Thyrotropin-releasing hormone (TRH) elevation of inositol trisphosphate and cytosolic free calcium is dependent on receptor number. Evidence for multiple rapid interactions between TRH and its receptor

Thyrotropin-releasing hormone (TRH) increases rapidly two potential intracellular signals, inositol trisphosphate (IP3) and free cytosolic calcium ([Ca2+]i), for stimulated prolactin release and synthesis in GH4C1 rat pituitary cells. We have examined the temporal relationships between TRH-enhanced formation of inositol phosphates and TRH-elevated [Ca2+]i. TRH-enhanced IP3 content was closely paralleled by the initial phase of TRH-elevated [Ca2+]i. To investigate receptor-effector coupling for these rapid actions of TRH, we examined their dependence on receptor number in five GH4C1 variant strains containing 0-2.6 X 10(5) receptor sites/cell. We found that receptor number (up to 1.7 X 10(5)/cell) was limiting for TRH-enhanced IP3 formation as well as for both the initial burst and plateau phases of TRH-elevated [Ca2+]i. The ED50 for rapid (5 s) TRH-stimulated IP3 formation was higher than for other sustained TRH actions in these cells, and we postulated that the initial TRH receptor interactions occur with rapid dissociation kinetics. To test this hypothesis, we performed rapid dilution experiments following a 1-s stimulation and found that TRH-stimulated IP3 formation decreased within 4 s of dilution and disappeared within 60 s at which time fresh TRH could restimulate IP3 formation. We conclude that receptor occupancy is the limiting step for TRH-stimulated IP3 formation and elevated [Ca2+]i and that maximal TRH action requires multiple rapid interactions between TRH and its receptor.     

Cross-talk between thyroid hormone receptor and liver X receptor regulatory pathways is revealed in a thyroid hormone resistance mouse model

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor beta (TR-beta) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-beta knock-out mice, generalized expression of a non-ligand binding TR-beta protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7alpha-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-beta and liver X receptor alpha (LXR-alpha), which both bind to a direct repeat + 4 (DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-beta antagonized induction of this promoter by LXR-alpha. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-beta. A mechanism for cross-talk, and potential antagonism, between TR-beta and LXR-alpha is proposed.     

The expression of the 35- and 67-kDa calcimedins is dependent on thyroid hormone

We have investigated the expression of the 35- and 67-kDa calcimedins and calmodulin during fetal and neonatal periods and in adulthood in rat liver, muscle, and brain. The 35- and 67-kDa calcimedin expression in liver and muscle increased during the perinatal period and correlated with the thyroid status of the developing rat. In fact, animals treated with thyroxine demonstrated a precocious appearance of the 35- and 67-kDa calcimedin in liver and muscle. Animals treated with methylthiouracil, an inhibitor of T4 and T3 synthesis, strongly suppressed the synthesis of the calcimedins in these tissues. Neither treatment influenced the levels of either the 35- and 67-kDa calcimedins in brain. In contrast, each tissue examined produced a unique pattern of calmodulin expression during development. None of the tissue calmodulin concentrations changed during hyper- or hypothyroid states. Collectively, these data support the concept that the intracellular calcium signal possesses multiple, independent molecular pathways of mediation. In addition, the variety of these pathways is influenced by hormonal preconditioning in that the cellular response to elevated cytosolic calcium is dependent upon the thyroid status of a tissue.