Muscarinic responses of gastric parietal cells

Summary Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 m) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC₅₀ of 13 m; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC₅₀ of 110 m; and by the M1/M3 antagonist, diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC₅₀ of 35nm. The three antagonists displayed equivalent IC₅₀ values for the inhibition of carbachol-stimulated production of¹⁴CO₂ from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 m La³⁺. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC₅₀ of 1,7nm, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (>30nm), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca] _i transient. Displacement of³H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca] _i response. Elevation of steady-state [Ca] _i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca] _i is necessary but not sufficient for acid secretion.     

The effect of propylthiouracil on thyroid-stimulating hormone-induced alterations in iodothyronine secretion from perfused dog thyroids

The aim of this study was to see whether the inhibitory effect of propylthiouracil on thyroidal secretion of 3,5,3′-triiodothyronine (T₃) and 3,3′,5′-triiodothyronine (rT₃) could be reproduced in intensively stimulated thyroids, and to elucidate whether an increase in the fractional deiodination of thyroxine (T₄) to T₃ and rT₃ during iodothyronine secretion might be responsible for the transient fall in the T₄/T₃ and T₄/rT₃ ratios in thyroid secretion seen in the early phase after stimulation of thyroid secretion.For this purpose T₄, T₃ and rT₃ were measured in effluent from isolated dog thyroid lobes perfused in a non-recirculation system using a synthetic hormone free medium. 1 mmol/l propylthiouracil induced a significant reduction in thyroid-stimulating hormone (TSH) stimulated T₃ and rT₃ release while the release of T₄ was unaffected. This supports our previous conclusion that T₄ is partially monodeiodinated to T₃ and rT₃ during thyroid secretion. Infusion of 1 mmol/l propylthiouracil for 30 min or 3 mmol/l propylthiouracil for 120 min did not abolish the transient fall in effluent T₄/T₃ and T₄/rT₃ induced by TSH stimulation. Thus, this phenomenon seems not to depend on intrathyroidal iodothyromine deiodinating processes.     

Studies on the involvement of calcium and calmodulin in the action of growth-hormone-releasing factor

A possible role for Ca 2⁺ and calmodulin in the action of growth-hormone-releasing factor (GHRF) was investigated . Low extracellular Ca²⁺ (<100 M), methoxyverapamil, flunarizine, cinnarizine, and Co²⁺ decreased both basal and GHRF-stimulated growth-hormone secretion, but did not totally inhibit GHRF-stimulation secretion. A calmodulin antagonist, W7, abolished GHRF-stimulated GH secretion, with no effect on basal secretion. It is suggested that GHRF may act primarily by elevating cellular cyclic AMP, which may then modulate calcium mobilization or flux; the increased intracellular Ca²⁺ concentrations may then activate calmodulin.     

Investigations on myelinogenesisin vitro: II. The occurrence and regulation of protein kinases by thyroid hormone in primary cultures of cells dissociated from embryonic mouse brain

The occurrence and regulation by thyroid hormone of four protein kinases (cyclic AMP independent and dependent, calcium/calmodulin stimulated, and calcium/phosphatidyl serine stimulated protein kinases) was studied in primary cultures of cells dissociated from embryonic mouse brain. Serum from a thyroidectomized calf, which contained low levels of L-3,5,3'-triiodothyronine, T₃ (<25 ng/100 ml), and thyroxine, T₄ (<1 g/100 ml) was used in the culture medium in place of normal calf-serum (T₃, 130 ng/100 ml; T₄ 5.9 g/100 ml) to render the cultures responsive to exogenously added T₃. Cultures grown in hypothyroid calf-serum containing medium had less cAMP dependent and independent protein kinase activity than control cultures grown in normal calf-serum containing medium. However, this activity was restorable to a considerable degree if the cultures grown in hypothyroid calf serum containing medium were supplemented with L-3,5,3'-triiodothyronine (T₃). The presence of calcium/calmodulin stimulated protein kinase was also distinctly observed. In comparison, the activity of calcium/phosphatidyl serine stimulated protein kinase was less than the other protein kinases.     

