calcimycin and Hypothyroidism

The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.

Topics: Adenosine Triphosphate; Aminoquinolines; Animals; Ca(2+) Mg(2+)-ATPase; Calcimycin; Calcium; Calcium-Transporting ATPases; Fluorescent Dyes; Hypothyroidism; Ion Channels; Male; Muscles; Rats; Rats, Inbred Strains; Sarcoplasmic Reticulum; Spectrometry, Fluorescence

The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcimycin; Cell Line; Colforsin; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Hypothyroidism; Iodide Peroxidase; Phorbol Esters; Pituitary Neoplasms; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyroid Hormones; Thyrotropin-Releasing Hormone; Thyroxine; Trifluoperazine; Triiodothyronine; Triiodothyronine, Reverse

Postnatal development of glycogen phosphorylase activation by the cAMP-independent pathway was examined in isolated rat hepatocytes from control and propylthiouracil-treated congenital hypothyroid rat pups. At 5 days postnatum there was complete phosphorylase activation by beta-adrenergic stimulation, glucagon, and the calcium ionophore A23187, but no activation by alpha-adrenergic stimulation. Activation of phosphorylase by angiotensin or vasopressin was less than in hepatocytes from adult rats (P less than 0.01). At 28 days postnatum activation by all of these hormones was complete. In the propylthiouracil-treated group hormone responsiveness was similar to the control at 5 days postnatum. However, alpha-adrenergic (P less than 0.025), angiotensin, and vasopressin (P less than 0.05) activation was decreased at 28 days postnatum, and beta-adrenergic, glucagon, and A23187 activation was complete. The attenuated responses were restored by thyroxine replacement from 15 days postnatum. [32P]Pi incorporation into phosphatidylinositol by epinephrine and vasopressin in 28-day propylthiouracil-treated rats was lower than the control (P less than 0.01). We speculate that the diminished phosphorylase response of hepatocytes to alpha-adrenergic, vasopressin, or angiotensin stimuli in the early neonatal period could be related to low receptor numbers and the weaker phosphoinositide response during this period. Also, the depressed phosphorylase response to alpha-adrenergic, vasopressin, and angiotensin stimulation in congenital hypothyroidism at 28 days postnatum could be related to a decrease in number of plasma membrane receptors for these agonists.

Topics: Angiotensin II; Animals; Animals, Newborn; Calcimycin; Cyclic AMP; Dose-Response Relationship, Drug; Epinephrine; Female; Glucagon; Hypothyroidism; Liver; Phentolamine; Phosphorylase a; Phosphorylases; Pregnancy; Rats; Rats, Inbred Strains; Time Factors; Vasopressins

TSH responses to adrenergic agonists have been measured in 3-day monolayer cultures of euthyroid and hypothyroid male rat anterior pituitary (AP) cells. Responses were qualitatively similar in that (-)epinephrine and (-)norepinephrine had the same ED50 in each culture (ED50 = approximately 6 and 16 nM, respectively) and demonstrated the same alpha 1-adrenergic specificity. Hypothyroid cultures secreted approximately twice as much TSH per cell as euthyroid cultures over the 2-h experimental period. (-)Epinephrine produced a 95 +/- 8% (mean +/- SE) release of TSH relative to basal secretion in euthyroid cultures and only 62 +/- 7% release in the hypothyroid cultures (P less than 0.01). The comparable figures for (-)norepinephrine were 62 +/- 7% and 38 +/- 5%, respectively (P less than 0.05). In absolute terms, adrenergic agonists released the same amount of TSH from euthyroid and hypothyroid cultures. In contrast, TRH (and the Ca+2 channel ionophore A23187) released twice as much TSH from the hypothyroid cells as in the euthyroid cultures. Epinephrine-induced TSH release was significantly impaired (P less than 0.001) when either euthyroid or hypothyroid cells were cultured without thyroid hormones. In contrast, TRH-induced TSH release was enhanced (P less than 0.001) in the euthyroid cultures. [3H]Dihydroergocryptine [( 3H]DHE) was used to quantify alpha 1-adrenoreceptors on the same cell preparations as those used to derive the functional data (see above). Prazosin (1 microM) was used to define nonspecific binding of [3H]DHE. Specific binding to euthyroid cells had a Kd of 5.8 +/- 4 nM and a maximum binding capacity of 2.2 +/- 0.4 fmol/10(5) cells (n = 5). In parallel cultures of hypothyroid cells, the Kd (6.2 +/- 5 nM) was not significantly different, whereas the maximum binding capacity (1.4 +/- 0.3 fmol/10(5) cells) was significantly reduced (P less than 0.05). Adrenergic compounds showed a rank order of potency of prazosin greater than (-)epinephrine greater than or equal to (-)norepinephrine greater than or equal to yohimbine greater than clonidine against the binding of 5 nM [3H]DHE to euthyroid and hypothyroid cells. The amount of [3H]DHE binding per cell that each adrenergic compound was able to displace at saturating concentrations was less in hypothyroid cells than in euthyroid cells. There was no change in the ED50 values of these compounds in the same experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Calcimycin; Cells, Cultured; Epinephrine; Hypothyroidism; Isoproterenol; Kinetics; Male; Pituitary Gland, Anterior; Prazosin; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Thyroid Gland; Thyrotropin; Thyrotropin-Releasing Hormone; Yohimbine

