R. D. Hesch

Human thyrotropin receptor gene: expression in thyroid tumors and correlation to markers of thyroid differentiation and dedifferentiation.

Human thyrotropin (TSH) receptor steady-state transcript levels were analyzed by Northern blot analysis in thyroids of patients with thyroid carcinoma, with hyperfunctioning adenoma and in normal controls. In control tissue and benign tumors expression levels of TSH receptor mRNA were high whereas in anaplastic carcinomas no normal TSH receptor mRNA was detected. In papillary and follicular tumors it varied from normal to markedly reduced levels. Thyroid peroxidase (TPO) and thyroglobulin (Tg) mRNA were strongly expressed in normal tissue and in hyperfunctioning adenomas but were completely lost in all anaplastic tumors. In papillary tumors expression of TPO and Tg mRNA varied from normal to a complete loss of expression of either TPO, Tg or both. Tg and TPO steady-state expression did not correlate to TSH receptor transcript levels. C-myc mRNA was highly expressed in anaplastic carcinomas, very variable in normal controls and in differentiated thyroid tumors and low in hyperfunctioning adenomas. In summary, TSH receptor mRNA is persistently expressed in all differentiated thyroid tissues and tumors but lost in undifferentiated carcinomas. Its persistence far along the transformation pathway further supports the concept that this gene which inserts the thyrocytes in the physiological regulatory network is almost constitutively expressed in this cell.

Increase of vertebral density by combination therapy with pulsatile 1-38hPTH and sequential addition of calcitonin nasal spray in osteoporotic patients

SummaryCombination therapy with the biologically active (1–38) human parathyroid hormone peptide and calcitonin using pulsatile and sequential activation of the skeleton for 14 months in patients with low-turnover osteoporosis resulted in an increase in trabecular bone mass. These favorable responses were observed without any significant changes in cortical (forearm) bone mass content.

Results of a stimulatory therapy of low bone metabolism in osteoporosis with (1-38)hPTH and diphosphonate EHDP. Protocol of study I, osteoporosis trial Hannover.

SummaryIn contrast to prevention, the therapy of manifest osteoporosis remains a clinically significant problem. So far all therapeutic attempts have yielded unsatisfying results. For this reason we have tried to achieve a positive bone balance by sequential stimulation and inhibition of the osseous metabolism. The therapy consisted of six 14-day courses with 400 units (1–38)hPTH per day and, in addition, starting with the 2nd week of PTH therapy, EHDP 5 mg per kg body weight per day for a total of 2 weeks. Already the initial therapeutic course resulted in a stimulation of decreased bone metabolism which could be documented by an increase in the calcium-47 accretion rate (six patients). An increase of the alkaline phosphatase could be noted (four patients); this, however, did not correlate with the calcium accretion. A positive calcium balance could, nonetheless, only be attained in four of eight patients within this period, while neither the alkaline phosphatase nor the kinetics would allow a prediction of this effect. Changes of the balance coincided with equal changes in the net calcium absorption. The urinary calcium excretion increased temporarily during the therapeutic phase. We were not able to detect an influence on the vitamin D metabolites. Histomorphometric studies did not demonstrate an increase in bone mass in the iliac creast after six therapeutic courses. Nevertheless, progressive deformations of vertebral bodies did not occur. We conclude that already after 2 weeks this therapeutic concept can lead to a stimulation of bone metabolism.

Human Parathyroid Hormone: Antibody Characterization

PTH antibodies were raised in two sheep (S 469 and S 478) by immunizing with porcine and bovine parathyroid extracts. Both antisera were characterized with various PTH preparations and fragments. Both antisera cross react with human, bovine and porcine PTH, one antiserum also binds rat PTH. Region specificity could be attributed to the mid region of the PTH molecule with particularly high affinities of both antisera for the fragment 44-68 hPTH. S 478 has similarly high affinity for intact hormone (affinity constants 0.6 x 10(13) l/mol), while S 469 has much higher affinity for the 44-68 fragment (affinity constant 0.84 x 10(13) l/mol) than for intact hormone. The antibodies are useful not only for clinical radioimmunoassay, but also for experimental work. They have been distributed to many laboratories.