Paracoccidioidomycosis in the region of Botucatu (state of São Paulo,Brazil). Evaluation of serum thyroxine (T4) and triiodothyronine (T3) levels and of the response to thyrotropin releasing hormone (TRH)

T₄, T₃ and TSH serum levels were measured in 25 patients with paracoccidioidomycosis. Thyroid T₃ reserves were measured on the basis of the increase in T₃ (T₃) 2 h after intravenous injection of 200 g TRH, and pituitary TSH reserves were measured on the basis of TSH increase (TSH) 20 min after the same injection. Twenty healthy volunteers with no history of thyroid disease were used as controls. When the two groups were compared, the following results were obtained: (a) there was no significant difference in mean T₄, T₃, TSH between groups; (b) reduced T₃ levels were detected more frequently in patients with paracoccidioidomycosis, especially among those with the acute form of the disease or with the severely disseminated chronic form. The results suggest the occurrence of a reduction in peripheral conversion of T₄ to T₃, but do not indicate the occurrence of hypothyroidism in any of its forms (thyroid, pituitary or hypothalamic).     

Genetics of heat tolerance and thyroid function in Athens-Canadian randombred chickens

Summary Five experiments were conducted to assess the genetic variation in thyroid function (T₃, T₄), body weight and heat stress survival time in chickens. Thyroxine (T₄) levels were found to be elevated in response to 4 and 8 g bovine thyroid stimulating hormone (TSH) in experiment I. In experiment II, 4 g of TSH was injected into chickens from 30 sire families of the Athens-Canadian Randombred population. The heritability of T₄ levels after TSH injection was high. In experiment III, families identified as having innate high or low T₄ levels after TSH injection and a group of control birds were subjected to a heat Stressor of 50 °C for up to 240 min at six weeks of age and heat stress survival time was studied. The groups did not differ from each other in heat stress survival time. Experiment IV was similar to experiment I except triiodothyronine (T₃) was also measured after TSH injection. Both T₄ and T₃ levels after TSH injection were moderately heritable. In experiment V birds were reared to six weeks of age and heritability calculated for body weight, T₄, T₃, and heat stress survival time. Heritabilities were high for body weight, moderate for T₄ and heat stress survival time, and low for T₃. Phenotypic correlations were significant and negative for heat stress survival time with body weight and T₄, and for body weight with T₃ after TSH. Significant positive correlations were found for T₄ with T₃ after TSH and also T₄ and body weight. Analysis of genetic correlations suggested that none of the traits studied would be an adequate selection parameter for achieving heat tolerance without reducing body weight.Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia     

Effect of chronic desipramine treatment on neurotransmitter-thyrotropin releasing hormone—thyrotropin interactions in the rat

Long-term administration of the antidepressant drug, desipramine (20 mg/kg/day, orally for 28 days), decreased the stimulatory effect of the ₂-adrenoceptor agonist, clonidine (250 g/kg, i.p.) on thyrotropin (TSH) secretion in the rat, but did not alter basal TSH secretion. -Adrenoceptor-mediated inhibition of TSH secretion by isoproterenol (1 mg/ kg, i.p.) was unaffected by chronic desipramine treatment, as were the stimulatory effect of TSH-releasing hormone (TRH, 5 g/kg, i.v.) on TSH release and its inhibition by the -adrenoceptor antagonist, phentolamine (2 mg/kg, i.p.). These findings suggest that chronic desipramine treatment induces subsensitivity of ₂-adrenoceptors which modulate TSH secretion in the rat while not affecting -adrenoceptor-mediated inhibition of TSH release. These findings suggest that pituitary TRH receptors are unchanged but that changes occurred at the hypothalamic level in ₂-adrenoceptor-mediated stimulation of TRH release. Although cerebral -adrenoceptors have been shown convincingly to be down-regulated after chronic desipramine treatment, their function in the hypothalamic TRH system after 28 days of treatment with desipramine appears to be unimpaired.     

Effects of ATP and UTP in pheochromocytoma PC12 cells: Evidence for the presence of three P2 receptors,only one of which subserves stimulation of norepinephrine release

Summary 1. In pheochromocytoma PC12 cells ATP and, to a lesser extent, 2-methylthioATP stimulate phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, leading to stimulation of norepinephrine release. In contrast, although UTP also stimulates phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, there is no stimulation of norepinephrine release.2. 2-MethylthioATP, presumably acting at P_2y receptors, and UTP, presumably acting at P_2u receptors, in combination elicit a phosphoinositide breakdown greater than that elicited by either alone. Intracellular levels of calcium measured with Fura-2 increase to greater levels with ATP than with UTP and are sustained, while the UTP intracellular levels of calcium rapidly return to basal values. Both ATP and UTP cause a similar influx of⁴⁵ Ca²⁺ presumably by stimulation of a P₂ receptor directly linked to a cation channel.3. It is proposed that PC12 cells contain two distinct G protein-coupled P₂ receptors that activate phospholipase C and a P₂ receptor linked to a cation channel. The P_2y receptor sensitive to ATP (and to 2-methylthioATP) causes the depletion of a pool of intracellular calcium, sufficient to activate so-called receptor-operated calcium entry. The sustained elevation of intracellular calcium after ATP treatment is proposed to result in stimulation of norepinephrine release and activation of calcium-dependent potassium channels and sodium-calcium exchange pathways.4. The P_2u receptor sensitive to UTP (and to ATP) causes only a transient elevation in levels of intracellular calcium, perhaps from a different pool, insufficient to activate so-called receptor-operated calcium entry. Further sequelae do not ensue, and the functional role of the UTP-sensitive P_2u receptor is unknown.     

