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ABSTRACT
The microheterogeneity of pituitary thyroid-stimulating hormone (TSH) is dependent on variations in the hormone's carbohydrate moieties. In this study, changes in the pattern of heterogeneity have been assessed by chromatofocusing, which separates the isospecies on the basis of their isoelectric points (pI). Rats (n = 6 per group) were either untreated or rendered hypo- or hyperthyroid by including in the drinking water either propylthiouracil (0·05% for 8 weeks) or thyroxine (T4; 4 mg/l for 6 weeks) before they were killed at 16 weeks. On autopsy, serum TSH and total T4 were (means±s.e.m.): 2±0·3 μg TSH/l and 64±5 nmol T4/l (control); <1 μg TSH/1 and 133±6 nmol T4/l (hyperthyroid); 58±6 μg TSH/1 and 32±6 nmol T4/l (hypothyroid). The pituitaries were individually homogenized and the TSH isoforms separated by chromatofocusing over a pH range of 7–4. Fractions were assayed for TSH by radioimmunoassay. TSH from the control group was distributed into seven major peaks with pI values of (means ±s.e.m., n=6) 6·9±0·1, 6·6±0·1, 6·2±0·1, 5·8±0·1, 5·5±0·1, 5·2±0·1 and 4·8±0·1; 7±3% of the TSH had a pI of <40. Six peaks of TSH were conserved in the hypothyroid group (with pI values of 6·8±0·1, 6·5±0·1, 6·2±0·1, 5·8±0·1, 5·4±0·1 and 5·2±0·1), and 11±4% of the hormone had a pI of <40. In contrast to the other two groups, only one major peak (with a pI of 5·8±0·1) was detected in the pituitaries from the hyperthyroid group; 13 ± 5% of the TSH had a pI of <40. In the pH range of 5·5– 60, the per cent distribution of TSH was 58±15 (hyperthyroid) compared with 17 ± 3 (hypothyroid) and 22±3 (euthyroid). Above pH 6, only 25±13% of the TSH (hyperthyroid) was present compared with 46±5% (hypothyroid) and 45±5% (euthyroid). Below pH 5·5, the per cent distribution of TSH was 19±5 (hyperthyroid), 37±5 (hypothyroid) and 35±3 (euthyroid). In conclusion, both hyper- and hypothyroidism are associated with changes in the composition of pituitary TSH. This change was most marked in the hyperthyroid group, where there was a selective loss of several isoforms of TSH.
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ABSTRACT
The relationship between the immunological and biological activities of thyroid-stimulating hormone (TSH) isoforms present in the three human pituitary preparations 68/38 (1st IRP), 80/558 (2nd IRP) and 63/14 (MRC Research Standard A) was investigated. The isoforms were separated by chromatofocusing. Six peaks of immunoactivity were detected in 80/558, with pI values (means±s.e.m.) of 6·6±0·1, 6·2±0·1, 5·9±0·1, 5·5±0·1, 5·2±0·1 and 4·9±0·1. Four peaks, with pi values of 6·8±0·1, 5·9±0·1, 5·5±0·1 and 5·2±0·1, were observed for 68/38. Standard 63/14 had five peaks, with pI values of 6·9±0·1, 6·4±0·1, 5·9±0·1, 5·4±0·1 and 4·9±0·1. For each standard, six fractions around the peak areas and at the top and bottom of the gradient were pooled and microconcentrated to <1·0ml. Microconcentrated TSH samples were assayed in three TSH bioassays based upon FRTL-5 thyroid cells, utilizing cyclic AMP accumulation, iodide and thymidine uptake as end-points and standard 80/558 as reference preparation. The more acidic forms of TSH showed a higher biological: immunological (B: I) ratio for cyclic AMP accumulation with, for example, 63/14 having a maximum of 3·7 (pI 4·9) and a minimum of <0·7 (pI 6·9). In contrast, the maximum and minimum B: I ratios for iodide uptake for 63/14 were 3·8 (pI 6·9) and <0·8 (pI 4·6), and for thymidine uptake, maximum and minimum ratios were 7·2 (pI 6·9) and 1·1 (pI 4·6) respectively. In conclusion, the acidic forms of TSH stimulated cyclic AMP accumulation more than the alkaline forms, whereas the opposite was shown for iodide uptake and thymidine uptake.
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ABSTRACT
pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO3− ions, FRTL-5 cells regulate intracellular pH (pHi) by an Na+/H+ antiport but HCO3−-dependent exchangers cannot operate. We have investigated pHi regulation (by microfluorimetry and the pH sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6′)-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO3− ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na+/H+ antiporter and the stilbene derivative disodium 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) for HCO3 −-dependent mechanisms), ionic substitution and by NH4 +/NH3 (10mm) acid loading. Basal pHi was lower in the presence (7·3±0·058, mean±s.d., n= 14) than in the absence (7·59±0·078, n=10) of HCO3 ions. In HCO3 −-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pHi of 7·35±0·06. This recovery was Na+-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO3 −-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na+ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na+ from HCO3 −-containing media acidified the cells (ΔpH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl− alkalinized the cells (ΔpH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na+ and presence of amiloride, alkalinization was still seen on the removal of Cl−, albeit at a diminished rate (i.e. ΔpH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pHi by two Na+-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na+-independent, Cl−/HCO3− mechanism.
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ABSTRACT
We have studied the effects of acute administration of tri-iodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to <1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80mM KC1, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150±56nM (mean ± SD, n=14) by 127±47nM on depolarization with 80mM KC1. Application of T3 (1-100nM) led to an increase (P<0.05) in [Ca2+]i (mean amplitude of 152±35nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when lOOnM T3 was applied (median and range; 6.8, 0-14 min) than when 1nM T3 was used (16, 7.0-56.1 min) (P<0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.