Time series prediction of plasma hormone concentration. Evidence for differences in predictability of parathyroid hormone secretion between osteoporotic patients and normal controls.

Recent evidence links osteoporosis, a disease of bone remodeling, to changes in the dynamics of parathyroid hormone secretion. We use nonlinear and linear time series prediction to characterize the secretory dynamics of parathyroid hormone in both healthy human subjects and patients with osteoporosis. Osteoporotic patients appear to lack the periods of high predictability found in normal humans. Our results may provide an explanation for why an intermittent administration of parathyroid hormone is effective in restoring bone mass in osteoporotic patients.

Pulsatile secretion of parathyroid hormone and its action on a type I and type II PTH receptor: A hypothesis for understanding osteoporosis

Parathyroid hormone (PTH) acts on bone to modulate bone turnover [1] but we still do not know how it exerts its action at the organ-specific and cellular milieu. In the last few years we have learned much about the hierarchical order of hypothalamic-pituitary networks which modulate the function of peripheral glands in order to express their biological signals at the organ and tissue level. It became obvious that this dynamic regulation occurs through pulse amplitude and frequency modulation of hormonal signals transporting biological information at various levels in the hypothalamus, pituitary, and peripheral glands. This has been demonstrated for gonadotropins [2], growth hormone [3], thyroidstimulating hormone (TSH) [4], and ACTH [5]. The expression of the biological function of these hormonal systems depends ultimately upon the pulse amplitude and frequency modulation of the hormone receptor unit. It has been known for many years that the dynamic up and down regulation of the hormone receptor unit by the delivery of dynamically encoded hormone signals to the hormone receptor unit will determine the subsequent signal transduction to the main cellular pathways [6]. Two main such pathways are to be considered: a receptor type I-operated pathway which acts through stimulation of adenylate cyclase to produce cAMP and subsequent phosphorylation of regulatory cellular proteins by protein kinases [7, 11]; and a second pathway operated by type II receptors wherein, after binding of the hormone to its receptor, a regulatory Gp-protein is activated in a way that inositolphosphates are cleaved by phospholipase-C (PLC) to produce inositoltriphosphate (IP-3) and diacylglycerol (DAG) [8]. IP-3 acts to control intracellular-free calcium either by intracellular exchange of calcium between various corn-

AUTOANTIBODIES TO PARATHYROID HORMONE RECEPTOR

Autoantibodies which block the binding of parathyroid hormone to membrane receptors for the hormone were detected in the sera (especially in the IgG fraction) of 49 out of 50 uraemic patients with secondary hyperparathyroidism (patients with high levels of C-regional parathyroid hormone). These antibodies are species-specific. Their presence in the serum in unaffected by dialysis. Inhibition of binding appears to be related to the rise in C-regional parathyroid-hormone levels and the duration of uraemia. The production of cyclic adenosine monophosphate by parathyroid-hormone-stimulated adenyl cyclase was reduced by the blocking antibodies. The findings show that secondary hyperparathyrodism in uraemia is another example of a receptor-antibody disease, but it is not known whether the antibodies act by modifying the affinity of the receptors for the hormone or by reducing the concentration of receptors available.

RAPID DESENSITIZATION OF PARATHYROID HORMONE DEPENDENT ADENYLATE CYCLASE IN PERIFUSED HUMAN OSTEOSARCOMA CELLS (SAOS-2)