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the inhibitory effect of veratridine (a Na⁺ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 M veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P₄ formation from Ins 1,4,5-P₃.Abbreviations used Ins 1,4,5-P₃ inositol 1,4,5 trisphosphate - Ins 1,3,4,5-P₄ inositol 1,3,4,5-tetrakisphosphate - PMA phorbol 12-myristate 13-acetate     

Acute effect of thyroid hormones in mimicking photoperiodically induced release of gonadotropins in Japanese quail

Summary Photoperiodic induction occurs in Japanese quail after exposure to a single long day and this leads to a wave of pituitary LH secretion which lasts for up to 10 days. Pharmacological doses of thyroid hormones mimic this photoperiodic response if given to quail on short days, the magnitude and duration of the rise in LH and FSH output being dose-dependent. Thyroxine (T₄) is some 7 times more potent than tri-iodothyronine (T₃). There is no effect of T₄ on LH secretion in quail already on long days although such birds can increase LH output markedly if treated with Gn-RH. Testosterone prevents the initial rise in LH secretion following T₄ but does not block the long-term effect, suggesting that T₄ acts high in the photoneuroendocrine chain to mimic long days. The first rise in LH secretion following T₄ injection takes place about 24 h after the injection and the time-scale of secretion is quite similar to that seen when quail are exposed to a long day. T₄ elicits a rise in LH secretion even if the quail are maintained in darkness. However, T₄ does not act simply as light for if it is given at the exact time when birds are in a photoinducible state (i.e. 12–16 h after dawn) the rise in LH secretion still occurs 24 h later.Abbreviations FSH follicle stimulating hormone - Gn-RH gonadotropin releasing hormone - LH luteinizing hormone - T ₄ thyroxine - T ₃ tri-iodothyronine     

The effect of selective noradrenergic denervation on thyrotropin secretion in the rat

The effect of DSP₄ [N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine], a neurotoxin which selectively lesions noradrenergic projections from the locus coeruleus, on thyrotropin (TSH) secretion was investigated in the rat. DSP₄ treatment (60 mg/kg injected i.p. 10 days prior to experimentation) significantly decreased the noradrenaline (NA) content of the hippocampus, frontal cortex and hypothalamus of the rat brain. DSP₄ treatment did not affect the clonidine (250 g/kg, i.p.) or TSH-releasing-hormone (TRH 5 g/kg i.v.) induced stimulation or the isoproterenol induced inhibition of TSH secretion in the rat. These results suggest that the noradrenergic projection from the locus coeruleus to the hypothalamus does not play a significant role in the regulation of TSH secretion. Furthermore, the noradrenergic deficiency did not give rise to the development of the abnormal TSH response to TRH administration which is frequently observed in depression.     

Effects of ovine prolactin on iodide metabolism and thyroid activity in the eel

Summary In the eel, ovine prolactin (oPrl) treatment (0.018 IU/day·g body weight), for 8 to 13 days modifies neither iodide absorption from the water nor excretion, extrathyroidal metabolism and plasma level of iodide.Thyroid activity, evaluated by epithelial cell height, radioiodine uptake and absolute iodide uptake is approximately twice that of controls. However, the amounts of total iodine, thyroxine (T₄) and triiodothyronine (T₃) in thyroid are unaltered by oPrl. Therefore, the decrease of plasma T₄ and the increase of plasma T₃, previously observed in oPrl-treated eels, do not result from a preferential thyroidal secretion of T₃, but only from a stimulation of peripheral conversion of T₄ to T₃. Furthermore, the increased thyroid activity probably originates from a decreased feedback inhibition following the fall of circulating T₄ induced by oPrl.Abbreviations oPrl ovine prolactin - T ₄ Thyroxine - T ₃ 3.5.3 triiodothyronine - TRH thyrotropin releasing hormone - TSH thyroid stimulating hormone - PBI protein bound iodine     