Anterior pituitary cells from euthyroid and hypothyroid male rats have been cultured as monolayers for 3 days with or without 5 nM T3 and stimulated with either human pancreatic GH-releasing factor 1-40 (hpGRF), TRH, or the Ca2+ channel ionophore A23187. Basal GH secretion was reduced in the hypothyroid cultures (P less than 0.001) and basal TSH secretion increased (P less than 0.001). Culture with T3 increased GH secretion and intracellular GH content in euthyroid and hypothyroid cultures but suppressed TSH secretion with no effect on intracellular TSH content in either euthyroid or hypothyroid cultures. hpGRF released more GH from euthyroid [3.52 +/- 0.2 (SE) micrograms/6 h X 10(5) cells] than hypothyroid cultures of (0.17 +/- 0.01 micrograms/6 h X 10(5) cells, P less than 0.001) without a change in ED50 (approximately 0.02 nM). The reduction in hpGRF-induced GH release remained significant when corrected for the reduced intracellular GH content in the hypothyroid cultures. hpGRF-induced GH release also declined relative to A23187-induced GH release in hypothyroid cultures. Culture with 5 nM T3 doubled maximum hpGRF-induced GH release in euthyroid cultures and increased maximum release 10-fold in hypothyroid cultures without altering the ED50 of hpGRF action. In contrast, T3 suppressed TRH-induced TSH release in euthyroid cultures but was without effect on TRH-induced TSH release in the hypothyroid cultures. T3 did not effect the ED50 of TRH action (2-5 nM). In summary, hypothyroid rat anterior pituitary cells in culture have a reduced maximal GH response to hpGRF, but the same ED50. hpGRF activity can be partially restored by physiological concentrations of T3 in vitro.

Topics: Animals; Calcimycin; Cells, Cultured; Cytological Techniques; Growth Hormone; Growth Hormone-Releasing Hormone; Hypothyroidism; Male; Peptide Fragments; Pituitary Gland, Anterior; Rats; Thyrotropin; Thyrotropin-Releasing Hormone

Topics: Angiotensin II; Animals; Arginine Vasopressin; Calcimycin; Epinephrine; Female; Hypothyroidism; Kinetics; Liver; Liver Glycogen; Prazosin; Quinazolines; Rats; Rats, Inbred Strains

Topics: Adenosine; Adipose Tissue; Animals; Calcimycin; Calcium; Catecholamines; Hypothyroidism; Lipid Mobilization; Male; Rats

Adipocytes isolated from normal, hypothyroid, and hyperthyroid rats were characterized with respect to their lipolytic activity (assessed by glycerol release) and beta-adrenergic receptors (assessed by binding of (--) [3H]alprenolol). Fat cells from hypo- and hyperthyroid rats showed the same affinity (K = 1.4 X 10(10) M(-1) and binding capacity (N = 1.21 X 10(-13) mol/microgram DNA) toward alprenolol as those from normal animals. Adipocytes from hypothyroid rats were unresponsive to epinephrine in a concentration range of 0.1-10 micron, with moderate responses at higher concentrations; injection of T3 in hypothyroid rats restored lipolytic responsiveness of the adipocytes to normal levels. Quabain (1 mM) inhibited lipolytic responses to epinephrine by 40--45% in normal and hyperthyroid rats; the lipolytic increment due to the hyperthyroid state was uninfluenced by ouabain. The lipolytic refractoriness to epinephrine of hypothyroid adipocytes was restored to normal levels by theophylline (1 mM) or EGTA (1 mM); the theophylline and EGTA effects were not additive, suggesting that they stimulated lipolysis via a common mechanism. Epinephrine-induced lipolysis in all groups was progressively inhibited by increasing concentrations of Ca2+ in the medium. The Ca ionophore, A23187, showed a concentration-dependent inhibitory action. Theophylline (1mM) almost completely overcame the inhibitory action of the ionophore; in the presence of lower concentrations of theophylline, the inhibitory effect of the ionophore was least in hypothyroid and greatest in hyperthyroid fat cells. The findings suggest that the differences in the lipolytic response to epinephrine observed in hyperthyroid, euthyroid, and hypothyroid adipocytes are not due to alterations in the number or affinity of beta-adrenergic receptors nor to a membrane mechanism that might show differential ouabain sensitivity, but may be related to altered cellular Ca2+ concentrations which may indirectly alter cellular phosphodiesterase activity.

Topics: Adipose Tissue; Alprenolol; Animals; Calcimycin; Calcium; Epinephrine; Hyperthyroidism; Hypothyroidism; Kinetics; Lipid Mobilization; Male; Propylthiouracil; Rats; Receptors, Adrenergic, beta; Thyroid Gland; Triiodothyronine