2+ response. Ca2+ influx in whole or part is activated by a capacitative Ca2+ entry mechanism.

Continuous vs. pulsatile administration of thyrotropin-releasing hormone (TRH) in the model of the chronically cannulated rat: long-term effects on thyroid function.

Thyroid-stimulating hormone (TSH) belongs to a family of glycoprotein hormones secreted by the anterior lobe of the pituitary gland in a pulsatile fashion. Although this pulsatile pattern of release has been characterized by several groups, its exact physiological relevance remains to be elucidated. We established a model of a chronically cannulated rat allowing us to apply quantified pulses of thyrotropin-releasing hormone intravenously and to monitor the biological response on the anterior pituitary as well as on the thyroid level. Serum rTSH, T3 and T4 levels were measured as functional parameters. During the five day infusion period, TSH increased under both continuous and pulsatile TRH stimulation, but this increase was only maintained following pulsatile TRH but not after continuous application. No differences between the continuous and the pulsatile infusion regimen could be observed in terms of TT3 release, whereas TT4 decreased following an initial stimulation under continuous application, but remained stimulated during pulsatile application. Our data indicate that the model of the chronically cannulated rat appears to be a valuable tool to study the impact of the temporal pattern of TRH/TSH on thyroid physiology.

Functional and morphological changes of the thyroid gland following 5 days of pulsatile TRH stimulation in male rats.

The aim of the present study was to evaluate in vivo the selective effects of a small increase in plasma TSH levels on thyroid function, proliferation and morphology. Chronically catheterized male Sprague-Dawley rats were stimulated i.v. over 5 days either with TRH (2 micrograms TRH in 100 microliters 0.9% (w/v) NaCl (TRH-P) or the NaCl carrier alone (P), both given as pulses every 2 h. Control groups were cotreated i.v. with 10 micrograms thyroxine (T4)/100 g body weight per day (TRH-P + T4) starting 2 days before pulsatile stimulation. TSH plasma levels were approximately doubled by TRH-P (P < or = 0.001), T4 plasma levels significantly increased (P < or = 0.001) but tri-iodothyronine plasma levels did not change compared with treatment with P. No significant changes between groups were found in thyroid weight and in intrathyroidal iodine content, but the percentage of 5-bromo-2'-desoxyuridine-labelled thyrocytes as a marker of proliferation in TRH-P-treated animals was significantly increased over P or TRH-P + T4 (P < or = 0.001). Ultrastructural analysis of the thyroid evaluated by electron microscopy revealed a significant increase in the number of lysosomes (P < or = 0.001). The size of the endoplasmic reticulum (ER) in relation to the cytoplasm was significantly increased when treated with TRH-P compared with P or TRH-P + T4 (P < or = 0.001). Post-embedding immunogold staining revealed Tg as a major product within ER cisternae. Immunogold labelling was moderate in controls and higher densities of gold particles were obtained in TRH-P-treated animals (P < or = 0.001). In conclusion, short-term pulsatile TRH stimulation increasing the plasma levels of immunoreactive TSH only twofold is capable of inducing hypertrophy of the thyrocytes by gross ultrastructural changes which are paralleled by an increase in circulating T4. These data underscore the dominant role of TSH on thyroid ultrastructure within the narrow boundaries of normal physiological regulation.

Effects of continuous and pulsatile administration of pituitary rat thyrotropin and recombinant human thyrotropin in a chronically cannulated rat.

Pulsatile secretion of thyrotropin (TSH) is a well characterized phenomenon in the human, yet only limited data are available from animal studies. We propose a combined model of chronic catheterization and suppression of endogenous TSH release in the rat allowing us to apply quantified pulses of different preparations of thyrotropin intravenously with minimal interference with endogenously produced hormone. Sprague-Dawley rats treated with drinking water containing 3,5,3'-triiodothyronine (T3) showed a significant 6-fold decrease of the rat TSH (rTSH) level after four days. Free thyroxine (FT4) levels were 9-fold below unsuppressed levels; FT4 response to exogenous TSH application above this suppressed baseline level was selected as an endpoint. Infusion studies compared pulsatile with continuous TSH administration. A low and a high dose of rTSH and recombinant human TSH (rec-hTSH) were administered. Levels of FT4 were compared after two and four days and responsiveness to a standard high bolus of rTSH 6 h after end of infusion was assessed. With regard to its potency for FT4 release, differences indicating a general trend as to superior efficiency of pulsatile TSH stimulation of the thyroid gland in comparison to continuous stimulation could be observed in all four experiments. More pronounced effects were seen after 2 and 4 days of high rTSH infusion and after 4 days of low rec-hTSH infusion. Comparison of FT4 responses to rTSH boli showed a 2- and 6-fold higher response in the pulsatile group compared to the continuous group after low and high rTSH infusion, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)