Calcium- and calmodulin-independent modulation of calmodulin-sensitive hypothalamic cyclic nucleotide phosphodiesterase activity by the (11–19) fragment of thymosin β4

A fragment (11–19) of thymosin 4 was found to stimulate phosphodiesterase activity even in the absence of calcium and calmodulin. Half-maximal enzyme activation occurred with 10 nM peptide, and was further increased by phospholipids such as phosphatidylserine. The mechanism of stimulation is an increase in the V_max of cAMP degradation without a substantial change in the K_m for the substrate. In the presence of calcium ions and calmodulin the peptide was also stimulatory.     

Measurement and regulation of thyroidal status in teleost fish

Summary We have reviewed the stages in teleost thyroid function and its regulation, from the initial biosynthesis of the TH to their eventual interaction with putative receptors.TH biosynthesis depends on an adequate plasma iodide level, determined partly by dietary iodide and partly by active branchial iodide uptake from the water, Pulse-injected radioiodide can be used to evaluate thyroidal iodide uptake, aspects of TH biosynthesis and TH thyroidal secretion. However, owing to variable plasma iodide levels, care is required in interpretating these parameters. TH biosynthesis, thyroglobulin properties and intrathyroidal secretion mechanisms have received limited recent attention. Histological indices of thyroid tissue changes, while useful in many situations, do not always correlate with more direct estimates of thyroidal secretion and can be misleading.Thyroid function is regulated by the hypothalamo-pituitary-thyroid axis, but neither the identities of the hypothalamic factors nor a reliable immunoassay for TSH have been established. Currently, activity of the hypothalamic-pituitary axis is usually determined by pituitary thyrotrope histological appearance or bioassay of pituitary TSH. Plasma free T₄ feeds back at both the pituitary and hypothalamic levels and inhibits TSH release. Thyroidal T₄ secretory activity is presumably adjusted to maintain a constant plasma T₄level according to physiologic state.Plasma T₄ is probably the most commonly used index of thyroidal status. However, (1) T₄ is probably not the active form of TH, (2) the T₄ plasma level may be influenced by the binding properties of plasma proteins, and (3) the T₄ concentration alone makes no provision for the rate of T₄ turnover in plasma. The most practical way to measure thyroidal T₄SR is to determine plasma T₄DR, and assuming steady-state conditions, equate it to T₄SR. The T₄DR is determined from kinetic studies employing^*T₄, which also enable estimates of sizes of vascular and extravascular T₄ pools and their rates of exchange. Excretion of T₄ or its derivatives in urine or bile can be determined also. A high proportion of T₄ is enzymatically monodeiodinated in liver and other tissues, generating T₃ for local (intracellular) and vascular systemic compartments.Bothin vivo andin vitro methods have been used to quantify T₄ deiodinase activity, which is highly responsive to physiologic state and environmental variables. T₃ production is inhibited by a moderate T₃ excess indicating an autoregulatory system, whereby tissue T₃ levels are maintained at a set-point appropriate for a particular physiologic state. The rate of T₃ production provides an informative measure of thyroidal status in a given tissue. However, other pathways also contribute to the maintenance of T₃ homeostasis at a particular set-point. These include the rate of T₃ degradation to 3,3-T₂, the rate of T₄ substrate diversion to rT₃ (an inactive isomer) and by the excretion of parent compounds or conjugates in bile and urine. Potential losses across branchial or integumentary surfaces have yet to be evaluated.The most fundamental measure of thyroidal status is represented by the amount of T₃ saturably bound to receptors/nucleus for the cell type of interest. This is estimated most accurately in double isotope studies in which T₃ contributions from both vascular and intracellular compartments are evaluated. Less satisfactory but meaningful indices of T₃ availability to receptor sites may be obtained from the plasma T₃ (or free T₃) level and from the tissue T₃ level. The former is appropriate if the cell type in question obtains its T₃ primarily from plasma; the latter should be measured if the cell type derives its T₃ mainly through intracellular deiodinase activity. If the proportion of vascular T₃/intracellular T₃ bound to receptors is known, it may indicate the degree of receptor activation. However, even cytosolic T₃ levels may not vary in proportion to nuclear T₃ levels.Differences in thyroidal function between teleosts and homeotherms can be attributed to distinctive strategies in iodide economy and to fundamental differences in control of thyroidal status. Owing to more certain iodide availability (branchial iodide pump and plasma iodide-binding proteins), teleosts are probably more liberal in their iodide use and have less efficient mechanisms for recovery and retention of hormonal iodide than homeotherms. Also, primary control of teleost thyroidal function appears peripheral. It is the finely regulated conversion of T₄ to T₃ in tissues which may largely determine the T₄ secretion rate. Thus, T₄, as a prohormone, may be produced more to satisfy the substrate needs for T₄ conversion rather than to drive T₃ production. Because TH are mainly implicated in tissue- or cell-specific processes involved in development, growth and reproduction in teleosts, it may be advantageous for their thyroidal status to be determined locally through T₄-to-T₃ deiodination. In homeotherms, primary control is mainly central through the hypothalamic-pituitary axis, which regulates thyroidal secretion of T₄ and significant amounts of T₃. The level of T₄ (free T₄) is believed to drive the production of T₃ in most peripheral tissues. Because TH are extensively involved in the systemically integrated adjustment of basal metabolic rate in homeotherms, it may have been advantageous to evolve a system leaning towards central control by the hypothalamus, the brain centre associated with thermoregulation.     