The pulsatile but not the continuous application of parathyroid hormone (PTH) increase bone mass in vivo. To study the effects of intermittent hormonal administration on bone-derived cells in vitro, we established a perifusion system using the human osteosarcoma cell line SaOS-2. Cells were grown in suspension culture attached to collagen beads and were then loaded into a 3 ml syringe for perifusion experiments. The application of PTH(1-34) resulted in a dose-dependent increase of cAMP release by SaOS-2 cells into the effluent medium. Cyclic AMP accumulation was rapidly desensitized by approx. 80% after 30 min of continuous exposure to PTH(1-34) (10(-7) M), while cells remained responsive to forskolin. The recovery of PTH responsiveness required at least 2 h of hormone-free perifusion. Desensitization in the experimental setting was dose-dependent (EC50 = 1 x 10(-10) M PTH(1-34)). Neither 8Br-cAMP (2 x 10(-4) M) nor PMA(1 x 10(-7) M) had an effect on the PTH(1-34)-induced desensitization of the adenylate cyclase. Radioreceptor assays showed that [125I]-[Tyr36]hPTHrP(1-36)amide binding to SaOS-2 cells was decreased by 60-70% by PTH(1-34) (1 x 10(-6) M), bPTH(1-84) (1.8 x 10(-6) M) and bPTH(3-34) (2 x 10(-6) M), whereas 8Br-cAMP (2 x 10(-4) M) had no effect on radioligand binding. PMA (1 x 10(-7) M) appeared to slightly increase [125I]PTHrP binding. This observation is consistent with a small (3-fold) increase in PTH-induced cAMP release as a result of PMA pre-treatment. Receptor internalization was dose-dependent EC50 = 3 x 10(-7) M PTH(1-34)). The maximal effect occurred after 10-30 min and was largely reversible within 2 h. Monensin (3 x 10(-5) M) inhibited the recovery from receptor internalization. We conclude that a perifusion system using SaOS-2 cells is a suitable model to study the effect of discontinuous application of PTH on cAMP release. A rapid, homologous desensitization of PTH(1-34) stimulated cAMP accumulation has been observed that does not appear to involve protein kinase A or C.

Direct radioimmunoassay for human atrial natriuretic peptide (hANP) and its clinical evaluation

A direct radioimmunoassay for the rapid and accurate detection of human ANP from unextracted plasma is described. The sensitivity was approximately 50 pg/ml, respectively 2.5 pg/tube, the intra-assay variation 4%, and the inter-assay variation less than 12%. Rat ANP (1-28, 5-25, 5-27 and 5-28), oxydized and reduced hANP as well as plasma samples from various patients run in parallel to the 1-28 hANP standard curve. These findings imply, that the antibody primarily recognizes the mid-region (amino acids 6-25) of the intact ANP, that the C-terminal portion further increases the immunoreactivity, and that circulating plasma hANP is reliably measured. Plasma hANP ranged from 50-166 pg/ml (mean +/- SD: 98.3 +/- 44.6) in healthy individuals, there was no significant difference between samples were drawn in upright or lying position, the apparent half-life of injected hANP was 5.65 minutes. Patients with liver cirrhosis revealed significantly higher hANP levels of 244.5 +/- 173.5 pg/ml. Patients with various forms of cardiac disease had hANP concentrations ranging from 50 to 1744 pg/ml, depending at least partially on the right atrial pressure. No difference was observed if the samples were drawn from either right or left intracardial locations. Our findings with this system demonstrate that hANP is reliably measured even without prior extraction.

Radioimmunoassay of thyroxine-binding globulin in human plasma.

A highly specific and precise radioimmunoassay for thyroxine-binding globulin (TBG) has been developed. Crossreactivity with albumin and prealbumin was excluded. The normal range in young adults was 0.97 mg/100 ml. In childhood TBG was elevated (1.34 mg/100 ml) also in old age (1.28 mg/100 ml). In normals and in childhood there was a good correlation of TBG with thyroxine (T4). T4 did not correlate with TBG with regard to age. Triiodothyroxine (T3) did not correlate with TBG in any group. T4 and T3 exhibited a progressive decrease with age. No correlation between age and TBG was found. In mild thyrotoxicosis (T3 : 4.5 ng/ml, with normal T4 and negative TRH-test) TBG was slightly increased (1.20 mg/100 ml), whereas in more severe hyperthyroidism (T3 : 6.4 ng/ml, T4 : 16.3 mug/100 ml) TBG concentration was not significantly different from normals. In hypothyroidism TBG was elevated (1.26 mg/100 ml). The conclusion from these data is that TBG does not follow the progressive decrease of T4 and T3 with age. Thus, age-dependent euthyroid ranges for thyroid hormone concentration, including TBG concentration, must be established for better clinical discrimination of thyroid function. Possible dependence of TBG on the nature of thyroid hormone concentration must be considered in the production and peripheral kinetics of thyroid hormones.