Evidence for the involvement of calmodulin in natural cytotoxicity using a range of calmodulin antagonists of varying potency and improved specificity

The calmodulin antagonist W7 and 4 of its analogues were examined for their ability to inhibit human NK cell mediated cytotoxicity. With the exception of one of these compounds, which is extremely hydrophobic, there was a good correlation between the ability of drugs to inhibit human NK antitumour cytotoxicity and calmodulin-dependent phosphodiesterase activity in vitro. The most potent of the compounds, 5-iodo-l-C₈, an analogue of W7, has an IC₅₀ of 3 M upon biological and biochemical assay. This particular compound is both more potent and specific than the parent compound W7, is non-toxic to cells over the range used and is also capable of inhibiting the biological activity of NK cells upon pre-treatment of the effector cells, inferring the mechanism of NK cytotoxicity to be calmodulin dependent.     

Interaction between perchlorate and nifedipine on insulin secretion from mouse pancreastic islets

In order to elucidate the mechanisms responsible for the stimulatory effect of perchlorate (ClO ₄ ^– ) on insulin secretion, we have investigated the interaction between this chaotropic anion and the organic calcium antagonist nifedipine. This drug, known as a blocker of L-type calcium channels, was chosen as a tool to test the idea that ClO ₄ ^– acts on insulin secretion by stimulating the gating of voltage-controlled Ca²⁺ channels. ClO ₄ ^– amplified the stimulatory effect of D-glucose on insulin release from perfused pancreas (first and second phases) as well as from isolated islets incubated in static incubations for 60 min. This indicates that ClO ₄ ^– amplifies physiologically regulated insulin secretion. Nifedipine reduced D-glucose-induced (20 mM) insulin release in a dose-dependent manner with half-maximum effect at about 0.8 M and apparent maximum effect at 5 M nifedipine. In the presence of 20 mM D-glucose, the inhibitory effects of 0.5, 1 or 5 M nifedipine were only slightly, if at all, counteracted by perchlorate. When 12 mM ClO ₄ ^– and 20 mM D-glucose were combined, calculation of the specific effect of ClO ₄ ^– revealed that nifedipine produced almost maximum inhibition already at 0.05 M. Thus, the perchlorate-induced amplification of D-glucose-stimulated insulin release shows higher sensitivity to nifedipine than the D-glucose-effect as such. This supports the hypothesis that perchlorate primarily affects the voltage-sensitive L-type calcium channel in the -cell.     

Purinergic agonists stimulate the secretion of endothelin-1 in rat thyroid FRTL-5 cells

The aim of the present study was to investigate the mechanisms regulating endothelin-1 (ET-1) secretion in rat thyroid FRTL-5 cells. ET-1 was found to be secreted after stimulation with adenosine and ATP. The release of ET-1 was sensitive to pertussis toxin, indicating a role of G-proteins in the stimulus-secretion coupling. The stimulation evoked by ATP or adenosine was inhibited by the P₁-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and in the presence of adenosine deaminase the adenosine- and ATP-mediated ET-1 secretion was abolished. These evidences suggest a role of a P₁-adenosine receptor in the secretion of ET-1. Increasing cyclic AMP with forskolin decreased the adenosine-mediated secretion. In addition, the intracellular calcium chelator BAPTA or inhibition of calcium entry with Ni²⁺ prevented the response. Protein kinase C (PKC) is also partly involved in ET-1 secretion in FRTL-5 cells. Activation of PKC with the phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated the secretion of ET-1 in a time- and dose-dependent manner. Furthermore, downregulation of PKC decreased the secretion of ET-1 stimulated by adenosine. In conclusion, ET-1 secretion in FRTL-5 cells is stimulated via a pertussis toxin-sensitive P₁-receptor pathway which is modulated by several signal transduction mechanisms including cAMP, Ca²⁺, and PKC. © 1996 Wiley-Liss, Inc.     

SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kγ Activity through Deacetylation of Specific Lysine Residues in Mammals

Background

SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there.

Methodology/Principal Findings

Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)γ was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kγ and enhanced PIP5Kγ enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kγ knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kγ, PI(4,5)P₂, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice.

Conclusions/Significance

Our findings indicated that the control of TSH release by the SIRT1-PIP5Kγ pathway is important for regulating the metabolism of the whole body.     

Further studies on the regulation of the Harderian glands of golden hamsters by the thyroid gland

Summary Long-term increased or decreased circulating levels of thyroid hormones significantly modify porphyrin concentrations and morphology in the Harderian glands of male and female hamsters. Administration of T₃ reduced porphyrin concentrations in females; this treatment or decreasing thyroid hormone levels with KClO₄ suppressed the post-castration rise of porphyrins in males. Hypophysectomy led to increased porphyrins in the Harderian glands of males; this rise was suppressed in hypophysectomized males by T₃ or T₄. In females, hypophysectomy reduced porphyrins which were further reduced by daily administration of T₃ or T₄. These modifications in the normal females were identical in castrated males. Mitotic activity in the Harderian glands of females was stimulated by KClO₄ and by hypophysectomy with or without exogenous T₃. In males, castration increased mitotic activity which was suppressed by T₃ and exacerbated by KClO₄. Increased mitotic activity seemingly follows loss of tissue mass. The data show that thyroid hormones act directly on the Harderian glands rather than indirectly through modification of TSH synthesis/release. Female type glands in males are a consequence of loss of gonadal androgens by castration, or by suppression or loss of thyroid hormones by hypophysectomy or by treatment with KClO₄. However, male type glands in females are the result of androgen treatment, and/or increased levels of thyroid hormones via reduced ambient temperatures or of photic input. We conclude that regulation of the Harderian gland appears to be different in the two sexes.Abbreviations T ₃ Triiodothyronine - T ₄ Thyroxine - TSH Thyroid Stimulating Hormone - KClO ₄ Potassium Perchlorate - h hours - ml milliliter - mg milligram - g gram - male - female - castrated male - AP hypophysectomized - CON Control - ALA delta aminole-vulenic acid - HG Harderian Gland     

The inhibitory effects of calmodulin antagonists on TSH-stimulated thyroid hormone release by mouse thyroid lobes

Calcium ions have been shown to play a mojor regulatory role in the release of various hormones from a wide variety of endocrine organs. More recently, in vitro evidence suggests that a calcium-binding protein, calmodulin, is also involved in the release of many hormones. So we examined the effects of several types of calmodulin antagonists on TSH-stimulated thyroid hormone release in vitro. Mouse thyroid lobes (one thyro-tracheal unit/tube) were incubated in Krebs-Ringer bicarbonate buffer at 37 degrees C for 4h. Free thyroxine (fT4) released in the incubation medium, thyroidal cAMP and calmodulin content were measured by RIA. TSH (5 mU/ml) and dibutyryl cAMP (DBC) (200 micrograms/ml) caused a 2-4 fold increase in thyroidal release of fT4. The stimulatory effects of TSH on fT4 release were significantly inhibited by trifluoprazine and prenylamine lactate at the concentration of 5 X 10(-5) M. More specific calmodulin antagonists, W-7 and W-13, were also shown to inhibit TSH stimulation of fT4 release at the concentration of 5 X 10(-5) M. In contrast, TSH stimulation of fT4 release was not depressed by non-specific antagonists, W-5 or W-12, at the same concentration as 5 X 10(-5) M. Further, W-13 also markedly inhibited DBC-stimulated fT4 release. Neither TSH nor PGI2 altered the thyroidal calmodulin content, dissociating with a marked increase in the cAMP concentration. These results suggest that calmodulin plays an important role in TSH-stimulated thyroid hormone release and further that this mechanism exists, at least in part, at the site subsequent to the generation of cAMP.